New Aspects of the Ultrastructure of Frog Rod Outer Segments

New Aspects of the Ultrastructure of Frog Rod Outer Segments J ~ G E NROSENKRANZ

Lehrstuhl f u r Zellmorfologie der Ruhr-Universitat Bochum, Bochum, West Germany I. Introduction . . . . . . . 11. The Rod as a Constituent of the Retina . . A. Implantation of the Rods in the Optocoelium B. Frequency and Dimensions of the Rods . C. TheRodasPartoftheRodCell . . . 111. Chemical Composition of the Rods . . A. Water . . . . . . . . B. Proteins . . . . . . . C. Lipids . . . . . . . D. Saccharides . . . . . . . IV. Ultrastructure of the Light-Adapted Rod . . A. TheCellMembrane . . . . . B. The Connecting Cilium. . . , . C. Apical Microvillous Processes . . . D. The Lamellar B o d y . . . . . . E. TheRimsoftheLamellae . . . . F. Rod Cytoplasm. . . . . . . V. The Dark-Adapted Rod. . . . . . A. Details of Dark-Adapted Rods and Location of Fuscin . . . . . . . . B. Ultrastructure of the Dark-Adapted Rod . C. Results Obtained from Isolated Rhodopsin . VI. Changes in Rod Ultrastructure with Time . . A. Development into a Mature Rod . . . B. Constant Renewal of a Rod in the Adult Frog C. Diffusion ofRhodopsin . . . . VII. The Green Rod . . . . . . . A. Characteristics . . . . . . . B. Ultrastructure . . . . . . C. Renewal . . . . . . . VIII. Summary . . . . . . . . Appendix 1: Extreme External Influences and Their Consequences . . . . . . . . A. Changes in Rod Structure Affected by Osmotic Shocks . . . . . . . B. The Behavior of the Rods in a Magnetic Field Appendix 2: On the, Limitations of the Experimental Techniques Used . . . . . . A. Electron Microscope Preparation Techniques . B. Diffraction Methods . . . . . . References . . . . . . . . .

.

.

.

.

.

.

.

25

26 27 27 29 32 32 32 34 42 47 47 49 55

56

57 114 117 119 119 120 124 125 125 129 129 136 136 136 137 137 139 139 142 147 147 151 154

26

P R G E N ROSENKRANZ

I. Introduction

Rod cells are those cells in the frog retina that, together with the less numerous cone cells, form the sclerad border of the retina. The part of the cell directed toward the pigment epithelium and situated outside the actual neuroretina has a cylindrical or rodlike shape and is therefore called the rod outer segment. In the frog there are two kinds of rod cells: red ones, which are more numerous, and green ones. They differ in the following ways: (1)the inner segment of a red rod cell has a diameter comparable to that of the outer segment, while the inner segment of a green rod cell is much thinner than its outer segment; (2) in the retina the green rod cells are situated more sclerad than the red ones; and (3)the (initially dark-adapted) green rod cells appear grayyellow when observed from above in dim white light (color temperature of daylight), while the red ones appear red-orange. In the following discussion the term rod is used for the red rod outer segment of the frog, and green rod refers to the green rod outer segment of the frog. The general expression frog instead of Rana catesbeiana, R . esculenta, R . pipiens, or R. temporaria seems justified in this connection, as our electron microscope investigations of ultrathin sections did not show significant differences in the supramolecular ultrastructure of the rods of these four species. Morphological differences are given in Table 11. The only recent review that deals primarily especially with the fine structure of the rod outer segments of the frog was written by Worthington (1974).While in Worthington’s article stress is laid on x-ray diffraction as the investigation method, the following workers describe studies of outer segments of various vertebrates by complementary methods, essentially biochemical and electron microscopical: Young (1969), the whole rod cell (structure and function); Borovjagin et al. (1971),the photoreceptor membrane (structure and function); Cohen (1972), supramolecular morphology; Abrahamson and Fager (1973) and Daemen (1973), biochemistry; Ebrey and Honig (1975), visual pigment. The work on the frog rod outlined in the following discussion refers to experimental results from the last 5 years (ca. 1970-1975). In this article we have tried not only to collect observations on rod ultrastructure but to examine them critically, present them in a balanced and understandable form, and interpret them synoptically.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

27

11. The Rod as a Constituent of the Retina

A. IMPLANTATION OF THE RODS IN THE OPTOCOELIUM

The rods are located exclusively in the primary optocoelium and a t least parts of their surface are therefore in contact with the cerebrospinal fluid. Other parts of their surface may have closer contact with the numerous microvillous projections than with the fluid. A marked circulation around the rod seems unlikely, because only about 10% of the optocoelium is free of light receptors and microvillous projections. This figure is obtained when dense, hexagonal packing of the projections is assumed for the estimation. This assumption is justified judging from scanning electron micrographs by Steinberg (1973) and micrographs of ultrathin sections of this area by Rohlich (1970).The projections effect about a 200-fold increase in the pigment epithelium surface in contact with the optocoelium. On illumination the melanin pigment, fuscin, which is present in the pigment cells, passes into the microvillous projections but leaves them when illumination is removed (Section V,A). In electron spin resonance experiments Cope et al. (1963) found a reversible increase in radicals after illumination of fuscin granulas in a cattle melanin suspension. The radicals were stable at pH 2 7.0. Because of the polyquinone structure of the melanins, these investigators assume that the radicals are semiquinone radicals. The optocoelium has no direct contact with blood cells; it is separated from the aorta ciliaris in the lamina choriocapillaris by the lamina basalis and the pigment epithelium, and it is separated from the aorta hyaloidea by the membrana limitans interna and the layers of the actual retina. The optocoelium contains, besides rod outer and inner segments and microvillous projections, a compound of mucopolysaccharides and proteins as a matrix which, together with the cerebrospinal fluid, fills all gaps between the cell parts previously mentioned. Mucopolysaccharides form an essential part of this compound, as shown b y the results of electron microscope and histochemical investigations b y Rohlich (1970), who treated frog retinas with collodial ferriammonium glycerate (pH 1.0-1.2) as well as phosphotungstic acid (PTA) (pH 0.5). In both cases, the above-mentioned gaps became stained. This did not occur when the staining process was blocked by methylation. The results of the first treatment (collodial ferriammonium glycerate) indicate the existence of anionic groups of acidic

28

J~~RGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

29

mucopolysaccharides in the matrix; and the unpublished observation was found, suggests sulfomucopolyof Young ( 1969), that saccharides. The results following treatment by PTA suggest the presence of glycoproteins, the hydroxyl groups of which are responsible for this reaction (Rambourg, 1967). Ocumpangh and Young (1966) found that in the rat 70% of the sulfopolysaccharide is soluble in hyaluronidase; but as the hyaluronidasesoluble fraction varies in different classes, this result capnot be extrapolated to the frog without further experiments. This is also the case when considering the presence of the mucopolysaccharide in the optocoelium; in the rat its half-life is 2.5 days. The staining of this matrix with uranyl acetate is certainly due to the carboxyl and hydroxyl groups incorporated in the pol ysaccharide structure (Rothstein and Meier, 1951).

B. FREQUENCY AND DIMENSIONS OF THE RODS The distribution of the light receptor cells (Fig. 1) in the retina is neither homogeneous nor radially symmetric, for example, in the pa-

pilla optica; but if one averages the number of single receptor cells along each radius, the present author states that the number of red rods is smaller in the central part of the retina than in the peripheral area, while the opposite is true for green rods and cones. The normal average number of red and green rods and cones is listed in Table I. The deviations are caused by differences in the number of receptors from section to section (thickness ca. 5 km). The total number of light receptors in one retina was calculated to be (2 f 0.2) x 106 on the basis of data from R. esculentu. The outer dimensions of the cylindrical rods differ depending on their position, for example to the papilla optica. In Table I1 the respective values are listed for light-adapted frogs. Based on data from R. esculentu, the expected value from Tables I and I1 for the length of the fixed rods is 1' = (0.89)(28) + (0.11)(18.5)= 27pm, and for the rod diameter is d ' = (0.89)(5.75)+ (0.11)(4.9) = 5.6 pm. These values are based on measurements of rods fixed with Bouin stain. If there has been no shrinkage during preparation, the expected values should be equal to experimentally determined average values of 1 and d of rods in Ringer's suspension containing red as well FIG.1. Three dimensional view of part of the neuroretina of a bullfrog. In the receptor layer a cone (sc),a green rod (gr),a red rod (=),and a double cone (dc) are shown, followed vitread by the outer nuclear layer (on), the outer plexiform layer (op), and the inner nuclear layer (in). Glutaraldehyde fixation, critical-point drying, plated with gold. Scanning electron micrograph (Kent-CambridgeS4), 20 kV; the X2300 magnification is approximate, as is the case with all scanning electron micrographs. Bar, 5 pm. Reproduction from Steinberg (1973).

TABLE I PERCENTAGE OF LIGHT RECEPTORS

0

Red rods

Green rods (%)

Cones

-

58.5'

12.1'

29.4'

-

77.5

2.5

20.0

215 4566

50.7

80 ? 3

14.4 10?4

Species

Counted

Kana temporaria

0

Rana pipiens Rana esculenta

(%)

" Throughout this article an asterisk

34.9 10 & 3

IN THE

RETINA''

Section of retina Area centralis retinae Upper part, near era serrata Posterior pole Mean value for whole retina

Reference Krause (1892) Krause (1892) Nilsson (1964a) J. Rosenkranz (unpublished results, 1975)

(') denotes data measured or calculated from a figure or table in the reference cited.

TABLE I1 DIMENSIONS O F THE RODS

Species

Rana fusca Rana pipiens Rana esculenta

t2

Rana catesbeianu Rana pipiens Rana temporaria T:

Preparation for

Electron microscopy Light microscopy Bouin, Mayer’s hemalum erythrosin Ringer’s solution (227 mosM, pH 7.2)

Number of measurements

Distance from section to papilla (pm)

-

Various Various f1580 p m

-0 pm -1150 pm -2950 pm Suspension

2 10

10 10 10

Red rod Length

Diameter (pm)

Length (pm)

54-60 27-45

38 & 1

6-7 6.0-8.0 6.0 5.8 i0.2

34.4 20-30 -

28 2 1 28 f 1 19 f 1 45 f 7ra

5.8 i0.3 18 f 1 4.9 f 0.2 5.7-0.2 19 1 4.9 2 0.2 3.7f0.1 6.8 0 . 1 ~30 f 4T 5.8 3 Z O.lT

(pm)

-

Rod length 116 50

50 50

Suspension Suspension Suspension Suspension

calculated from the following line and Table I.

Green rod

*

43 0.6 64 1.0 58 1.0 48 f 0.5

* *

Diameter (pm)

6.1 5.5-6.5

-

*

Rod diameter

6.7 k 0.08 7.3 f 0.2 6.9 k 0.1 6.9 0.1

*

Reference Krause (1892) Nilsson (1965)

J. Rosenkranz (unpublished results, 1975)

32

JURGEN ROSENKRANZ

as green rods; in suspensions, the two types cannot be distinguished from one another by the light microscope after illumination. We have measured the dimensions of 116 rods of R. esculentu in amphibian Ringer's solution, finding 1 = 43 f 0.6 pm and a! = 6.7 0.08 pm. This means that considerable shrinkage occurred as a result of the Bouin fixation. From these measured values the length and diameter of the red and green rods (in uiuo) were calculated assuming a constant length ration of both rods, r in Table 11.

*

c.

THE ROD AS PART OF THE ROD CELL

In Fig. 2 the essential parts of a red and a green rod cell together with their surroundings are shown half-schematically but in true dimensions. For details see the figure legend. 111. Chemical Composition of the Rods For a deeper understanding of the rod structure knowledge of the kind and quantity of chemical compounds in the rods is necessary. Thus far chemical analysis has been performed only on isolated receptors. Criticisms of the method are that (1) the chemical analysis does not differentiate between the outer segments of red and green rod cells and cone cells; (2)probable differences in the chemical composition of the cell membrane and lamellar membrane are not taken into account; and (3) sometimes the ellipsoids of the inner segments with their mitochondria still stick to the rods, thus possibly leading to impurities in the preparation. These factors must certainly be considered in future attempts at rod description. Here it is assumed that the results of chemical analysis can be ascribed only to the lamellar body. Chemical analysis has shown that in the rod the following substances are represented: proteins, lipids, and cholesterol. The carbohydrates in the rod are assumed by different investigators to be bound to different substances: to a protein, glycoprotein (Heller, 1969); to a lipid, glycolipid (Eichberg and Hess, 1967; Masonet al., 1973),or possibly to a lipoprotein, glycolipoprotein (Young, 1969). The quantities of the most frequently occurring substances in the rod are listed in Table 111. This table clearly shows a special property of the lamellar membrane: The molar ratio of cholesterol to phospholipids here is only 0.1 and thus strongly deviates from that of an average cell membrane, which is z 0.5 (Kom, 1967).

A. WATER The major part of the rod volume V consists of water; its volume Vw is calculated by subtracting the lipid volume VL and the protein vol-

FIG.2. Two frog rods, a red one on the left and a green one on the right, shown half schematically but true to scale together with their immediate surroundings: a pigment epithelial cell (Porter and Yarnada, 1960) sclerad; dendrites presumably of horizontal and bipolar cells (Evans, 1966; Dowling, 1968) vitread; microvillous projections of pigment epithelial cells lateral. Large parts ofrod nucleus regions, the rod fiber and the pedicle are surrounded by Muller cells, and smaller parts by other receptor cells (Nilsson, 1964a; J. Rosenkranz, unpublished results, 1975).

34

J f h G E N ROSENKRANZ

TABLE I11 PROTEIN, LIPID, AND CHOLESTEROL CONTENT IN PERCENTAGE OF TOTAL ROD DRY MASS Lipids Protein

Phospholipids

Glycolipids

Cholesterol

Species

59.4 60.4

26.6 29.5

9.5 10.1

1.7 2.18

R . pipiens R. catesbeiana

Reference Eichberg and Hess (1967) Mason et a[. (1973)

ume Vp from the total volume V. For this purpose we take the following rod dimensions from Sections II,B and IV,D,3,a: average length I = 45 pm; average diameter d = 6.8 pm; lattice constant a = 300 A. And, from Sections III,B,5 and C,5, VL= 250 pm3

Vp = 130 pm3

This leads to V = 1.52 x 103 pm3 and

Vw = V

-

(V,

+ Vp) = 1.140 pm3 = 0.75V

This means that ca. 75% of the rod volume consists of water. B. PROTEINS 1. The Amount of Protein As Table IV shows, the protein content of the rods is mainly determined by the visual pigment, rhodopsin. The importance attributed to this special protein is expressed also by the large number of studies on rhodopsin as compared to the very few studies concerned with the remaining nonrhodopsin proteins. As yet, proteins of this kind in the frog have been reported only sporadically; Bownds et al. (1971) write: “If isosmotically prepared M EDTA, outer segments are extracted sequentially with water, and 0.8 M NaCl . . . ,no measurable amount of protein material is removed.” Hall et d.(1969) state that, except for 3-5%, the nonrhodopsin proteins are soluble in hexadecyltrimethylammonium bromide (CTAB). Representative of the protein fraction in the frog rod that has not been investigated are the ATPases, which have already been found in other species. Measurements of the total protein content and especially the amounts of rhodopsin are compiled in Table IV.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

PROTEINS OF T H E

Total protein

ROD

Rhodopsin

TABLE IV IN PERCENTAGE OF

35

TOTALROD DRY MASS

Species

Reference

R. pipiens R . pipiens R . catesbeiana R. catesbeiana

Eichberg and Hess (1967) Hall et al. (1969) Bownds et al. (1971) Mason et al. (1973)

~~~

59.4 X Z

60.4

(0.80-0.85)x (0.82 0.1)~ > 50

Table IV shows that on the average 80% of all rod protein is rhodopsin. As the remaining protein has not yet been definitely identified, only rhodopsin is considered in the following estimations. Whether rhodopsin is a glycoprotein or a glycolipoprotein depends on whether it is regarded structurally as a chemical compound or as a functional unit in the visual process. In the latter case, the experiments of Shichi (1971) with cattle rhodopsin isolated with digitonin show clearly that in uitro only a phospholipid-rhodopsin complex has properties known to exist in rhodopsin in d u o . One must also consider that rhodopsin can be separated from this strong lipoprotein complex only by detergents that destroy the whole membrane. Among these detergents are Emulphogene BC-720 and Triton X-100 (Abrahamson and Fager, 1973). 2. Rhodopsin and its Components Chemically, rhodopsin is a glycoprotein, as the experiments of Heller (1969) have shown and as has been suggested by Robinson et al. (1972). The kinds and quantities of its amino acid residues are listed in Table V. Robinson et al. (1972) found 469.2 amino acid residues per retinyl group; this is almost twice as many as the 237 observed by Heller (1969).The difference of a factor of 2 may be due to a systematic error since, except in two cases (Val and Cys) Robinson et al. found twice as many residues of each amino acid as Heller. As Robinson et al. obtained equal values from different experimental methods, their values are perhaps more acceptable. Both investigators, however, confirmed a ratio of 1: 1 between polar and nonpolar amino acid residues, which could be of importance regarding the position of rhodopsin in the lamellar membrane. Heller (1969), furthermore, found that each rhodopsin molecule also contains three glycosamines and three neutral sugars and, as a prosthetic group, one retinylidene group; the latter was confirmed by Abrahamson and Fager (1973). The vitamin A, derivative retinylidene is bound to the apoprotein scotopsin as a pro-

TABLE V THE AMINO ACID RESIDUES OF THE RHODOPSINMOLECULE"

G h Leu Val

Ala

Phe

Ser

Thr Asp

Gly

Ile

Pro

Tyr

Lys Met Cys Arg His

20

18

18

17"

16'

15

156

14

13

10

1g6

1g6

15

gd

6d

6

4

42.5 37.5 27.6 35.5 31.8 39.3 30.5 35.9 31.8 23.7 22.0 23.2 22.2 16.0 18.0 14.5 9.2

Trp

Specks

Reference

Heller (1969p 8.0 R. catesbeiana Robinson et al. (1972)

4e

R. pipiens

Values are reported as residues per molecule. Duplicate samples were hydrolyzed in 6 N HCl at 110°C for 24, 48, and 72 hours. to infinite time. Values extrapolated to zero time. CyS0,H and MetSO,, determined on separate samples after performic acid oxidation (Moore, 1963). Determined on separate samples by titration with N-bromosuccinimide at p H 4.0 (Patchornik et al., 1958). Reprinted with permission from Biochemistry 8, 675 (1969). Copyright by the American Chemical Society. Glucosamine, determined on the long column of the analyzer after hydrolysis in 4 N HCl at 100°C for 6 to 10 hours. Neutral sugar, determined by the phenolsulfuric acid method (Dubois et al., 1956) with a mannose-galactose (2:1) mixture as standard. "

* Values extrapolated

'

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

37

tonated Schiff base (Pitt et al., 1955); the binding site is the €-amino group of a lysine residue. This has been shown in cattle rhodopsin by Bownds (1967). The coupling is thoroughly discussed by Ebrey and Honig (1975). 3. Molecular Weight and Extinction Constant of Rhodopsin Robinson et al. (1972) are the only workers who have recently newly determined the molecular weight of frog rhodopsin (R. catesbeiana). This was carried out using several different experimental methods. They state the probably most reliable value to be M R = 40,000. This value has generally replaced the value M k = 28,000 for R. pipiens (Heller, 1969), which was determined experimentally by only one method. Special difficulties involved in the determination of the molecular weight of the rhodopsin molecule are discussed by Abrahamson and Fager (1973). The value of the molar extinction constant €(A) of the frog rhodopsin has also been discussed. The molar extinction constant E (or absorption constant, since the scattering can be neglected when compared to the absorption) is important for calculation of the molar concentration cRof rhodopsin in the rod according to =

(2.30/cRd)log,,

@in/l@out)

where d = layer thickness of the solution investigated, Win = incident radiation, and WOut= emerging radiation. The constant E(l/cmat which in a state mole) is measured for the maximal wavelength,,A of dark adaptation maximal extinction occurs. According to studies by Liebman (1962) and Sidman (1958),,,A is 510-511 nm or about 508 nm in the isolated rod and about 502 nm in a solution of rhodopsin in digitonin (Sidman, 1958). While Heller (1969) assumed for the molar extinction constant e' = ~ ' ( 5 0 0=) 23,000 & 1000 for R. pipiens, the value E = 42,000 for the same species is preferable, this number resulting from the detailed investigations of Bridges (1971),who determined it in different ways (rhodopsin dissolved in digitonin and CTAB) and obtained approximately the same result. 4. Secondary Structure of Rhodopsin

Thus far there has been no agreement concerning suggestions as to the shape of the rhodopsin molecule. Until a few years ago the general opinion was almost exclusively in favor of a spherical shape. The diameter of the rhodopsin molecule is approximately 40 A, according

38

P R G E N ROSENKRANZ

to Blasie e t al. (1969) (this diameter probably represents the nonpolar nucleus of the visual pigment molecule); Nir and Pease (1975) assume 50-55 8, and state that: “really accurate measurements are not as yet feasible, because the interface between globules and the surrounding material was not as clearly and exactly defined with present techniques as would be desirable.” This statement is more thoroughly analyzed in Section IV,D. It may be sufficient to say here that it applies to numerous similar views which are partly simply assumptions concerning the shape of the rhodopsin molecule in order to interpret, for example, sedimentation constants. As is known, there is a theoretical basis for the evaluation of such data only for the most simple cases such as a sphere or an ellipsoid; but what is to be done when the object of the investigation is shaped differently must also be considered. Recently the existence of the rhodopsin sphere has been questioned by several investigators. Instead of a sphere, a mathematically more complicated body presenting rotational symmetry, an ellipsoid with two axes, is discussed as a possible shape. Wu and Stryer (1972)labeled cattle rhodopsin at sites A, B, and C of the rhodopsin molecule with different fluorescent chromophores; these labeling reagents exchanged energy as donors with the acceptor ll-cis-retinal. Site A of the rhodopsin was, for example, a sulfhydryl group to which the three following fluorescent compounds were acid, its bound: N-(iodoacetamidoethyl)-l-aminophthalene-5-sulfonic 1,8 isomer, and 5-iodoacetamidosalicylic acid. These investigators perhaps should have shown how the energy U of the dipole-dipole interaction between an excited molecule (a‘) and a molecule in the ground state (b),represented by (a’,b) + (a,b’), may be expanded to a multipole series. If only the first two terms, the dipole-dipole terms, are taken into account because Rab is large, the potential energy of dipole ma in the field of dipole mb is obtained:

where n = refractive index of the surrounding medium, Rab = distance between molecule a (donor) and b (acceptor), ma = transition moment [[map oscillator strength of transition between the ground and excited states], and aa = angle between ma and Rab. If

-

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

39

Forster (1965) showed that, in the case of predominant dipole-dipole interaction with weak coupling between molecules a and by the energy transition rate [Eq. (1)l is

Presumably Wu and Stryer started with Eq. (3)in order to obtain the efficiency J for the energy transfer between molecules a and b lying R a b apart from each other:

where w is the transition probability. For na’-.b = nbl-a, it follows that J = 1/2 and that there is a special value for R a b = Ro. From Eqs. (2) and (3) we obtain 1

.I = 1 + (RadR,)6

(4)

However, the efficiency is

J

=

( A E a - AEb)/AEa

(5)

where AE; = maximal transferable energy in the dark-adapted state, and AEb = energy radiated by the acceptor molecule b. As A E T = h, with h = Planck‘s constant and T = lifetime of the excited state, we obtain from Eqs. (4) and (5):

-

Equation (2) implies Rab 3dk;for sufficiently fast Brownian rotation Forster uses k 2 = %. This value has also been assumed by Wu and Stryer. They measured the lifetimes on the right side of Eq. (6)for Rab, for example, as the distance between 114s-retinal (R) and label (A) situated farthest away from R. For this distance they obtained values from 73 to 77 8,. At right angles to AR there are two energy donors B and C, 30 8, apart from each other. These values suggest the existence of an ellipsoid with avolume equal to that of a sphere with a 41-A diameter. A third suggested shape is put forward by Poo, Cone, Worthington, Dratz,Stryer, and Hubbell (Po0 and Cone, 1973). These investigators

40

N R G E N ROSENKRANZ

suggest a dumbbell-shaped model, fitting the idea that rhodopsin acts as a light-controlled pore. Rhodopsin would in this case consist of two ellipsoid parts of not exactly equal size, linked by a helix. The larger part of the molecule pointing toward the interlamellar space would contain the energy donor (A) and the saccharide complex (S) of the rhodopsin. As the former is assumed to be 75 A away from the retinylidene (R) and the latter 51-65 A, the retinylidene must be assumed to be situated in the intralamellar part of the rhodopsin dumbbell. Determination of the distance SR was made by means of an energy transfer, similar to the determination of AR; for this purpose the saccharides of rhodopsin were first oxidized b y periodate. Fluorescent molecules like fluorescein carbohydrazide or N-methylanthranilate hydrazide were then coupled as donors to the aldehyde groups obtained in this way by Renthal et al. (1973). Independent of these considerations we also suggest a dumbbellshaped model for rhodopsin as the synopsis in Section IV,D,l,a,iv shows. We propose a single rhodopsin peptide chain coiled up at both ends to form two tangles with a distance of 50 A between them and with their peptide chain ends pointing toward their neighbors. There are three differences between this dumbbell model and the first: (1) the peptide chain between the two tangles is not assumed to be coiled up in form of a helix but to be straight; (2)the retinylidene is assumed to be situated on the interlamellar side of the rhodopsin spanning the membrane, and the saccharide group is assumed to be attached at the end of the peptide chain also on the interlamellar side (see Section 111,D); (3) we are inclined to conclude on the basis of our freezeetching and x-ray diffraction experiments (Section IV,D,S,b) that six such small dumbbells lying together form a large dumbbell, a rhodopsin aggregate; see Fig. 28c (Rosenkranz, 1976b). With this configuration a pore is also formed, as shown in the same figure. The importance of this pore lies in its considerably larger diameter through which, for example, hydrated Ca2+could pass.

-

5. Spatial Proportion of the Rhodopsin in the Rod To permit further development of the lamellar membrane model and a discussion on its structure in the next section, more data on rhodopsin are required and are therefore compiled below. At present the only reliable estimation of the rhodopsin mass m Rcan be obtained, in our opinion, by the indirect method via the molar concentration. According to Liebman (1962), the rhodopsin concentration in the rod is cR = 2.5 & 0.5 mM. This leads to mR = V C R = 1.52 X lo-'' gm.In order to determine from this value the volume of the rhodopsin VR and that of the total protein Vp, we need the densities pR and pp

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

41

which are still unknown. One can merely approximate pRand pR-l as compared with the specific volume of other glycoproteins of similar molecular weight (41,000-45,000). The average specific volume of a,-glycoproteins, trypsin-a,-inhibitor, zinc-a,-glycoprotein, mucoprotein, and a,-seromucoid (all human) isp-' = 0.68 ml/gm (Sober, 1968). On the basis of the certainly justified assumption pkl = p-I = 0.68 ml/gm the rhodopsin volume is

vR= mRp-I

=

10, pm3

and the protein volume is

Vp = 0.8-'mRp-'

=

1.3 x 10, pm3

that is, nonhydrated protein occupies about 9% of the rod volume, about half the volume of the lipids (Section III,C,5).This means that the lamellar membrane, provided it is continuously covered by rhodopsin, contains a rhodopsin layer of about 10 A thickness. The number nRof rhodopsin molecules per rod is

where L = 6.02 x lV3 molecules/mole. The area each rhodopsin molecule is, accordingly, FR

=

2fsn/nR = 4400 k

FR

governed by

(7)

where n = 1500 lamellae per rod, and fs = area of the rod cross section = 34 pm2. If one assumes that rhodopsin binds a h,xdrate layer that approximately corresponds to its dry volume and that it is shared by both sides of the membrane (see Fig. 44a and b), the hydrated rhodopsin molecule occupies about half the area FR. According to Abrahamson and Fager (1973), a rhodopsin molecule consists of a single peptide chain, the length of which can be estimated to be maximally 2000 A on the basis of the preceding data and measurements using molecule models designed by Stuart and Briegleb [see Briegleb (1949/1950)1. Finally, to permit evaluation of the diffraction experiments, a knowledge of which and approximately how many atoms a rhodopsin molecule contains is required. This is shown in the following composition: H,3.66 x 103; C,2.42 X 103; 0,0.711 X 103; N,0.595 X 103; S, 0.034 x 103.

42

JuRGEN ROSENKRANZ

C. LIPIDS 1. The Mole Fractions of Various Lipids In the last few years, four different groups of workers have investigated the phospholipid composition of the rod; their results are in good agreement, as Table VI shows. This table shows only eight phospholipids but, since they represent 90-95% of the total amount of phospholipid, further compounds have been neglected. 2. Fatty Acid Composition of Some Lipids Table VII gives information about the type, frequency, and position of the fatty acid residues in the phospholipids. Here the agreement between the various investigators is not as good as that for the data shown in the previous table; Anderson and Risk (1974) do not offer sound arguments to explain the differences. According to Mason et al. (1973),the ratio of unsaturated to saturated fatty acids of all lipids in the rod is about 1.56 : 1. Thus, for every two lipids with straight fatty acid residues, there are three with more-or-less bent hydrocarbon chains. 3. The Molecular Weight of an Average Lipid in the Rod If there are really glycolipids in the rod, in contrast to the results of Heller and Lawrence (1970),their sugar component is still not known exactly. Since this point has not yet been thoroughly investigated, we assume, for further estimations, that all the lipids are represented by phospholipids. About 90%of the total phospholipids are represented by phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. We confine our considerations to these four phospholipids, which together amount to three-quarters of all the lipids in the rod, as only these have been closely analyzed (see Table VII). From data already known exactly, we can calculate data for the average lipid molecule, which then will represent the lipid molecule in estimations made in subsequent sections. The molecular weight ML of the average lipid is, in this case, M L = 770. 4. Geometry and Density of the Lipids Apart from their different chemical composition, the shape of the phospholipids also varies widely; the two saturated fatty acid residues can elongate the phosphate group, thus leading to a thin phospholipid more than 40 A long. The fatty acid residues can also be bent perpendicularly to the phosphate group or form semicircles, depending on number and site of their double bonds. These considerations, when

TABLE VI DISTRIBUTION OF MORE THAN90% OF

THE

ROD PHOSPHOLIPIDS",~

Eichberg and Hess (1967)

Hall et al. (1973)e

Anderson and Risk (1974)

Rana pipiens

-

Rana pipiens

Mason et al. (1973)" ~~

~~

Phospholipid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Alkyl ether phospholipid Sphingomyelin Cardiolipin

I,

49.4 24.6 9.5 1.4 3.0 3.5 1.8 0.6

45.3 37.3 11.3 2.4

-

1.6

*

45.3 1.0 34.6 2 1.1 12.8 k 1.3 2.2 & 0.5

-

1.9

* 0.9

Rana catesbeiana

Mean value

~~

44.6 26.1 15.1 2.1 5.3 6.4

46.1 30.6 12.2 2.0 4.1 2.9

Reprinted with permission from Mason et al., Biochemistry 12,2147 (1973).Copyright by the American Chemical Society. Values given in moles x I@*. M. 0. Hall, Basinger, and D. Bok (1973). Unpublished data (cited by Daemen, 1973).

FATTYACn>

COMPOSITION OF THE

TABLE VII FOURMOST FREQUENTLY OCCURRING

Phosphatidylcholine (lecithin) Fatty acid residueb

&

10:o ll:0 12:o 13:O 14:O 15:O 16:O 16:l 17:O 18:0 18:0, DMA 18:O + 18: 1, DMA 18:l 18:2 18:3 19:o 20:o 20:l 20:2 20:3 20:4 20 :406 20:5 20 :5w3

PI-IOSPHOLJPIDS IN THE

Phosphatidylethanolamine

A-R

A-R

ROD'

Phosphatidylserine

1

2

M

1

2

M

A- R

M

-

-

0.12 T 0.21 2.07 0.55 2.23 3.96 2.45 0.25 13.8

-

1.8 0.3 5.7 2.4 0.3 -

0.73 0.42 4.96 5.29 3.93 7.24 11.26 6.64 1.67 5.90 3.28 2.26 13.88 0.89 1.04 1.72 1.60 1.10 -

1.3 1.5 4.0 2.4 19.3

0.34 3.12 2.46 4.15 4.20 13.65 2.78 1.16 7.61 4.28 2.17 5.61 0.41 -

1.4 2 0.7 0.1 k 0.1 49.6% 1.7 T 0.1 & 0.1 37.1 2 0.7

-

5.6 2 0.3 0.6 2 0.5 0.7 2 0.9 T 0.3 2 0.5

-

1.1% 0.4

T

1.4 i 1.1 0.1 2 0.1 17.6 k 1.7 8.4 2 0.6 0.1 % 0.1 1.4 & 0.8 11.8 & 0.7 0.8 2 0.1

-

0.4 k 0.4 T 0.2 2 0.3 3.1 2 0.2 -

-

6.10 4.03 17.36 T T 1.36

-

8.23

-

0.7 0.7 14.0 0.7 0.4

-

0.6 28.1 11.8 1.0 1.4

-

0.9 4.4

-

-

2.5 4.0 0.4

-

0.9

-

7.3

-

0.6

-

3.4 0.6

-

0.1

-

2.2

-

-

1.14 1.37 2.04 -

Sphingomyelin

M

2.03 2.36 3.81 6.33 4.23 14.14 1.38 4.40 0.31

-

2.66 13.57 12.19 0.23 1.43

-

1.26 4.30

-

0.71 -

21:o 22:o 22:4 22 :406 22:5 22 :506 22 :5w3 22 :603 22 :6w6 22:6 24:0 24: 1 Unknown

&

0.3 & 0.7

-

0.3 2 0.3

T

2.7 t 0.4 -

-

-

T 0.1 0.1 0.4 i 0.3 53.8 3.1

* *

-

11.0 4.91 3.93 6.84 6.55 1.41

-

11.8 4.0 4.5 16.1

0.4

-

0.3 73.0

T 9.18 4.24 0.94

-

4.16 1.32 1.10

-

-

6.8 4.5 3.1

-

46.4

-

4.0

0.56 3.12 5.43

-

4.12

-

24.16 3.20 1.07 -

0.87 1.06 3.14

3.90 6.69 0.41

-

All values are given in moles x 10-*A-R, Anderson and Risk (1974), R. pipiens; M, Mason et al. (1973), R. catesbeianu (reprinted with permission from Biochemistry, copyright by the American Chemical Society). 1, Fatty acid residue at the first carbon atom in the phosphoglyceride; 2, fatty acid residue at the second carbon atom in the phosphoglyceride. T, trace. The first number indicates the number of carbon atoms, and the second the number of double bonds. DMA, Dimethylacetal. 0 6 denotes that the sixth carbon atom from the methyl group takes part in the last double bond.

46

flRGEN ROSENKRANZ

applied to the Stuart-Briegleb molecule model, have been investigated in the experiments of Demel et al. (1972);they determined the minimal area needed by a lecithin molecule with two saturated Cle fatty acid residues (18:0/18:0)to be FL = 41 Az. A 40-A long, straight phospholipid molecule leads to a maximal (mechanical) density of phospholipids of pLmav= 1.67MJ40FL = 0.8 gm/cm3

Note, for comparison, that triglycerides like depot fats have pfat = 0.91 gm/cm3 (Sober, 1968). Demel and his co-workers furthermore determined a maximal area requirement of 115 A2 per molecule of moder= 0.5 gm/cm3 if ately unsaturated lecithin, which corresponds to pL.mln the length of the lipid molecule is in this case assumed to be about 22 A. Owing to the extraordinary proportion of unsaturated fatty acids in the rod, one must preferably consider pL = 0.5 gm/cm3 rather than the double value for volume estimations. 5. Volume Proportion of Lipids in the Rod To calculate the volume proportion VL of lipids in the rod one must know, besides pL, the total lipid dry mass content mLof the rod. The only direct weighing of mL,found to be 2.3 x 10-lo*gm by Eichberg and Hess (1967), is too high by a factor of 2 as shown by our calculations based on the values in Table I11 and assuming a rhodopsin concentration cR of 2.5 mM.' If cR = 2.5 mM is assumed to be correct, mL= 1.27 x 1W'O gm. With this value and with pL = 0.5 gm/cm3 the lipid volume is calculated to be VL = 250 pm3; this corresponds to 17%of the rod volume and, on the basis of the following values, fairly well to a continuous monomolecular lipid layer in the lamellar memM; (mechanbrane of 25 A: rhodopsin concentration, cR = 2.5 x ical) density of the lipid,pL = 0.5 gm/cm3;number of lamellae per rod, n = 1.5 x 109; and area of rod cross section, fs = 3.4 x 10 pm2. The number of nL of lipid molecules amounts to nL = 1P4mJ1.67ML= 9.9 x 1Olo

For later calculations of charge densities and scattering length densities, the type and number of the various atoms found in an average lipid molecule in a rod are: H, 80; C, 44; 0, 8; N, 1; P, 1. Throughout this article an asterisk denotes data measured or calculated from a figure or table in the reference cited.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

47

D. SACCHARIDES Saccharides are components of either only rhodopsin or also of some lipids. Heller (1969) found three glucosamines and three neutral sugars per rhodopsin molecule, while Eichberg and Hess (1967), as well as Mason et aZ. (1973), report that approximately 10%of the total rod dry mass is made up of glycolipids. In all cases the analyzed saccharides were hexoses. If one assumes that the experimentally demonstrated statement by Heller and Lawrence (1970), that all the saccharide in the bovine rod is in the rhodopsin molecule, applies to the fiog also, a difference of opinion is revealed which has not yet been clarified. Heller and Lawrence also found that the six sugars form a single hydrophilic oligosaccharide which is coupled to the polypeptide chain by means of an N-aspartylglycosamine bond. Whether the oligosaccharide is located on the intra- or interlamellar side of the membrane is still open to question; the findings of Renthal et al. (1973) and Steinemann and Stryer (1973) indicate that the oligosaccharide is attached near a membrane surface and in a distance of more than 50 A from the prosthetic group. According to P. Rohlich (personal communication, 1975), PTA contrasts preferably the intralamellar space at low pH, which is a reason to assume the presence of glycoproteins. On this subject our findings may also be of interest, that is, that the membranes of a lamella appear separated from each other in the intralamellar space after treatment of the rods with phospholipase D (- 10 mg/ml tris buffer; incubation 1 hour at 39°C); they are no longer parallel but separated by large irregular distances. The explanation for this could be that the saccharide groups are split from glycolipids fixed in the intralamellar space. A similar assumption has been made by Debuch (1965)for phospholipids.

IV. Ultrastructure of the Light-Adapted Rod In the following discussion we consider the ultrastructure of the rod as far as it is accessible for direct or indirect optical observation. As most investigations at the supramolecular level (applying to a reciprocal resolution of about 10 A) have been carried out on light-adapted rods, their ultrastructure is dealt with in detail in this section, while in Section V only the structural changes that occur in the dark-adapted state are described. Since the experiments of Chabre (1975b), we know that the structure of the rods during the first minutes after illumination resembles neither that of the light-adapted nor of the darkadapted state. The final stages of light and dark adaptation are there-

48

flRGEN ROSENKRANZ

fore by no means the two only possible states of the frog rod; perhaps the most important modification has not yet become accessible to experimental investigation. For this study therefore we still have to assume a rod ultrastructure that is more static than dynamic. The assumed rod structure is a combination of that observed in the light- and dark-adapted states. Light-adapted, in morphological investigations, means that a rod has been exposed to daylight or a similar light source prior to or during the investigation, whereas dark-adapted rods generally are from frogs kept about 24 hours in total darkness before enucleation and whose rods have been investigated either under infrared or red light. Looking closely one can differentiate among the following parts of the rod (see also Fig. 3): the microvillous processes fitted against the indentations of the rod, the connecting cilium, the cell membrane, the cytoplasmic space, the space containing the lamellae without their

FIG.3. Schematic longitudinal section (a) and cross section (b) of a rod. a, Lattice constant; bb, basal body; cc, connecting cilium; cm, cell membrane, CS(1h cytoplasmic space; CS(*), interlayer; cx, ciliary matrix; la, lamella with thickness b; 11, interlamellar part of the lamellar membrane; l,, intermediate layer; la, intralamellar part of the lamellar membrane; l,, intralamellar space; lr, loop region, rim region; mp, microvillous process. AB denotes the fracture face.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

49

rims, that is, the lamellar body, and the rims or the surrounding loop region of the lamellae. In the following discussion these parts are described separately. A. THE CELL MEMBRANE A detailed description of the cell membrane of the rod is not yet available, partly because there is still no exact definition that applies generally to all cell membranes, and also because experiments on isolated rod cell membranes, in analogy for instance to the erythrocyte membrane, have not yet been carried out. Concerning the overall chemical composition of this cell membrane, one therefore has to go back to averaging and interpolating the data obtained from, for example, liver, kidney, and erythrocyte cell membranes of various species. Roughly, a cell membrane consists mainly of three substances with the following mass proportions: 50-80% protein, 1 4 % carbohydrate, ATPase (possibly as glycoprotein), and 10-40% lipid (having a ratio of unsaturated to saturated hydrocarbon chains of about 0.5: 1 and a molar ratio of cholesterol to phospholipids of 0.5:1 or more). The surface area of the rod outer segment cell membrane is 1% of the total surface area of all the lamellae included. It corresponds to the surfaces of about 18 lamellae.

1. The Cell Membrane Following Freeze-Etching Figure 4 shows the cell membrane of a light-adapted rod following freeze-etching from the extracellular side. There, embedded in a plane membrane surface lie the same truncated, hexagonal pyramids described extensively in Section IV,D,l,b,i. The distance between two opposite edges of a hexagonal base isfGF = 140 A; the height of the truncated pyramids can only be roughly estimated to be about 50 A. The pyramids can be divided into two classes according to their height: those showing marked elevations and others which are mere indications of hexagonal bodies of negligible height; the latter seem to cover the whole remaining extracellular membrane surface. The truncated pyramids emerge quite irregularly from the membrane and cover one-quarter of the total extracellular membrane surface. The thickness of the cell membrane CmGF generally cannot be determined however, is a exactly in freeze-etched preparations; CmGF = 150 reasonable estimate. 2. The Cell Membrane in Ultrathin Sections The appearance of the cell membrane in ultrathin sections strongly depends on the kind of fixation and dehydration agent and on the

50

JURGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

51

electron stain used. For instance, the cell membrane does not appear at all when fixation involves glutaraldehyde followed by ethyl alcohol dehydration, when uranyl acetate in ethyl alcohol is used as electron stain, even when hexylene glycol is used for dehydration, or when the fixation agent consists of glutaraldehyde and hydrogen peroxide. In the last-mentioned case it seems probable that the cell membrane has been fixed but not stained, as the neighboring cytoplasm ends abruptly in a straight line. The existence of cell membranes in the rod can always be demonstrated to different extents: 1. For example, when fixation with glutaraldehyde alone is followed by hexylene glycol dehydration and aqueous uranyl acetate as an electron stain (Fig. 5). After fixation with glutaraldehyde alone the cell membrane always appears in negative contrast; two noncontrasted layers are connected by an intermediate layer which is strongly stained by uranyl acetate. All three layers are approximately of the same thickness. Sometimes the dark, central layer is somewhat thinner than the bright, outer layers. The total thickness of the cell membrane cannot be stated exactly, as the limiting lines appear fiinged and one cannot be sure whether further positively contrasted layers follow toward the interior or exterior of the cell. The total width of the three layers just described is cmm = 130-140 A. The kind of buffer (phosphate or cacodylate buffer) and the embedding agent, for example, Epon, water-soluble Durcupan (Rosenkranz, 1976b) or glutaraldehyde urea resin (Godfiey, 1973), do not play a decisive role in the appearance of membrane structure. FIG.4. Extracellular surface of a rod cell membrane (cm) with attached or sunken truncated, hexagonal pyramids (hx). cs, Rod cytoplasm. Platinum-carbon replica of a deep freeze-fracture. This rod was treated for 3 hours with 25%glycerol in Ringer's solution before freezing, but there was no difference in the ultrastructure of the cell membrane in this and an untreated rod. In this and the following micrographs a bar always indicates 500 A if not otherwise marked. FIG.5. Part of a rod longitudinal section with a negatively stained cell membrane (cm). Glutaraldehyde fixation (300 mosM, pH 7.2), dehydration with hexylene glycol, staining with aqueous uranyl acetate (pH 4-5). See also Table IXC. FIG.6 . Part of a rod longitudinal section with a negatively stained cell membrane (cm). Rana temporaria. Fixed in 1%glutaraldehyde, dried in an argon stream at 18"-20°C for 18-24 hours, stainedwith 2%aqueous ammonium molybdate. See also Table IXC for data, partly provided by Borovjagin. Reproduction from Borovjagin et al. (1973). FIG.7. Part of a rod longitudinal section with a positively stained cell membrane (cm). Rana temporaria. OsO, fixation, dehydration by freeze-substitution, modified after Hereward and Northcote (1972).See also Table IXA for data, partly provided by Borovjagin. Reproduction after Borovjagin et al. (1974).

52

JORGEN ROSENKRANZ

2. A cell membrane is also apparent when, after fixation with glutaraldehyde, dehydration is performed by drying in a steam of argon and staining is carried out with aqueous ammonium molybdate; in this case embedding is not necessary (Fig. 6). 3. Cell membranes are observed after treatment with Os04 no matter whether it is used as a fixation or postfixation agent. In both cases the membrane appears positively stained, either in a longitudinal section as a black ribbon about 65 in width after hypotonic fixation (90 mosmoles of glutaraldehyde in collidine buffer, Os04, ethyl alcohol dehydration), or as a trilaminar layer with a total width of cmUL= 150 after (use caution) freeze-substitution (Fig. 7). If dehydration and embedding are performed at temperature SO’C, but not fixation (Fernindez-Morsin, 1961), a cell membrane becomes visible which is comparable to a lamellar membrane. A similar effect can be obtained following many other preparation methods (e.g., Nilsson, 1965).

a

a

3. Histochemical Investigations of the Cell Membrane The following histochemical reactions of the cell membrane are as expected. The cell membrane is completely dissolved by pronase P (Streptomyces griseus) within less than 60 minutes and remains unaffected by phospholipase D (Rosenkranz and Hauser, 1972); in both cases the cell membrane behaves like a lamellar membrane. The following two observations, however, indicate different structures for the two membranes. (1) Treatment with hydrogen peroxide during fixation causes the appearance of lamellar membranes, but not of cell membranes. (2) After OsOl fixation of the rod and treatment with tris all rod structures seem to vanish except for the cell membrane, the loop regions, and the connecting cilium (Falk and Fatt, 1969). Furthermore, treatment with PTA leads to marked staining of the extracellular side of the membrane (or of the immediately adjacent extracellular space?) (Rohlich, 1970). Under the same conditions the interand intralamellar sides of the lamellar membrane are also stained, although to a somewhat weaker extent (P. Rohlich, personal communication, 1975). The question whether or not the cell membrane contains rhodopsin has so far been investigated in the frog by light microscopy only (Dewey et al., 1969).On the basis of these experiments (coupling of a fluorescent visual pigment antibody to visual pigments of the rod) the existence of rhodopsin in the cell membrane is “strongly suggested” for light- and dark-adapted rods. The electron microscope and histochemical experiments of Jan and Revel (1974) with mouse rods

ULTRASTRUCTUFW OF FROG ROD OUTER SEGMENTS

53

yielded stronger evidence for the existence of visual pigment in the cell membrane; by means of bovine visual pigment a specific immunoglobin IgG was produced in rabbits and attached to a peroxidasecoupled goat IgG specific for rabbit IgG. The result was, in addition to labeled lamellae, a thick precipitation of IgG-peroxidase complexes on the extracellular cell membrane of the rods and a thinner precipitation on the intracellular side.

4. Synoptic Znterpretation of the Results Concerning the Structure of the Cell Membrane The traditional view of the cell membrane is based on careful observations such as those of Fernhdez-MorAn, Nilsson, and others. These investigators described a cell membrane with structures and dimensions that are also valid for lamellar membranes. The findings of Falk and Fatt and the cell membrane shown in Figs. 5-7 seem to contradict this interpretation. As far as the findings of Falk and Fatt are concerned, our observations revealed that in cross sections as well as in longitudinal sections the rod cell membrane was missing from approximately 70% of the circumference shown, while in the remaining 30% the cell membranes of the rod outer segments were observable but were considerably more weakly stained than those of the microvillous processes directly adjacent to the cell membranes.. As already mentioned, it is technically rather difficult to show both cell membranes and lamellar membranes of a rod at the same time with the same optimal quality, therefore Figs. 5-7 cannot be regarded as truly representative of the natural state. However, one can speculate from these micrographs that the actual thickness of the cell membrane is approximately 150 8, including a positively stained layer of 10 8, width on both sides of the cell membrane, which appears after fixation with glutaraldehyde. In Figs. 5 and 6 there appears to be no difference between the cell membrane and a whole lamella in the interior of the rod, neither in degree of staining nor in widths. If this is so, it means that the rod cell membrane is a double membrane and that, considering antibody experiments and the results of PTA staining, the cell membrane is morphologically a giant lamella, although it also has additional properties. This assumption, based on the results of various preparation methods which are not yet understood, contradicts the hypothesis that the lamellae develop through invaginations of the cell membrane. Possible explanations accounting for the different staining patterns are dealt with in Appendix 2,A,1. It is astonishing that antibodies cou-

54

WRGEN ROSENKRANZ

55

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

pled to peroxidase (total molecular weight k130,000), as well as the PTA complex, can penetrate the cell membrane and enter the intracellular space of the rod, although Cohen (1968, 1970) showed for R. pipiens that at least lanthanum nitrate, barium sulfate, and ferritin ( 100 diameter) in phosphate buffer cannot penetrate as far as the intracellular space of the rod. These findings lead to the conclusion that the cell membrane of the rod remains intact only after careful preparation and that, under other circumstances, larger artificial openings in the membrane also allow large molecules (diameter > 100 A) to pass into the intracellular space.

-

B. THE CONNECTINGCILIUM The connecting cilium is the organelle that connects the rod inner segment with the rod outer segment and which, in the frog, represents the only cytoplasmic bridge between these two segments. It contains cytoplasm and a modified cilium which originates from a centriole eccentrically situated about 0.5 pm away from the plasma membrane of the inner segment; these as well as the following data refer to R. escuZenta. At right angles to the first centriole or basal body, the origin of the cilium, the second centriole is situated between the basal body and the center of the inner segment. The two cylindrical centrioles each have an average diameter of 0.17 ym; in a figure published by Young (1968), however, it was 0.3*ym. The basal body contains nine microtubule triplets which pass over into nine microtubule doublets 0.2 pm below the beginning of the connecting cilium. The maximal diameter of such a doublet is 370 & 10 A. After reaching the connecting cilium the microtubules begin to diverge up to an angle of 23" from their original direction. The greatest divergence is always perpendicular to the line from the basal body to the center of the inner segment. As the cell membrane lies parallel to the microtubules, the FIG.8. Obliquely cut connecting cilium with microtubule doublers (mt) and centriole (ct). Fixation with 2.5% glutaraldehyde and 0.35% HzOnin 0.15Msodium cacodylate buffer (340mosM), posffixation with 1% OsO, solution (33mosM). FIG.9. A connecting cilium obliquely sectioned but at an angle of 90"to the section in Fig. 8 . mt, Microtubule doublets. Preparation as in Fig. 8. FIG.10. Cross section of a ciliary matrix. r, and r, are explained in the text. Preparation as in Fig. 8. FIG. 11. The microvillous processes (mp) of the bullfrog rods seem to establish a connection between the rod inner segment (ris) and outer segment (ros). From this scanning electron micrograph one can imagine the compactness of the transitional zone between the rod inner and outer segments. Preparation as described in Fig. 1. x 9640. From Steinberg (1973).

-

56

NRGEN ROSENKRANZ

connecting cilium has an elliptical cross section and a slightly funnel-shaped longitudinal section. The cross section at the vitreal end of the connecting cilium has an average radius of 0.15 pm, also according to Young, and at the scleral end, 0.17 pm (short semiaxis) and 0.3 pm (long semiaxis). The length of the connecting cilium is 0.5 pm or up to 0.6* pm, according to Young (1968);see Figs. 8 and 9. In spite of numerous attempts to follow the microtubules into the rod area by means of serial sections the microtubules could not be found more than 0.1-0.2 pm sclerad of the end of the connecting cilium. Therefore we are justified in assuming that the length of the microtubules is not more than about 0.8 pm, measured from the basal body. On top of the microtubules the lamellae gradually approach the cell membrane (Fig. 10).The lamella-free scleral continuation of the connecting cilium is called the ciliary matrix; it can be regarded as a bar-shaped, more-or-less abruptly ending body with a rectangular to elliptical cross section. From the micrographs of Young (1968) and Rosenkranz and Hauser (1972)the following data for the ciliary matrix are obtained: a length not exceeding 8 pm, and semiaxes with an elliptical cross section of rI = 0.3-0.5 pm and r2 = 0.17-0.3 pm. The modification of the connecting cilium in comparison to a motile cilium is not only that it diverges like a funnel and seems to be generally shorter, but also that the two single microtubules in the center are missing. The function of the connecting cilium is still uncertain. Thorough investigations concerning this problem have not been made. It is, however, certain that amino acids (or their residues) like histidine, methionine, leucine, and phenylanine are transported through the connecting cilium, as autoradiographic experiments by Young (1968) have shown. C. APICAL MICROVILLOUSPROCESSES A very minor part of the surface of the rod is not in direct contact with the mucopolysaccharide matrix of the optocoelium but with the apical microvillous processes. These processes are tube-shaped evaginations of the inner segment (Fig. ll), which in preparations fixed with Os04fit against the rod incisures with a constant distance of 180*(Nilsson, 1965) or 50-100 A (J. Rosenkranz, unpublished results, 1975).The cross sections of the processes are approximately elliptical. They contain about 20 microfibrils, each having a diameter less than half the microtubule’s diameter; the data are compiled in Table VIII. The microfibrils are directed vitread in the inner segment between the cell membrane and ellipsoid (cf. Fig. 2; Fig. 12);in addition to the

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

57

TABLE VIII DIMENSIONS OF MICROVILLOUSPROCESSES" Species

Rana pipiens Rana esculenta Rana catesbeinna

g1

(w) gz (v)1 (,urn)

i 0.30 I0.25

50.15' 50.07

-

-

2 15'

7

' 5

gs

(A)

Reference

20-30 Nilsson (1965) 80 40 J. Rosenkranz (unpublished results, 1975) Steinberg (1973)

*

6,. Large diameter of the ellipse (as cross section); g2, small diameter of the ellipse; 1, length of the microvillous process; g,, diameter of the microfibrils.

microfibrils, approximately hexagonally shaped bodies (diameter ca.

200 A) are found in R. esculenta (Fig. 13).Participation in the develop

ment of rod structure seems to be one of the functions of the microvillous processes. Other functions still are a subject for speculation.

D. THE LAMELLARBODY 1. Electron Microscope Aspect a. Ultrathin Sectioning of the Lamellar Body. On the basis of electron microscope investigations of the frog rod the following supramolecular picture of the lamellar body can be drawn. The lamellar body, representing 90% of the rod volume, contains two kinds of layers in longitudinal section, that is, when cut parallel to the rod axis (see Fig. 3): layers with 300-A repeat (la) and interlayers between them The first layer forms, together with the surrounding thicker rim, the lamella (disc) of the rod. The interlayer between every two lamellae is often called the interlamellar or cytoplasmic matrix or simply part of the cytoplasm in the rod. This means that it has neither specific properties nor a specific function, and that there is no difference between it and the C S ( ~ space ) in Fig. 3, the actual cytoplasmic space. This simplified view is contradicted by more recent results (Section IV,D, l,a,iii) which justify the separation into cytoplasmic space and interlayers. As these layers do not enclose the lamellae completely but extend only toward the surrounding rims, the interlayers are not directly connected to one another and thus do not represent a matrix proper. In longitudinal section the lamellae have a periodicity of a = 300 A. This was found to be constant over the whole cross section of the rod; therefore a can be referred to as a lattice constant. The contour of a cross-sectioned lamella, that is, cut perpendicular to the rod axis, appears circular (Fig. 15),but a closer inspection shows

58

J~~RGEN ROSENKRANZ

ULTRASTRUCTURE O F FROG ROD OUTER SEGMENTS

59

n’ incisures which extend from the rim toward the center of the lamella and have different lengths. For R. pipiens, n‘ is -20 (Nilsson, 1965), and for R. esculenta, n’ is 20 to 28; in any case the number of incisures is sufficient to guarantee that on the lamella sulface there are only a few points that are more than 0.5-0.8 pm away from a rim. The form and size of the incisures of neighboring lamellae are, on the whole, similar. Those segments of the lamellae in contact with the ciliary matrix do not show changes in this region, indicating that rims also exist here. The majority of the lamellae are most probably not connected with the cell membrane, as concluded by Cohen (1968, 1970) from numerous experiments. Only at the vitreal base of the rod, some lamellae, u p to five in the case of R. pipiens (Nilsson, 1965), are connected in such a way with the cell membrane that they must be recognized as indentations of this membrane (Moody and Robertson, 1960). Cohen (1970) is impressed by how seldom he found open lamellae at the rod base. The question whether or not the lamellae are connected to each other is answered differently by various workers. While Cohen (1972) tends to regard the lamellae as separated from each other on the basis of the experiments previously mentioned, we are of the opposite opinion. Indications for connections between neighboring lamellae parallel to the rod axis are (1) the anastomoses that appear after fixation with glutaddehyde-0sO4 (Rosenkranz and Hauser, 1972), (2) the many fibrils parallel to the longitudinal axis appearing after fixation with glutaraldehyde (Rosenkranz, 1973), (3) the nearly three dimensionally presented anastomoses in Fig. 14. Finally, the connection between the ends of the two deep incisures shown in Figs. 16 and 17 can be explained only by the assumption that a short tubulus connects two successive lamellae or, more exactly, the surrounding rims of two lamellae. These findings are confirmed by a reh G . 12. Part of a rod cell longitudinal section: The microfibrils (mt) pass by the mitochondria (mi) of the inner segment and into the microvillous processes (mp). Preparation as for Fig. 8. FIG.13. Part of a rod cell longitudinal section. Because of a slightly modified fixation procedure the microfibrils (mf) seem to be thicker than in Fig. 12. hx, Hexagonal particles. Fixation with 2.5% glutaraldehyde in 0.5 M collidine buffer (480 mosM), postfixation with 1% OsO, solution. FIG. 14. Part of an oblique section through a rod. As a result of the special fixation procedure some details are lost, but others are shown more distinctly. an, Anastomose bemeen lamellae; tu, longitudinal view of a tubulus which connects the end of an incisure (es) to another incisure not present in this figure but observed in the tilted series in Figs. 16 and 17. Preparation as in Fig. 8.

60

J~~RGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

61

sult of Cohen (1968) showing that some lamellar sectors were completely surrounded by barium sulfate; Cohen assumed that these sectors were “connected to something not in the plane of section.” After this general morphological view of the lamellar body, which can only be gained fi-om electron microscope investigations, we deal with the structure of one lamella in cross-sectional and longitudinal view. The facts known about the interlayer connecting every two lamellae are then compiled. i. The lamellar membrane viewed from above. Good micrographs of the surface of a lamellar membrane are very seldom obtained for technical reasons. Generally a section contains more than one lamella because of its thickness and, as all details of the whole section are shown by the electron microscope, summation of the different staining densities leads to the picture of a relatively homogeneous surface. Therefore one can only hope to see a lamellar membrane surface if one succeeds in obtaining an ultrathin section with only one membrane. It is presumed that Robertson (1967) was the first to obtain a picture of the surface of a single frog lamellar membrane. He found a honeycomb structure of the membrane consisting of single hexagonal honeycombs with diameters between 50 and 60 A. If one considers fixation by KMn04 now known to be unsatisfactory, and the shrinkage caused by it, it is not difficult to imagine that the hexagonal honeycombs can be twice as large in vivo. This result would be in good agreement with our findings concerning the surface of the lamellar membrane of R. esculenta; in some cases we succeeded in demonstrating part of a membrane surface in an ultrathin section. Figure 18, for example, shows such a membrane of a developing rod. Here one can assume that the essential parts of one lamellar membrane which is by chance situated within the section are constructed of hexagonal particles surrounded by an unstained halo. The distancefULbetween two parallel sides of a hexagon is 134 ? 5 A (20 measurements). From

FIG. 15. Rod cross-sectioned at the level of the first vitreal fifth. mp, microvillous processes; eb, indentations of the lamellae parallel to which the cell membrane passes; es, incisures not accompanied by the cell membrane. Preparation as in Fig. 8 . FIG.16. Part of a rod cross section with five incisures (es), two of which, lying one above the other, are connected to one another by a short tubule (tu); this was ascertained by a tilted series. The axis of the tubule on the right side lies perpendicular to the section plane. Fixation with 2.5% glutaraldehyde and 0.35% H202in 0.15M sodium cacodylate buffer, dehydration with hexylene glycol. FIG. 17. The same cross section as in Fig. 16 but tilted 21”: The tilt axis lies parallel to the incisures.

62

J~~RGEN ROSENKRANZ

Figure legends on page 64.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

Figure legends on page 64.

63

64

P R G E N ROSENKRANZ

Fig. 18 it follows that the average distance fin between two neighboring hexagonal particles is 236 & 72 A (175 measurements) and that the fluctuation width

This subjective description is supported by light diffraction experiments. With a light diffractometer (Rosenkranz, 1975a) the diffraction patterns of Fig. 18 were obtained. Inset (a) is a schematized drawing of Fig. 18, and inset (b) shows the diffraction pattern of the electron microscope negative for Fig. 18. Distance fi and fluctuation width are consistent with the subjectively determined values: exactly a firstreflection order exists (Hosemann and Bagchi, 1962). The approxiFIG.18. Part of a longitudinal section of a developing rod in which lamellar membrane fragment accidently lies in the plane of sectioning. hx, Hexagonal particles. P r e p aration as in Fig. 13. Inset (a) represents an abstraction of the electron micrograph. The light diffraction pattern in inset (b) results from the electron micrograph negative; the direction of view is toward the laser. Further explanations are given in the text. FIG.19. Part of an almost cross-sectioned rod with a lamellar membrane (lm) fragment tom out. The short fibrils (fb) (5250 Along and -30 %in diameter) in the extracellular space must be part of one of the rod layers. sp, Spherical particles with 40- to 45-A diameter. Fixation with 1.8% glutaraldehyde in 25 mM sodium phosphate buffer (N%HP04,NaH2P04;115 mosM; Durcupan embedding. FIG. 20a. Part of a relatively thin cross section with spherical particles (sp) which often seem to be connected by short fibrils (fb).Preparation and magnification as in Fig. 19. FIG.20b. Part of a 1-pm-thick section of a rod. Sections of this thickness can only b e observed in a scanning transmission electron microscope. As mentioned in the text, the upper surface of a section is seen more distinctly by this method than the lower one. Thus particles (hx) on the section s u r i c e can be shown, which correspond to those in Fig. 18; the lower electron optical magnification is caused by the lower resolving power. Fixed with glutaraldehyde in 0.5 M collidine buffer, posdixed with OsO,. JEM-1WB-Based analytical electron microscope, probe diameter 50 A, scanning time 50 seconds, beam voltage 100 kV. FIG.20c. High-resolution scanning electron micrograph of part of a lamella. The arrowhead indicates one of the humps discussed in the text. Fixed as in Fig. 48,then shadowed by rotational evaporation of carbon and gold. Stereoscan S410 (Cambridge), scanning time 100 seconds, beam voltage 10 kV. FIG.20d. Part of an ultrathin longitudinal section as observed with a scanning transmission electron microscope. The section was stained only with uranyl acetate and lead citrate; there is a marked similarity between this electron micrograph and Fig. 23, which was obtained from a transmission electron microscope. In this figure the same loci (re) are as heavily stained as those that stand out in Fig. 2 3 as a result of PtCl, postfixation. Because of their arrangement the dark stains are presumed to be retinylidene groups. Preparation as in Fig. 16.

-

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

65

mately hexagonal position of the particles as identified by the pattern is surprising. Such a degree of order would not be expected from mere observation. In Fig. 19 positively stained, spherical particles with a diameter of 40-45 A can be seen in a torn-out lamellar membrane; the extracellular space of this region is filled with short fibrils which apparently came out of one of the rod layers. Figure 20a shows part of a relatively thin cross section with the same spherical particles that fiequently seem to be connected in pairs by short fibrils. We also tried to take advantage of two other electron microscope techniques: the highly resolving scanning electron microscopy and scanning transmission electron microscopy. The latter permits a sharper view of the upper surface of a section than the lower one, because of the topbottom effect; the disadvantage is lower resolution than with the usual transmission electron microscope. Figure 20b therefore shows distinct particles (hx) on the upper surface of a common section; the contours of these particles are, however, fused because the particle diameter is only three times larger than the spot size of the scanning electron beam. Particle identification is not quite so satisfactory in the case of the scanning electron micrograph. Figure 20c demonstrates a lamellar surface as seen by a scanning electron microscope controlled to give as high a magnification as possible. As judged by the distances and sizes, the particles observed can only be hexagonal particles decorated by a comparatively thick evaporation layer, that is, those that stand out slightly more than others. A further indication that a main building block of the lamellar membrane is a cluster of electron-dense material is shown by experiments in which sections were tilted in the electron microscope (Rosenkranz and Hauser, 1972). In the process of these investigations we determined the scattering thickness of different membrane models as a function of the tilting angle. It was concluded that the electron-dense material was neither spread in a plane over the membrane nor predominantly in form of fibrils, but in clusters. It seems that the electron densities in Fig. 21a-d can only be explained in this way. A comparison of Figs. 21 and 22 indicates further that there is a surprisingly good correspondence of results if one assumes the clusters are dumbbell-like aggregates. ii. The cross-sectioned lamella. Though the number of really informative electron micrographs of the lamellar membrane surface is very small, a great number of electron micrographs of longitudinal sections, that is, parallel to the rod axis, exists. This indicates how skeptically the results of the ultrathin sectioning technique have been

66

JORGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

67

FIG.22. A cut through a stained rod longitudinal section at right angles to the axis of tilt K described in Fig. 21. If the electron-dense material is assumed to b e arranged dumbbell-like in the lamella (la) as indicated here, the electrons having passed the layer of thickness (sd)will produce the mass density distribution (mv); the distributions for the two angles az = - 18" and al = -42" are explicitly given. These distributions agree well with the darkening of the section (Fig. 21),which is proportional to the mass density distribution (microdensitometer measurements). The distances x' = 420 A and x" = 570 A correspond to those in Fig. 21d and c.

regarded. Now it seems justified to reduce this skepticism, because different investigation methods have begun to show agreement concerning the structure of the frog rod. In the last 5-6 years it has been realized that OsO, and KMnO, conserve rod ultrastructure not without artifacts; this is an important achievement in this field as compared to the decade from 1960 to 1970. Ten to fifteen years ago when, among others, Fernindez-Morin (1961, 1962), Robertson (1966), and Nilsson (1964a,b, 1965) published their classic studies on the rod structure, the best fixation was achieved with OsOl or KMn0,. Since 1970 glutaraldehyde has become the only, or at least the primary, fixation agent, together with oso4as postfixation agent. It became obvious that essentially two or three parameters decisively determine the FIG. 21. These four figures represent the same part of a rod longitudinal section tilted by different amounts around the axis K. The reader should compare this figure with Fig. 22 which is a schematic drawing of Fig. 21. The arrowheads show the direction of observation in Figs. (a) to (d). (a) Section tilted a, = +42" from the perpendicular about the axis of tilt, K. tu, Part of a tubule running parallel to the rod axis. The arrows indicate ring-shaped, osmophilic structures of which the tubule seems partly to be composed. (b) Angle of tilt as = + 18".This is regarded as the zero position. For further data see Table IXB;(c) Angle of tilt a2 = - 18".The values of the distances x" and x' are given in Fig. 22. (d) Angle of tilt a, = -42". Preparation as in Fig. 13. (Rosenluanz and Hauser, 1972.)

DIMENSIONS OF

THE

TABLE IX LAYERTHICKNESSES IN

THE

LAMELLARBODY^

A: Fixation by OsO, only

Buffer (mosM)b Cp' -200' 0 ?

ve ?

Osmium (%)

1 2' 1-2?

Total Period (mosM)" up -230' -60' ?

-220 -300 280"

Lamella"

Interlayer'

I,

1,

I,

I,

1,

93 150" 110'

123 150' -170"

-20' 25' -30'

-25" 25' -30"

-18' 25' -15"

0' 0' 0'

Embeddingh Fig.

-

vs

ep ma

7

-

Reference Nilsson (1965) Borojagin et ul. (1974) Fernindez-Morhn (1961)

B: Fixation by glutaraldehyde and OsO, or KMnO, (k)

'

Buffer (mosM)b Cp'

Glutaraldehyde

Osmium

Lamella'l

(%)

Total Period, (mosM)d ue

Interlayer'

1,

1,

loo'

200"

25"

35" 25'

190"

25'

30'

25'

I,

1,

1,

Embedding''

Fig.

Reference

20-30

gh

25

30'

ep

24

Nirand Pease (1973) Borovjagin et ul. (1971) Jones (1974) Nir and Pease (1975) Jones (1974)

ph

1

1

200-240

?

?

1

1

-230"

-300'

110'

120 -120'

ph ea

-1' 1

1 1 (k)

-230' -230'

-230 -260'

115' 65-85'

-115' 175-195'

15' 18'

20' 40'

15" 18'

20' -40'

ar gh

-

150 195 215 270

ph ph kk kk

-1' -1' 0.8 2.5

1 1 1 1

-260' -300' 300

105' 90" 60 80

115" 110' 130 150

16'

16' 18' 33 38

ar

33 38

19' 19' 0 0

13'

ar

-

540

220 200 190 230

480

kl

2.5

1

- 740

230

60

170

33

0

33

40

ep

21b

-120'

300"

18'

0' 0 0

ep ep

-

J. Rosenkranz (unpublished results, 1975) Rosenkranzand Hauser (1972)

C: Fixation by glutaraldehyde only or followed by PCI4 (p) Buffer

(mosM)b Cp" 220

kk

Glutaral- Total Period, dehyde (mosM)d a'

Inter layer'

Lamellag

1,

1,

1%

1,

0.8

300

200

55?

145?

lo?

50

10

1.6 +

240

190

35

155

21

50

- 13

-230' -230" -300' 300

235 -240' 165 220

IOO?' -40?* -35? s95?

140 200?' .-130?' 125?

20?' 17?" .16?' lo?

330 65' 35' 42

-330'

-210'

- loo?*

110?5

2 40?

170?

-90

ph

120 -120' 195 215

ph ea ph kk

-1.0 1.0 -1.0' 0.8

2220'

ph

1.o

270

kk

2.5

- 1.7 (P)

540

210

I,

Embedding"

-5

du

Fig.

23

0

du

20' 17' 16' 10

0' 0' 0" 0

ar gh ar eP

14?" 27'

14'

o4

6

16?

16

0

-

-

52

Reference J. Rosenkranz (unpublished results, (1975) J. Rosenkranz (unpublished results, (1975) Jones (1974) Nir and Pease (1975) Jones (1974) J. Rosenkranz (unpublished results, 1975) Borovjagin et al. (1973) Rosenkranz (1973)

A question mark indicates that the layer border was not discernible. Buffer as part of the fixative solution. c Kind of buffer: ea, Earle's physiological salt solution; kk, cacodylate buffer; kl, collidine buffer; ph, phosphate buffer; ve, Verona1 buffer. Total osmolarity of the fixative. Lattice constant, that is, the sum of the lamella and the interlayer thickness. Thickness of the interlayer. For abbreviations see Fig. 3. ar, Araldite; du, Durcupan; ep, Epon; gh,glutaraldehyde-urea; ma, methacrylate mixture; vs, Vestopal W. '1

f

70

J~~RGEN ROSENKRANZ

ULTRASTRUCTURE O F FROG ROD OUTER SEGMENTS

71

electron microscope picture of the rod in the ultrathin sectioning procedure: the fixation agent and its osmolarity and, in the case of fixation with glutaraldehyde alone, the electron stain. All other parameters such as the kind of buffer used, the dehydration agent, and the embedding medium, are of minor importance (but compare Appendix 2,A,1). Results of investigations concerned with the layers building up an elementary cell are presented most clearly when the findings of different investigators are compiled in tables. An elementary cell is a disc with a thickness of the period or lattice constant a situated at right angles to the rod axis. For a better understanding of Table IX it should be noted that after fixation with glutaraldehyde alone (or additional postfixation with a heavy metal such as osmium, platinum, or chromium) or after fixation with OsO, alone, the staining pattern can be assumed to be as shown in Fig. 3. Each lamellar membrane consists of two electron-dense layers, 1, and 1, in Fig. 3; often there is no significant difference in the staining and dimensions of 1, and 13, so one assumes in this section that 1, = 13. Between 1, and 1, is the electrontransparent layer 1,; l,, the intralamellar space, can be 20.When 1, is FIG.23. Part ofarod longitudinal section. On the lamellae, especially on their interlamellar side, small but heavily stained particles (re) are seen 40-60 A apart from each other. Because of their arrangement and size these particles may represent retinylidene groups. Fixation with 1.8% glutaraldehyde in 25 mM sodium phosphate buffer (115 mosM), Durcupan embedding. For data see also Table IXC. FIG. 24. Lamellar rims and parts of lamellae of a R. temporaria rod. The two triple-layered lamellar membranes (lm) and the intra- (l,) and interlamellar spaces are clearly visible. Fixed with 1% glutaraldehyde solution, postfixed with 1% 0 s . solution. For further data, partly communicated by Borojagin, see Table IXB. Reproduo tion after Borovjagin et al. (1971). FIG. 25. Lamellar rims and parts of lamellae in a longitudinal rod section. The cytoplasm proper and the cross-sectioned rims (hairpin loops) are more intensively stained than the interlayers and lamellae. Sample soaked for about 8 minutes in 0.1 M sodium phosphate buffer, fixed in 1% glutaraldehyde solution, postfixed with 1% OsO. solution, and polymerized in glutaraldehyde-urea. For further data, partly provided by Pease, see Table IXB. Reproduction after Nir and Pease (1973). FIG.26. Part of a rod longitudinal section from R. temporaria. In Jones’ opinion this rod, with a periodicity of 235 is neither swollen nor shrunk; this is questionable in view of the “true” periodicity of 300 A. Fixation with 1% glutaraldehyde in 50 mM sodium phosphate buffer (120 mosmoles). For details see Table IXC. Reproduction from Jones (1974). FIG. 27. Part of a rod longitudinal section. The heavily stained almost tubulelike lamellar rims (rw)are in contrast to the only badly preserved lamellae proper (lm). The “lumen” of the rims is more strongly stained than their walls. Fixation with 1.8% glutaraldehyde in 25 mM sodium phosphate buffer, postfixation in 50 mM Pt(IV)CI, solution, Epon embedding.

72

JURGEN ROSENKRANZ

reduced to zero, the summation of the staining of the neighboring layers 1, and 1, of a lamella leads to one heavily contrasted broader layer, so that the seven-layer lamella becomes a five-layer lamella. Fixation with oso4alone sometimes leads to only one stained layer for a lamellar membrane. Then one can assume 1, = 0, an assumption that is justified according to the investigation by Nilsson (1965). The following figures, representative of many others, may serve to illustrate Table IX: Fig. 7, Table IXA; Figs. 24 and 25, Table IXB; and Figs. 5, 6, and 26, Table IXC. The results in Table IX are classified in two ways: experiments with chemically identical fixation agents are compiled, and within these groups the experiments are listed with respect to increasing osmolarity. Jones (1974) was the first to investigate systematically the influence of the buffer used during fixation on the preservation of rod structure in longitudinal sections. H e found that, after fixation with glutaraldehyde as well as after fixation with glutaraldehyde and Os04 (but not after fixation with Os04 alone), the appearance of longitudinally cut rods depends on the osmolarity of the buffer. A 50 mM phosphate buffer with an osmolarity of 120 mosmoles, which is approximately half the osmolarity of the e y e liquid (Cohen, 1971), allowed the frog rods to appear “most normal.” By this expression Jones means (simplifying) that the order of the lamellae is essentially normal, that is, the lamellae are almost regularly ordered perpendicularly to the rod axis and are separated from each other by continuous electron-transparent spaces. The additional osmolarity caused by the glutaraldehyde (about 100 mosmoles per 1% glutaraldehyde) is irrelevant in the preservation of the structure because it does not change, at least when glutaraldehyde is used in the range 0.5-3%. A closer inspection of Table IX reveals a relation between buffer osmolarity and the lattice constant a only in the case of glutaraldehyde fixation followed by Os04 fixation (Table IXB); with decreasing hypoosmolarity of the buffer the lattice constant decreases. There is no difference, however, between hyperosmotic and isoosmotic buffers. After glutaraldehyde fixation alone the periodicity is = 220 A, even if the osmolarity is raised threefold. The following observations have been made concerning fixation agents. No matter which of the three chemical fixation methods is applied, the periodicity has in most cases an average value between 220 and 240 A, but in single cases up to 300 A. The thickness of the lamellae amounts to 130-160 A, independent of the three fixations. These fixatives lead to other similarities, for example, the three-layer staining pattern of the lamellar membrane and its relatively constant

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

73

thickness of about 75 8. Except for the fact that the proportions of the three membrane layers vary, the effects are similar for all three chemicals. The membrane is symmetric (11 = 13) after glutaraldehyde fixation followed by OsO, fixation (Table IXB), whereas the interlamellar part of the membrane (11) is always broader than 1, after postfixation with PtCl,; the same is true for OsO, fixation alone. In the case of glutaraldehyde fixation alone nothing can be accurately determined because of the indistinct contours of the layers. Differences also exist in the widths of the electron-transparent layers 1, and 1,. The latter does not appear after fixation with OsOl alone, or generally after fixation with glutaraldehyde alone or in combination with postfixation with platinum or chrome (which have similar effects). In the embedded material oxidizing stains show an intralamellar layer after fixation with glutaraldehyde alone and, for example, K M n 0 4 staining; the same applies to glutaraldehyde-Os04-fixedrods, independent of the osmolarity. After these quantitative considerations we now discuss the shape of the components that make up the lamellae. Borovjagin et al. (1971) maintain that a continuous mostly unstainable lipid bilayer is the basic skeleton of the lamellar membrane, with stained 40-45 A rhodopsin complexes penetrating into the lipid layer from both sides so that the rhodopsin is distributed symmetrically. In the 1960s, however, Robertson et al. (1964)and, still more decisively, Nilsson (1965)stated that the lamellar membranes of the frog rod are structured as follows. A linear array of unstained, globular subunits (diameter ca. 25 8, distance from each other ca. 50 8)separated by stained septa represents the central layer of the membrane; this is limited on both sides b y two symmetric, electron-dense layers. The beaded, stained outer layers are thickened where they make contact with the septa. Nilsson (1965) does not specify the exact position of lipids and proteins in this structure. While Nilsson (1965)was not sure whether or not the membrane described was artificially changed by the preparation procedure, later investigations by Godfrey (1973), Nir and Pease (1975), and J. Rosenkranz (unpublished results, 1975) showed that essential aspects of Nilsson’s description of the lamellar membrane are very close to the actual membrane structure. All workers, in spite of different preparation methods, found identical structures in the longitudinal section of the frog lamellar membrane. Godfrey (1973)describes the structure as “pale globular elements [which] appear to form an irregular band of unstained material against which small irregular masses of dark stain are precipitated.” Nir and Pease (1975) mention the electrontransparent middle layer (12) which consists of “globular structural

74

N R G E N ROSENKRANZ

subunits” of approximately 50-55 A diameter. On the basis of sections extracted by chlorofom-methanol they assumed that these unstained globular structural subunits contain protein, in contrast to Godfrey (1973),who observed lipids there. Perhaps the results obtained by Nir and Pease (1975) on sections treated with ionic stains can be explained in a different way: The electron-transparent globular subunits could appear globular owing to the fact that electron-dense, vertical bridges enlarging at both ends into strongly stained irregular structures span the membrane at intervals of 70-85 A (Fig. 28a, left). The

FIG.28. (a) Part of a rod longitudinal section. A Schematic representation of the staining pattern derived from Table IXB,appears on the right, with the abbreviations used in Fig. 3. Good preparations often show the pattern indicated on the left. Over an average distance b there are positively stained, approximately round particles (sp); these particles are frequently connected by narrow septa with similar but smaller particles on the other side of the membrane. Particles that appear round when viewed from above thus appear dumbbell-shaped when viewed from the side. (b) Part of hvo rod lamellae in a longitudinal section. We assume that this shape describes the arrangement

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

75

size of these structures is difficult to measure, as they do not have exact contours; they are, however, smaller on the interlamellar side and much smaller on the intralamellar side than the 55-A electrontransparent globular subunits. I n these micrographs, as well as in those of Godfrey, one can distinguish three gray tones, that is, three differently contrasted regions in the lamellar body: the lightest (most transparent) layer in the center of the lamellar membrane, a gray layer between the lamellae in the interlayers, and black, irregularly shaped particles attached to the light and middle layers. We have carried out investigations producing similar results (Figs. 20d and 23). Only the dimensions of the layers were found to differ, but this can be accounted for by the different preparation methods used. The positive staining of the attached particles on the interlamellar side was further increased by postfixation with PtC1, and less so by K2Cr07(J. Rosenkranz, unpublished results, 1975). iii. The interlayer. Besides these findings, which in principle agree with those of, for example, Nir and Pease (1975), Borovjagin et al. (1973), and Nilsson (1965),Rosenkranz (1973) found that fibrils seem to emerge from the stained lamellae and the interlamellar space in longitudinal sections. This is supported by the appearance of fibrils in the cross sections shown in Figs. 19 and 20a, as well as in Fig. 5. Because of recent experiments, however, we cannot maintain our earlier working hypothesis that the whole lamellar membrane consists essentially of fibrils. Rohlich (1971), Godfrey (1973), and Borovjagin et al. (1971) also found fibrils in the intermediate layer. Rohlich calls them of the rhodopsin (rh) and the retinylidene (re), the lipids (li), and the cell water containing some nonrhodopsin proteins (dotted area). Each rhodopsin molecule consists of one polypeptide chain coiled up from both ends to form tangles (kn), whose ends meet the corresponding ends of neighboring rhodopsin molecules, provided that the r h e dopsin aggregate (hx) consisting of six single rhodopsin molecules (rh) has not been destroyed. For clarity only the two tangles (kn) are indicated; the mass density at these loci is much higher. The dumbbell model shows further that by this special arrangement of the rhodopsin molecules a pore (pl and &) is established; the dashed circle indicates that larger particles could be positioned at pl; pore pz is open toward the interlamellar space. This is due to photoisomerization of the retinylidene (re), as suggested. The fibrils to the right of the rhodopsin aggregate (hx) are presumed to b e the ends of the polypeptide chain tangles; they are connected either loosely or not at all to the neighboring rhodopsin molecules; while this is a speculation, the arrangement of the components of the lamellar membrane model are in agreement with important experimental results obtained with different methods. (c) Enlarged details of (b). Here the geometry of the retinylidene group (re), which is responsible for the opening and closing of the pore, is shown more clearly. The ends of three fibrils of the lower membrane, which point to the interlamellar space, can also be observed.

76

JURGEN ROSENKRANZ

“vertical [to the lamellae] irregular structures in the cytoplasm,” Godfrey describes “globular and linear arrays of electron dense material,” and Borovjagin et aZ. just mention “fibrillar miniproteins.” Less specific studies on the structure of the interlayer have been carried out by Cohen (1968) and Borovjagin et aZ. (1973). Cohen (1968) found by infiltration experiments that barium sulfate could penetrate only into the cytoplasmic areas neighboring the rims (marginal cytoplasm csl; Fig. 3) and not into the interlayer (csz);for this reason he ascribed a “certain kind of structure” to the layer. For lanthanum nitrate, however, there is no difference between marginal cytoplasm and the interlayer, presumably owing to similar properties of La3+and Caz+.Borovjagin et a2. (1973) also mention differences in the behavior of marginal cytoplasm and of the interlayer. Thermal denaturation and treatment of the rods with 2-8 M urea before fixation with glutaraldehyde destroyed the interlayers; they were electron optically as empty as after fixation with O s 0 4 or KMn04; uranyl acetate dissolved in alcohol stained the interlayers more weakly than the marginal cytoplasm (Fig. 25); Pedler and Tilly (1967) found in the clawed toad (Xenopus Zaeuis) that a single lamella could be isolated by ultrasound only after immersion for at least 30 seconds on Os04 solution, but not in a glutaraldehyde solution. From this result one can draw two conclusions: (1)that in vivo there must be a connecting structure between the lamellae and (2) that this structure is at least partially destroyed after standard fixation. iv. Summary. Summarizing, the results obtained from ultrathin sections of rods can be interpreted as follows. If one takes into account the lattice constant a = 295 5 8, found by x-ray diffraction experiments on unfixed rods in vivo (Webb, 1972), it is most probable that the characteristics of the rod are optimally represented, that is, they are as similar as possible to those of the in vivo state, when the rods are fixed in glutaraldehyde with a 120 mosM buffer and then treated with oso4.A lattice constant of 300 8, is also obtained by freeze-substitution. A rod fixed in this way possesses a three-layer lamellar membrane, the outer layers (11 = l3 = 23 8,) of which are stained by oso4and uranyl acetate; these layers enclose the intermediate electron-transparent layer of about 30-8, thickness. The staining pattern 1, = l3may not represent the true material distribution; l1 # l3 cannot be completely excluded. The lamellar membrane in turn delimits an intralamellar space of 0- to 40-8,width (see, however, Appendix 1,A). The observation that all electron-transparent spaces become smaller with this fixation under increasing extracellular osmotic

*

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pressure, but not after fixation with glutaraldehyde alone, indicates a material soluble in water and oso4,which in this case can only be a protein compound. Accordingly, the continuous presence of electron-dense layers in the membrane suggests extensive-accumulation of fixed material. The observation that the layer stained by uranyl acetate does not extend as far into the interior of the membrane as that stained by OsO, leads to the conclusion (see Appendix 2,AJ) that the phosphate groups of the phospholipids are located in the outer interlamellar regions of the lamellar membrane, that their hydrocarbon chains point toward the center of the membrane, and that the water-soluble proteins are essentially located in both marginal areas. As for the building blocks of the membrane, an objective description of the ultrastructure of the layers mentioned above suggests that there are, strictly speaking, more than just the two possibilities of stained or nonstained as found in the 1960s. Recent experiments have revealed at least three staining grades: light, medium, and strong contrast; and in the electron-dense and transparent layers just described one also finds combinations of these gray tones. So the generally light intermediate layer of the lamellar membrane is traversed by narrow, stained bridges widening at their ends; this indicates a dumbbellshaped macromolecule. Some micrographs (Nir and Pease, 1975; Godfrey, 1973) suggest that the light areas between the dark bridges are spheres or globules. This interpretation is by no means convincing, and is less so considering that these globules have never been isolated; see also Fig. 5. Borovjagin et al. (1971),however, favor the view that the nonstained intermediate layer is a continuous lipid bilayer, an opinion that probably cannot be maintained in view of the many interruptions of this layer. As far as the dark, marginal layers limiting each lamellar membrane and the interlayer are concerned, more recent studies show a more differentiated staining pattern, especially on the interlamellar side (Nir and Pease, 1975; Godfrey, 1973; Borovjagin e t al., 1973; Figs. 20d and 23). Sometimes strongly stained, irregularly shaped particles, which are often continuations of the stained bridges, are attached to an interlayer of medium contrast at more-or-less irregular intervals. Some investigators have also described fibrillar material of medium contrast (Borovjagin et al., Godfrey, Rohlich, and Rosenenkranz). It is not quite clear why these, possibly only short, fibrils do not appear in all carefully treated preparations; presumably this is a problem of contrast reproducibility.

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An interpretation of the results previously mentioned, together with those mentioned later (see also Section III,B,4), allows a description of the lamellar membrane to be made. In taking account of the structural units and the formation of aggregates of electron-dense material (shown, for example, by the tilting experiments), as well as the probable short, fibrillar connections between the latter, a model was obtained as shown in Figs. 28 and 29. This model is certainly not the only possible one allowing an interpretation of the bulk of results obtained by the ultrathin section technique; it has, however, the advantage (as shown in Sections IV,D,l,b and c; IV,D,3; and V1,C) that it is

y?

sp' -

\

hx fb I

-

100A

FIG.29. (a) The packing density of single rhodopsin molecules (sp) in one lamellar membrane. This density has been calculated under the assumption that the rhodopsin concentration cR = 2.5 mM and that the length and diameter of the rod are as i n vivo. The rhodopsin molecules are shown to be almost circular and positively stained in the projection which was made at right angles to the membrane surface. Short fibrils (fi), not shown in all rhodopsin molecules in ultrathin section, are also often absent here. The almost statistical distribution of the single rhodopsin molecules (caused by the preparation technique?) corresponds to the experimental results if one assumes the positively stained particles (sp), for example, in Fig. 20a, to b e rhodopsin molecules. (b) This drawing represents the same packing density shown in (a). Contrary to that figure, here a high degree of order is assumed; every six rhodopsin molecules (sp) form an aggregate which can also b e described as a hexagonal particle (hx) because of its outline. The fibrils (fb)are interpreted as being the ends of the rhodopsin peptide chains which appear to b e loosely attached in pairs, p is the mean distance between neighboringrhoreflection of the small-angle x-ray difdopsin aggregates; b corresponds to the (55 fraction, the coordinate v is identical to that in the left part of Fig. 44b. Freeze-etching and x-ray diffraction experiments, however, show that this high degree of order is only approximately reached in small regions.

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fairly consistent with the findings from totally different preparations and investigation methods. As to the relationship between the differently stained parts of the lamellar membrane and the chemical compounds found in the rod, the general opinion (see also Appendix 2,A,1) prevails that the saturated fatty acid residues of the phospholipids are at the loci of weak or absent staining, while the major fraction of the proteins as well as hydrophilic parts of the phospholipids are located in the stained marginal areas. We, Borovjagin et al. (1973), and Jan and Revel (1974), who worked with the mouse and not the frog, also claim that the strongly stained parts of the marginal area of the lamellar membrane are due to rhodopsin embedded there. While Borovjagin et al. made their statement tentatively, Jan and Revel found marked precipitation of rabbit antibodies for bovine rhodopsin on both sides of the lamellar membrane of the mouse rod. The statement is also supported by a synoptic interpretation of the previous experiments, the x-ray and neutron diffraction experiments described later, and a consideration of staining mechanisms described in Appendix 2,A,1. The stained septa across the light membrane layer can be regarded as part of the 2000-Along rhodopsin molecule (if without secondary structure). It is considered that approximately equally large parts of a rhodopsin molecule are located three-dimensionally in both marginal layers linked by a short length of the rhodopsin molecule thread (see Fig. 28b). The appearance of stained septa is, however, partly caused by the stained unsaturated fatty acid residues of the lipids, which must be assumed to be located near the bridges in order to fit the pattern previously mentioned. On the basis of the ultrathin section technique alone, Nir and Pease (1975) came to an essentially different conclusion about the site of the rhodopsin in the lamellar membralle; they assume that the “globuli” represent at least the larger hydrophobic part of the rhodopsin molecule. b. Freeze-Etched Lamellar Body. AAer having described the lamellar body in detail in the preceding section we restrict ourselves to the two most essential aspects of the rod inner core: the longitudinal and cross sections of fracture. Appendix 2,A,2 should be consulted for the precautions necessary when interpreting freeze-etched replicas. i. Cross-fractured rods. Fracture faces of frog rods were published by Rosenkranz (1970,1976b) and Mason et al. (1974). As Mason et al. found great structural differences in isolated rods after light and dark adaptation, their results are also dealt with in Section V. In lightadapted rods, isolated or still part of the retina, these investigators found a smooth, hydrophilic intra- and interlamellar surface (ES and

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PS fracture faces), as well as a cleaved hydrophobic surface within the membrane and parallel to its outer surfaces, which they describe as rippled ( E F faces). [See Branton et al. (1975) concerning the nomenclature.] After illumination, this E F face has a great number of statistically distributed particles (diameter ca. 125-175 A) which appear to be aggregates of four to eight molecules and emerge about 20-30 A out of the surface. Taking into account the particle density (one molecule per 5400 &), the graded rhodopsin extraction, and the findings concerning illuminated rods fixed with glutaraldehyde, these investigators suggest that the particles on the intralamellar half of the membrane ( E F face) are rhodopsin molecules. According to this interpretation, the lamellar membrane seems to be a highly asymmetric protein-lipid double layer to which rhodopsin, in a state of dark adaptation, is bound only on the intralamellar ES face (see Section V,B,l,b). Rosenkranz and Stieve (1969) and Rosenkranz (1970) published the first micrographs of a cross-fractured rod. At that time we cautiously FIG.30. Part of a cross-sectioned rod not pretreated with glycerol. This standard platinum-carbon replica shows fibrils next to humps (hx) with diameters dl and d S which cannot, however, be pursued along their whole length. Compare with Fig. 32. Freeze-fracture without glycerol pretreatment (Rosenkranz, 1970). FIG.31. Parts of obliquely fractured rod lamellae with hexagonal particles (hx) connected at several points by short fibrils (fb). These particles are on the interlamellar side of the lamellar membrane; on the intralamellar side they are less distinct. Deep freeze-fracture, platinum-carbon replica. FIG.32. Obliquely cross-fractured rod not pretreated by glycerol as in Fig. 30. This time, however, the fracture was shadowed by tantalum tungsten alloy, producing a more detailed replica. The humps observed in Fig. 30 now appear as very distinct hexagonal, truncated pyramids (hx). Deep freeze-fracture (Rosenkranz, 1976b). Inset: Light-diffraction pattern. Contrary to what can be expected from mere observation of the micrograph, the membrane fragments covered with hexagonal particles (hx) shown in Fig. 32 produce this rhombohedra1 diffraction pattern which can also be described as a distorted hexagonal pattern; the obliquity is caused by the fact that the lamellar membrane does not run parallel to the plane of the negative. FIG.33. Part of a cross-fractured rod. The fracture face lies at the interlamellar surface of a lamella (Eface), mostly below the interlayer (El) but to some degree also above it (E.J. The El face therefore is the view fmm the interlamellar space ofa lamellar membrane; the larger, heavily contrasted particles (hx) are interpreted as being hexagonal particles, and the fibrils (fb)have a diameter of about 50 8, The & face represents a view of an interlayer. Here, too, fibrils (fb) occur with a diameter only slightly less than 50 8, Granules (gl) with the same diameter are predominant in the interlayer. These granules can also be found on the El face. [Nomenclature not according to Branton et al. ( 1 9 7 5 ) ~Deep freeze-fracture; retina soaked for 3 hours in 25% glycerol-Ringer’s solution and then frozen, via propane, in liquid nitrogen; platinum-carbon replica.

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interpreted the electron micrographs of freeze-fracture faces in the form of a working hypothesis, suggesting that the inner core of the rod consists of several classes of fibrils (diameter ca. 40-60 A) which are partly linked in a network and partly simply stacked, pointing in different directions. The distances between neighboring fibrils in the network were between 60 and 180 A. We continue to be convinced that an essential part of the lamellar body is of fibrillar nature, considering, for instance, the similarity of Figs. 6 and 37 which show a surprising amount of fibrillar material in spite of different preparations. With a better freeze-etching technique (Rosenkranz, 1976b), however, success was obtained in producing more conclusive pictures of cross-fractured rods (e.g., Fig. 32). The comparison of Figs. 30 and 32 immediately leads to a different interpretation. In the refined technique (Fig. 32) the spherical, irregularly arranged humps in Fig. 30 seem to be essential parts of an otherwise rather homogeneous surface. They no longer appear as cross-points of fibrils as in Fig. 30, but as truncated hexagonal pyramids on the interlamellar side of the lamella (Fig. 32). Structures that can almost only be interpreted as being fibrils are revealed with the improved technique too, but they no longer dominate; sometimes, as short fibrils, they seem to connect neighboring hexagonal particles (truncated pyramids) (Fig. 31), and sometimes they are located in the interlayers and appear as beaded fibrils (Fig. 33).The distance between two parallel sides near the base of such a truncated pyramid isf& = 139 -+ 22 A (Rosenkranz, 1976b), the mean distance between two such pyramids is ljcF = 192 & 65 A (97 measurements), and the fluctuation width is ApIpGF = 0.34. With respect to the rather similar arrangement and structure of the hexagonal particles in Figs. 18 and 32 a more detailed investigation of Fig. 32 by means of light diffraction, analogous that carried out for Fig. 18, seems to be advisable. The light diffraction pattern in Fig. 32 led to the diffraction pattern in Fig. 32a which, except for minor distortions, can be regarded as a hexagonal reciprocal lattice. In order to become more familar with these patterns Fig. 35 shows the reciprocal lattice of a schematic drawing of Fig. 32, and Fig. 34 shows the drawing itself. The absent long-range order of the hexagonal particles is expressed by the lack of higher (hexagonally arranged) reflection orders; the reduced intensity of two reflections in Fig. 32a and the slight shear of the reflections suggest particles that are not completely symmetrically arranged or a distorted electron microscope representation. The rod in Fig. 32 is not fractured exactly perpendicularly but slightly obliquely. ii. Longitudinally fractured rods. Longitudinal fracture faces of frog rods were rather extensively described by Korenbrot et al. (1973),

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FIG.34. This figure indicates the arrangement and size of the hexagonal particles in Fig. 32; it served as a specimen for the light-diffraction pattern in Fig. 35. FIG. 35. Light-diffraction pattern of Fig. 34. The light diffraction was performed under the same conditions as in Fig. 32.

Rohlich (1971), and Rosenkranz (1970); see Figs. 36-39. These faces, which are parallel to the longitudinal axis of the rod, have a period or lattice constant in this direction of 300 & 10 (Korenbrot et al., 1973) or 302 & 21 A (Rosenkranz, 1970). I n both cases the error is taken to be the systematic error in the length determination. According to Korenbrot et al., the height of the lamellae is 151 z t 15 A and thus equal to the height of the interlayer. These dimensions do not depend on preliminary treatment of the rods before freezing by 20% (vh) glycerol. We, however, found differences between rods treated with glycerol (Fig. 36), which look similar to those in Figs. 38 and 39, and rods that were directly frozen (without glycerol), which leads to electron micrographs like Fig. 37 in which the lamellae are less distinctly separated from the interlayers and the fibrillar material can be more easily distinguished. Rohlich has also described irregular structures at right angles to the disc (Fig. 38). His electron micrographs show humps mainly on the interlamellar sides of the

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membrane; these humps have a height of 110 k 6* A, a width of 95 k 5* A, and are at a distance of 160 k 15* A from each other. They also appear in the longitudinal fracture face as shown in Fig. 36, which is, however, not exactly parallel to the longitudinal axis. In addition, the dense granular material, filling the spaces of the interlayers in this figure, is remarkable. As shown by the fracture face in Fig. 33, the granulas could also be interpreted as strings of granulas, depending on the angle of view. iii. Summary. The results obtained by freeze-etching show that the distance between neighboring elementary cells is 300 A and that the thickness of the lamellae after treatment of the rods with glycerol is half this value. The influence of glycerol on the rod structure is, in our opinion, not yet clear. Some researchers agree that glycerol does not influence the structure at all. Three observations must, however, be considered: (1) rods that have been treated with glycerol, as is often the normal procedure for freeze-etching, and then fixed with glutaraldehyde and embedded for ultrathin sectioning, show a more disorganized lamellar structure in the electron microscope than rods not previously treated with glycerol; (2) rods treated with glycerol, and only these, have a much larger cytoplasmic space between the cell membrane and the lamellar body; and ( 3 )glycerol is used by some workers who claim that it does not change the structure or dimensions of the rods (then why use it at all?) for example, Korenbrot et al. (1973), whereas an essential advantage of the freeze-etching technique is that previous chemical treatment with glycerol can be omitted. However, we have also observed that glycerol has no strongly deforming influence on some parts of the rods such as the hexagonal particles. When glycerol is added, the cooling velocity is not so critical for structure FIG. 36. Part of a fracture face cleaved not exactly parallel to the rod axis. The interlayer is filled with granules (gl).The surface of the lamella facing the interlayer (El face) is covered with hexagonal particles (hx). Deep freeze-fracture; soaked for 3 hours in 25% glycerol-Ringer’s solution before freezing; platinum-carbon replica. FIG.37. Part of a longitudinal fracture of a rod not pretreated with glycerol. Fibrils (fb)are clearly seen. Compare this with the corresponding ultrathin-sectioned rod in Fig. 5. Freeze-fracture, platinum-carbon replica (Rosenkranz, 1970.) FIG.38. Part of a r o d longitudinal fracture from R. esculenta. Humps (hx) are attached to the lamellae. fb, Fibrils. “It is possible that the cytoplasmic proteins are oriented perpendicular to the plane of the lamella, and that their coarse structure is caused by the preparation method.” After soaking the isolated retina in 20% glycerol-O.l M cacodylate buffer, it was frozen, via Freon 22, in liquid nitrogen; platinum-carbon replica. From P. Rohlich with kind permission. FIG. 39. Part of a longitudinal fracture of isolated rods from R. catesbeiana. The rods had been kept in a 20% ( v h ) isoosmotic glycerol solution (232 mosM) prior to freezing. Standard freeze-etching. Reproduction from Korenbrot et al. (1973).

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preservation as when the preparation is directly frozen, thus the probability of obtaining better-quality replicas increases after glycerol treatment. Independent of the preliminary treatment and at high electron microscope resolution, longitudinal fracture faces show that the lamellae have an intralamellar space of 0 to about 25 A (Fig. 38) and that the lamellar membrane is constructed asymmetrically. It has marked hexagonal, truncated pyramids on the interlamellar side, but neither the height of these humps nor their distance apart can be distinguished so clearly on the intralamellar side. This is also observed after Os04 fixation or glutaraldehyde followed by postfixation with PtC14 (Section IV,D,l,a,ii); both treatments lead to more heavily stained P sides than E sides of the lamellar membrane. Short fibrils, parallel to the lamellar surface, can be found in the interlayer and between hexagonal particles in the cross-fracture face. In the interlayer fibrils of 50-Athickness appear to be constructed from globules of this diameter. The fibrils between the truncated pyramids have a diameter of about 50 A and seem to connect to neighboring hexagonal particles. As shown by light-diffraction experiments, there exists only a hexagonal short-range order of the particles and not a long-range order. A volume estimation (Rosenkranz, 197613) shows that a hexagonal, truncated pyramid is probably an aggregate of six rhodopsin molecules. This idea is confirmed by x-ray diffraction experiments (Section IV,D,3,b) and measurements of diffusion constants (Section V1,C). The results of Mason et al. (1974) are not compatible with those described above. The main criticisms of these results are that the magnification used was too small to investigate structure and that the particles in question were not defined, either in the text or in the figures. It is therefore difficult to decide whether the particles “clearly suggested to be rhodopsin molecules” are intralamellar (i.e., contrary to the previous situation) or in the lamellar membrane itself. It is also difficult to understand what is meant by “ripples.” Perhaps the aggregates of four to eight molecules are identical with the hexagonal structures described by Rosenkranz and the humps of Rohlich. Mason et al. (1974) and Abrahamson and Fager (1973) stated that the location of rhodopsin on the intralamellar side of the membrane is equivocal? c. Spreading ofRods. The term spreading is used here not only to describe the classic spreading procedure but also all drop preparation methods in which homogenized biological material is placed directly on grids for electron microscopy. The first experiments were carried After completion of the present article Corless et al. (1976) reported that the rhodopsin clusters (hexagonal particles),although seen in the fracture faces, are not found in oioo.

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out by Fernindez-Morin (1954) with frog rods fixed with Os04. In addition to publishing the well-known figures of single, whole lamellae he stated that the lamellar membranes “show a tendency to disintegrate into peculiar contorted threads reminiscent of submicroscopic myelin figures.” These threads have a width of about 150* Eleven years later, Blasie et al. (1965) carried out spreading and drying preparation experiments on rods of R. pipiens. They used sodium phosphotungstate, sodium silicotungstate, and KMn04 as staining and fixing agents. After treatment with sodium phosphotungstate and KMn04 they found dried fragments of lamellar membranes, partly connected by “tubular structures,” consisting of spherical light areas which they referred to as “particles.” These particles had a diameter of about 40 8, seemed to form square unit cells with an average dimension of about 70 and seemed to be interconnected along the base vectors. Another inspection of the micrographs published by Blasie et al. (1965) could lead to the following interpretation. Not the light areas (particles) but the dark stains between these areas may be particles, since the chemistry of the special negative stain has not yet been clearly determined and there is no evidence whether it stains negatively or positively. The dark areas are only somewhat smaller and less conspicuous compared to the light areas. The dark particles also seem to be connected by less intensely stained structures; this is due to the fact that the areas between the light and the dark particles are almost equally weakly stained and can be interpreted from two perspectives. A similar interpretation of the results from light-diffraction experiments with these membranes must also be made. The light-diffraction experiments are of decisive significance in the interpretation of Blasie’s experiments because they do not exclude the same hexagonal short-range order of the particles already detected in rods investigated by ultrathin sectioning and by freeze-etching. The light-diffraction pattern of the lamellar membrane negatively stained by phosphotungstate (Blasie e t al., 1965, Plate IIIa) clearly indicates a square array of the particles in the membrane, but the diffraction pattern of the similarly treated membrane shown in (Blasie e t al., 1969, Plate IV) can be regarded as a degenerate square or hexagonal lattice; this has been shown by our light-diffraction experiments. The lattice type seen depends on the area selected for the light diffraction. Negative staining by silicotungstate led to a different representation of the structures. The membrane appeared to disintegrate into ribbonlike structures of 130- to 150*-Awidth, which seemed to be composed of polygons (hexagons?), each showing a depression or pore in its center filled with stain. Rosenkranz and Hauser (1972) also demonstrated a ribbonlike

A.

A,

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structure in disintegrating PTA-stained membranes in drop and spread preparations. These ribbons had a width of 169 16 A and contained light, that is, negatively stained, possibly hexagonal areas. These stained areas also appear on other membrane surfaces. Figure 43 more clearly shows hexagonal particles after spreading on ammonium acetate. A comparison between light- and dark-adapted but otherwise similarly treated rods leads to the conclusion that the average diameter of the hexagonal particles increases after the rods have been illuminated. The distance fNF between adjacent, parallel sides of the hexagonal base is, in the light-adapted state, 270 & 74 A, the 74 A being the standard deviation (Rosenkranz, 1976b). The results of this section can be summarized as follows. In contrast to ultrathin sectioning and freeze-etching techniques, spreading and drop techniques conserve not the whole object but only the small parts to be investigated. In the rod these are the lamellar membrane fragments and their structural subunits. When handling larger fragments, such as membranes, one cannot expect the position of the structural subunits after spreading to correspond to their position in uiuo, because surface tension effects large forces in a direction away from the specimen while opposite forces operate during the drying process on the grid. Distances such as the basis vector length of a square unit cell in the membrane therefore cannot be accurately estimated. This is not the case when investigating the ribbons of about 150-A width mentioned by Femhndez-Morhn (1954), Blasie et al.

*

FIG.40. X-ray diffraction pattern of three pieces of neuroretinas placed in Ringer’s solution (4°C) and irradiated with their long axis parallel to the beam. h is the only reflection. The large horizontal bar and the spikes are artifacts caused by beam f e cusing. The cylindrical chamber had a diameter of 6 mm and a thickness (in the d i e o tion of the beam) of 2 mm. Irradiation for 9200 seconds with synchrotron radiation at DESY in Hamburg: 6.5 GeV, fiveeighths of the maximal available intensity; distance ~ 5(Rosenkranz, . fmm chamber to film 80 cm; magnification of the reproduction 1976a.) FIG.41. Oblique view into an indentation (eb) of a rod. rw indicates clearly discernible segments of two lamellar rims. The construction of the rims of ring segments is seen here as well as after freeze-etching. Fixation as in Fig. 16. FIG.42. X-ray diffraction pattern of the rods of an intact, unfixed retina (R. pipiens). The first eight orders of a lattice constant a = 296 A are distinctly visible; the ninth to twelfth orders observed by Worthington also appear on the negative but are too faint to be printed Irradiation time 2 hours. Reproduction from Worthington (1973). FIG.43. Parts of spread, dark-adapted rods. These hexagonal particles (hx) differ from those of light-adapted rods only in their mean size. Spread on 0.1 M aqueous ammonium acetate (194 mo&, pH 7.0); stained with 1% PTA (12 mo&, pH 7.5). (Rosenkranz, 1976b.)

-

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JORGEN ROSENKRANZ

(1965)and Rosenkranz and Hauser (1972).The frequency and consistency of their occurrence after different preparation techniques strongly suggest that here structural subunits of about 150-A width are closely connected with one another to form ribbons. An approximately hexagonal shape describes these elements better than a circle or a square. A hexagonal structure could explain the “contorted” ribbons described by Fernhndez-Morh (1954), which were unfortunately shown only at low magnification. The subunits of the ribbons shown by Blasie et al. (1965)are remarkably similar to the structures referred to as “hexagonal particles” in the ultrathin sections of a lamellar membrane in spite of totally different preparation techniques (Fig. 18). In both cases (and also in freeze-etched preparations, e.g., Fig. 32) the stained, hexagonal particles appear to be surrounded by a bright halo. When the hexagonal particles are totally isolated (Fig. 43), they appear to be of a different size. This difference cannot be expected to exist in viva unless several building blocks normally stick together. It could also be explained if the inner structure of one structural unit were loosened by the preparation technique. The former explanation can be excluded after taking other information and measurements into account, therefore only the latter explanation is possible. This view would be supported if traces of electron stain could be found in the larger, broken-up, hexagonal particles, but this is not the case. A clear definition is given by Blasie et al. (1965),who consider that “particles” is just a name for the light areas on “negatively” contrasted membranes. This description does not exclude the other possible interpretation that the dark, “positively” contrasted areas are particles. The term negative staining is therefore considered unsuitable in this case, since it implies that the size and position of the structural units are already known.

2. Neutron Diffraction Effected by the Lamellar Body Neutron diffraction has only recently been used to help clarify the description of rod ultrastructure; in the future it may prove to be a technique without which we can make no progress. The method is briefly described in Appendix 2,B,3. A short abstract by Yeager et al. (1974) reports that 10 retinas of R. catesbeiana were investigated by means of neutrons in the dark; in 100% D20the intensities of eight orders of 300-A periodicity were measured. We carried out neutron diffraction experiments with isolated rods, as described in Appendix l,A. Experiments performed by Chabre and co-workers have not yet been published but have been re-

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ported in a lecture (Chabre, 1975a).3These workers irradiated rods at right angles to their long axis; they registered the intensities of the diffracted neutrons two-dimensionally during a period of 1 minute by means of a position-sensitive detector. The rods were not isolated, in contrast to those used in our experiments, but were still connected to the retina and oriented magnetically. Intensity and sharpness of the reflections were thereby considerably increased. Reflections of the 300-A periodicity have been demonstrated up to and including the sixth order. Chabre has also investigated neutron diffraction patterns of rods embedded in different mixtures of DzO, HzO and Ringer’s solution. The main advantage of the neutron-diffraction method is that it is possible to adjust the scattering length density p n for neutrons of any compound by the addition of heavy water, thereby increasing the contrast of a certain other compound for neutrons, for example, protein or lipid. In other words, provided only two other compounds besides the cell water are in the system to be investigated, the addition of a certain quantity of D 2 0 makes the scattering length density of the cell water volume equal to that of one of the other compounds, so that the neutrons can no longer differentiate between the cell water mixed with heavy water and the other chemically different compound. Based on Fourier syntheses Chabre suggested that the lipids are predominantly situated on the outer surface of the lamellar membrane, while the protein sites are in the interior of the membrane. Chabre assumes (his investigations are still in progress) that the rhodopsin “bathes” in the lipids and emerges into the interlamellar space; the results of Fourier syntheses could suggest that the distribution of the three major components of the rod (water, protein, lipid) is as shown in Fig. 44a and b. Figure 44a represents a possible distribution of the water, rhodopsin, and lipid in the membrane; this suggestion is based on volume estimations reported in Section I11 and electron microscope observations of a triple-layered membrane (Section IV,D,l,a,iii). This distribution of water, lipids, and proteins would become more realistic if the schematic scattering length density distributions in Fig. 44b (right) could be confirmed; they were calculated for three D20-HzO mixtures on the basis of slides shown b y Chabre. The schematic aspect assumes that the cell water is completely exchanged by the Dz0-H20mixture, and that only cell water and no protein is found in the interlamellar space. a This work has since appeared as H. Saibil, M. Chabre, and D.Worcester (1976).NUture (London)262,266-270.

92

J f h G E N ROSENKRANZ

FIG.44a. Relative volume proportions oflipids, rhodopsin (protein), and water in the lamellar membrane (thickness Im). The volumes are from Section 111, and the arrange ments partly from Section IV,D,l and partly from a synoptical interpretation of other experimental results. The arrangement indicates that three different layers (u,/3,y)have to b e considered in the calculations (one, however, is very small). FIG.44b. On the left is a schematic sectional view through a lamellar membrane (lm) along the v axis described in Fig. 29b. hx, Hydrated hexagonal particle or rhodopsin aggregate assumed for the calculation of p&) and pn(x). Between the rhodopsin aggregates lie the lipids (li). The interlayer (csDJ is assumed to consist only of cell water; a further approximation should also consider the 12% nonrhodopsin proteins. The lower part of the figure represents the calculated electron density profile p.(x) of a whole lamella in the direction of the rod axis x. The left half of this profile has been drawn to represent exactly the p d r ) of the corresponding part of the membrane illustrated above it. The volumes were taken from Fig. 44a, and the electron densities from Table XV. The right side of the figure shows calculated scattering length densitiesp, of a lamellar membrane for thermal neutrons; they correspond qualitatively to the densities measured experimentally by Chabre (1975a). The arrangement of rhodopsin and lipid was taken to be the same as that for the pJx) calculation; the numerical data are from Table XV. 0% D20: pn profile when the rods are kept in Ringer’s solution. 11% D20: pnprofile when 11% of the Ringer’s solution is replaced by D20; this means that the neutrons are only aware of the proteins. 50% 40:p . profile when 50% ofthe Ringer’s solution is replaced by D,O; the neutrons are only aware of the lipids.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

93

It should be noted that Fig. 44b (left) can be regarded as a possible interpretation of the x-ray diffraction results in the rod, supporting the suggestion of a distribution of lipids, proteins, and water according to Fig. 44a. If these neutron diffraction results are confirmed, they could help to decide which of several possible models is the most feasible in explaining the ambiguous results of x-ray diffraction experiments. 3. X-Ray Diffraction Effected by the Lamellar Body

This section is Concerned with the interference phenomenon of x-rays and its usefulness in the analysis of rod ultrastructure. The x-ray diffraction involved is always small-angle x-ray diffraction, as the interesting structures in the rod have dimensions in the range of several 10 to several 100 A. The section is divided into two parts. The first deals with the information obtained from experiments on a rod oriented with its longitudinal axis vertical to the beam; in the second part results obtained when the longitudinal axis of the rod was parallel to the beam are described. a. The Rod in Longitudinal Section. Electron microscope investigations have shown that a rod consists of lamellae stacked in parallel; therefore x-rays striking these lamellae at a very small angle of inclination are diffracted by them. The diffracted x-rays interfere to form a “primary picture” (using the terminology of Abbe) of the structure traversed by the waves, that is, the diffraction pattern. This is recorded either photographically (see Fig. 42) or by means of a positionsensitive linear detector (see Fig. 45). As in the case of light diffraction at an optical lattice, the x-ray diffraction pattern reveals two pieces of information about the structure. First, it shows whether or not the structure is periodically ordered, and, if so, how great the periodicity or lattice constant is; additionally, it yields indirect information about the volume within the period. This information can be decoded by means of Fourier synthesis and represented as an electron-density distribution of the structure concerned. From electron microscope investigations it can be expected that x-ray diffraction should reveal a periodicity in the longitudinal direction of the rod, with a lattice constant of = 300 A; this is the case, as shown in Table X and Figure 42. It is, however, uncertain whether or not there is a periodic order of the structure with a respective lattice constant b when diffraction is carried out at right angles (see Table X). This second periodicity, if it exists, should be detectable when viewed with the longitudinal rod axis perpendicular to the beam, as well as when viewed as described in Section IV,D,3,b. Table X shows that the lattice constant for the longitudinal direction of the rod is 300

94

1

JURGEN ROSENKRANZ

x-raZeqcu0nta X-RAY

DIFFRACTION

RETINA IN RINGER -da& adoped 3 min after bleaching

_-

L

100 min nfter bleaching

3

2 1

0

1

2

3

~

5

6

7

a

g

i

o

H

FIG.45. Intensity distribution ofthe first 10 orders of a rod (rod axis oriented at right angles to the beam). The x-ray intensities deflected by the lamellae are shown on the ordinate. The abscissa represents the distance in the reciprocal or Fourier space in multiples of H = h = a-' = (300 A)-'. Reproduction, with minor modifications, from Chabre (1975b).

A; this also applies for the in vivo state and is independent of species and state of illumination. It follows from Table X that there must also be a certain order in the lamellar membrane itself, otherwise the established reflection b-l, corresponding to a distance b = 55 A, would not exist. This reflection is further discussed in Section IV,D,3,b. The data given in Table XI are very important when analyzing results of x-ray diffraction experiments and should therefore be carefully considered. It is regrettable that only one investigator has explicitly published experimental findings on integrated reflection intensities. The Patterson functions and Fourier syntheses are, however, dealt with in detail. If the measured reflection intensities vary as much as indicated in Table XI, the validity of exact computer calculations is affected. This observation implies that the calculations of Patterson functions and Fourier syntheses cannot be reproduced by others; and it is therefore difficult to judge the validity of the statements made. The next step in evaluation of the distribution of masses in the cross section of the lamellar membrane is determination of the Patterson function. As the structure parallel to the longitudinal axis x of the rod has been roughly determined by the lattice constant a, it is sufficient to concentrate on a description of the mass distribution within a period of length a , that is, - %a Ix I+ %a. The Patterson function

PERIODICITIESIN RODS Lattice constant

(1)

No. of orders

b

(A)

Reflection

State of adaptation Bleached

Unbleached

--50

295-300

10

295 320 300 f 30

8-11 2 1

295 & 5 308 296 296

7 8 19 19

- 55

Irradiation time (min)

IN

RINGER’SSOLUTION

Species

180- 1320 R. temporaria

Diffused 55 Diffused

290-300 310 299

TABLE X RETINAA N D

IN THE

-

+

Broad

-

+

+

+

+ +

0.17 780 80

105 120 300 300

Special technique

Reference

Blaurock and Wilkins (1969) Blaurock and Wilkins (1972) Orientation by mag- Chabre (1975b) R . esculenta netic field R. pipiens Corless (1972) R . pipiens Robertson (1966) R. esculenta Synchrotron radiaJ. Rosenkranz (untion; fluctuation published results, 1975) given by AA In oioo Webb (1972) R. catesbeiana Worthington (1973) R. pipiens R . pipiens

-

MAGNITUDE OF

THE

TABLE XI INTEGRATED REFLECTION INTENSITIES Z(H)" Order

Area -I(H)*

Z(H) 'I

1

2

53" 1840

4" 55

3 6" 168

4

5

6

7

8

10" 460

2" 76

44' 470

45" 858

128

Values are given in relative units. The magnitude of area proportional to the intensity. Area measured planimetrically from Fig. 4b of Chabre (1975b).

0"

9

10

11

Reference

6" 61

11' 39

-

Chabre (197513)' Corless (1972)

17

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

97

Pattr(x)is generally related to the electron-density distribution p,(x) of an infinite periodic structure: pm(xr)pm(xr + x) dx’ = p,(x)

* pm(-x)

(8)

where x = a vector between every two points within the period a , * p,(-x) = convolution square (see also Appendix 2,B,2) of p,(x). As we are interested only in the structure along the rod axis x, we can restrict our calculation to the case of one dimension. The Patterson function is a measure of the degree of overlap of two identical electron-density or charge distributions p,(x), with a periodicity a at a distance x. Apparently Patt’(x) has maxima at points where x = +ka (k = 0, 1,2, . . . ) and auxiliary maxima at x = ka k (x, - x,,); xm is the locus in the interval x I14/?al, where p,(x,) takes the mth maximum; x, is defined analogously. The usehlness of the Patterson function lies in the fact that (1)it leads to a value for charge distribution by way of trial and error, (2) all charge distributions obtained by other methods must agree with the experimentally determined Patt’(x), and (3)it can be determined without knowledge of the reflection phases. For convenience the Fourier series representation of the Patterson function is used:

x = Ixl/a, and p,(x)

Here F(h)is the structure amplitude of the mass included in the elementary cell. In our special case the Patterson function can only be approximated, because only 19 orders have been measured; furthermore, the form of the Lorentz factor L is not clear; L, however, is important for the transinto the formation of the measured, dimensionless intensities ZexP(h) structure amplitude F(h):

where IGI2 = lattice factor which describes the extent and shape of the total sample investigated (this is constant and is equal for all reflections as long as the assumption of a practically infinite crystal is regarded as valid), and L describes the finite divergence of the incident x-ray and its finite spectral width. For the case of plain, parallel

98

P R G E N ROSENKRANZ

lamellae (as in the rod) with a diameter d and an average distance a 4 d from each other, Hosemann and Bagchi (1962) determine

it = a h h

=

const. x

h-I

-L

(10)

where h = coordinate of the reciprocal lattice vector. With Eq. (10) it follows from Eq. (9) that

This Lorentz factor is also used by Corless (1972), while Blaurock and Wilkins (1969) use L' = hw2 without justification. Chabre does not state the Lorentz factor, and Worthington (1973) applies, perhaps without adequate justification, a Lorentz factor 1IL" = C (h) = exp (yh2)= 1. The application of these formulas leads to the following values for the structure factor IF(h = 7)p:

IF(h = 7)(2 = IF(h = 7)p = (F(h = 7)$ =

lexp(h= 7)

7Zexp(h= 7) 49Zexp(h= 7)

because (L")-'= 1 because L-' h =7 because &')-I h2 = 49

-

-

When h or h2 is used (Fig. 46a), the broken-off Patterson series

2 Patt(x) = 2

2 L1 Zexp cos ( 2 ~ h x )

h=l

where L = L(h) = Lorentz factor, appears surprisingly similar, although two of the three best known reflections differ considerably. Common to all three published Patterson functions Patt(x) (Fig. 46a)is the main maximum value at x = 0 (assumed to lie, for example, in the intralamellar space of the lamellae), two further marked maxima, and a third indicated auxiliary maximum at xl, x2, x3, as shown in Fig. 46a. Table XI1 shows the positions of these maxima. Chabre additionally reports another auxiliary maximum at xq = 50 A (personal communication). The interpretation of the Patterson function by Chabre and Cavaggioni (1975) is essentially similar to that of Blaurock and Wilkins (1969). According to this interpretation, x1 (Fig. 46a) is assigned to a triple-layered lamellar membrane in such a way that two opposite hydrophilic groups of the lipid layers are at a distance of x1 = 40 A. This would be possible assuming the existence of short, 16-carbon hydrocarbon chains which are bent or linked to each other. At the ends of

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

+

0.2 -0.0

--

-02-

- 0.L --

\

- 1.0

c o

99

15

5

20.10-6

10

(a) Reproductions of Patterson functions, Patt(x), from three different references: 1, Blaurock and Wilkins (1969);2, Chabre and Cavaggioni (1975); 3, Worthington (1973). The three distributions were calculated from x-ray diffraction experiments on the same type of specimen, that is, the frog rod; they are redrawn here with the same abscissa x which lies parallel to the rod long axis; the profiles have been shifted arbitrarily on the ordinate. x i (i = 1,2,3) indicates the distance mentioned in the text and listed in Table XII. (b) Fourier syntheses of the electron density distributions p,(x) partly derived from the Patterson functioris to the left. The syntheses reproduced here have been calculated by Blaurock and Wilkins (1969) (. . . .), Chabre (197%) (---), Corless (1972) (--), Kreutz (1972) (- - -), and Worthington (1973) (-). Only Worthington (1973) provided his distribution with absolute electron density values lying between 0.295. and 0.438. electrons As. x = 0 is assumed to be the middle of the intralamellar space. The half-widths taken from these distributions are listed in Table XIV. (c) Representation of Napier's logarithm of the relative intensity Z diffracted by the rods as a function of h' = (29A-1)gA-', where 29 = scattering angle and A = 1.5 A = wavelength of the synchrotron radiation used. The short, vertical bar indicates the locus and extension of the only discontinuity. A light-adapted retina was placed in the beam as described in Fig. 40. FIG. 46.

100

P R G E N ROSENKRANZ

TABLE XI1 LOCIOF THE AUXILIARY MAXIMAX i OF THE BROKEN-OFF PATTERSON FUNCTION

-

40 42 40 40'

85 90'

123 132'

83'

- 130

-

7 10 8-11 19

-

Blaurock and Wilkins (1969) Chabre and Cavaggioni (1975) Corless (1972) Worthington (1973)

Number of orders used for the calculations.

the 40-Avector the protein would be situated with the polar part of the lipids, thereby making the membrane symmetric. According to Blaurock, Wilkins, and Chabre, x 2 is the distance between neighboring membranes of a lamella; x3 is mostly regarded as being caused artificially by the break-off of the Patterson series; Worthington, however, includes x3 in the calculation of the charge distribution. A refined view of the structure of the membrane cross section is obtained from a knowledge of the charge distribution described by the Fourier synthesis: 1

h=+m

For a centrosymmetric structure and a finite number of h values Eq. (12) changes to the broken-off Fourier series

where "sign" = + 1 or - 1, depending on the phase belonging to the structure amplitude F ( h ) , which can be either exp (i+) = exp (i0)= + 1 or exp (i+) = exp (ir)= -1, and pz = mean electron density of the unit cell. Because of Eq. (9) experimentally measured intensities do not yield information about the phase belonging to the reflection amplitude The phase therefore must be determined from additional experiments, such as those on shrinkage and swelling of the rods in hyper- or hypotonic solutions; one can assume with reasonable certainty that the dimensions of the lamellar membrane itself are not altered by these procedures. Contradictory statements, however, have been published concerning the constancy of the

c.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

101

intralamellar space and the lattice constants obtained from such experiments. Korenbrot et al. (1973) clearly showed that the lattice constant is reduced to 230 A after hyperosmotic shocks, but that the thickness of the lamellae remains constant at -150 A, which consequently implies a constant intralamellar space. Chabre and Cavaggioni (1975) observed a comparable reduction in the lattice constant only for damaged rods; for intact rods, the lattice constant remained almost unchanged, even under high hyperosmotic pressure. Under hypoosmotic pressure, however, the lattice constant increased for all rods. In contrast, Blaurock and Wilkins (1972) resolved the phase combination from previously calculated charge density profiles since, after performing shrinkage and swelling experiments, they found that the interlamellar distance changed approximately proportionally to the lattice constant. We, however, prefer the results of the first investigators, because they described their findings in more detail. Worthington (1973) and Worthington et al. (1973) avoid the phase problem in the following way. They assume, especially in the ,case of the rod that the total charge distribution p,(x), apart from an additional fraction p,, is concentrated in a zone Y of the unit cell, the size of which is Y 5 [%a);fi, is always constant. The thickness of the rod lamella is exactly = %a. In this special case the Patterson function for infinite crystals can also be used to describe a sample of finite size; in our case p,(x) # 0 holds for only half the period length, thus

-

As the convolution square p,(x) follows that

* p,(-x)

had a period 2v = a, it

describes the charge distribution p,(x) of a rod elementary cell with a periodicity a if o e extracts the convolution root (see also Appendix 2,B,2) of Qo(x): Qox ) = p,(x). The term Qo(x) is called an “autocorrelation function” by Worthington. For the evaluation of this deconvolution operation Worthington et al. (1973) describe two methods which require only the four fundamental operations of arithmetic but which avoid the phase problem due to the special charge distribution in the rod. Worthington (1973) also determined the phases shown with those of other workers in Table XIII. Figure 46b shows five electron density distributions derived from Fourier syntheses. The similarity of most distributions is not sur-

&

102

flRGEN ROSENKRANZ

PHASES exp icp

OF THE

TABLE XI11 REFLECTION AMPLITUDES USED

FOR

FOURIER SYNTHESES

Order 1

2

3

4

5

6

7

8

9

10

11

Reference

+I

-1 -1 -1 +1

+1 +1 -1 +1

+1

+1 +1 -1 +1

-1 -1 -1 -1

-1 -1 -1 -1

-1 -1

+1 +1

+1 +1

+1

-

-1

+1

+1

Chabre (1975b) Corless(1972) Kreutz (1972) Worthington(1973)

+l +1 +1

+l -1 +l

-

-

-

-

-1

prising in view of the basic assumption of a lipid bilayer membrane with implanted or attached protein molecules. Information from Fig. 46b hits been compiled in Table XIV which shows half-widths corresponding to the electron microscope magnitudes in Table IX (see Fig. 28a). As these tables show, the patterns as well as the widths of the layers agree wel€. Additionally, the asymmetric electron density distribution calculated by Kreutz (1972) agrees with the asymmetric distribution of the platinum chloride in the membrane after glutaraldehyde fixation; the Fourier synthesis carried out by Worthington has no direct counterpart with regard to the heavy-metal staining pattern. The basic assumption of a lipid bilayer membrane does not come from x-ray diffraction experiments but, for example, from electron microscope observations. The x-ray diffraction experiments therefore do not independently represent evidence for a lipid bilayer membrane, but confirm an interpretation of light and electron microscope results. The main problem remaining unsolved is that of location of the protein, that is, essentially where the rhodopsin is located. As the two main maxima of the charge distribution of a lamellar membrane are almost equal in size, and since many investigators still favor the idea of a rhoHALF-WIDTHSOF Intralamellar space (A)

(A)

20 14' 4* 13' 10

23' 14' 28' 23' 23

1,

A

TABLE XIV LAMELLATAKEN FROM FOURIERSYNTHESES P&)

(A)

1,

(A)

of membrane cross section

Reference

20' 13' 20' 18' 19

21' 14' 30' 22' 28

Symmetric Symmetric Almost symmetric Asymmetric Asymmetric

Blaurock and Wilkins (1969) Chabre and Cavaggioni (1975) Corless (1972) Kreutz (1972) Worthington (1974)

1,

103

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

TABLE XV CALCULATED ELECTRON DENSITIES p,(x) DENSITIES p,(X) FOR

AND SCATTERINGLENGTH A ROD"

Molecule

P

z

M

PAX)

Phospholipid Water Rhodopsin Heavy water

0.5 1.0 1.47 1.11

429 10 2.86 x 104 10

770 18 4 x 104 20

0.17 0.33 0.63 0.33

P,,(X)

0.20 x -0.57 X 2.80 x 6.24 X

10'0 10" 10'" 10'"

" p , Mass density in gm/cm3;Z, number of' electrons in the molecule; M , molar weight of the compound; values of p&) given in electrons/As; values of p,(x) given in cm-*.

dopsin sphere, difficulties arise. Recently another idea has been suggested, namely, that the rhodopsin molecule is distributed (equally) on both sides of the lamellar membrane. If one accepts a charge distribution such as that in Fig. 46b, which is almost symmetric, the assumption seems to be reasonable. Blaurock and Wilkins (1969) tend to support this conclusion with a slight hesitation because the rhodopsin concentration is uncertain. Corless (1972) gives the existence of equal distribution of the rhodopsin on both sides as one of three possibilities but does not give it preference. Worthington (1974) clearly shows the beginning of a trend away from traditional ideas by two graphical representations: rhodopsin as a sphere on one membrane side, and spherical rhodopsin with a thin stem reaching to the opposite membrane side (like a toy balloon). Chabre (197513) interprets the charge distribution assuming that rhodopsin extends through the whole membrane thickness as a lengthy protein, similar to the model described by Po0 and Cone (1973). We suggest a distribution of lipids, protein, and water as shown in Fig. 44a. This suggestion results from an exact as possible determination of the charge densities of all the substances concerned (see Table XV) and a determination of the volumes occupied by these substances (see Section 111 and Fig. 44b). By considering the observation of hexagonal, truncated pyramids (particles) described in Section IV,D,l,a,ii and D,l,b,i, for these calculations a dumbbell-shaped rhodopsin model was assumed which together with five other rhodopsin molecules also forms a dumbbell-shaped rhodopsin aggregate (Fig. 44b, left). The calculated electron density distribution of the substance distribution (shown in Fig. 44b, left) in one lamella (two membranes) is renresented in the lower part of Fig. 44b. For this determination only data from Fig. 44a (Table XV) and from the Patterson functions (Fig. 46a) were used. As

104

flRGEN ROSENKRANZ

the calculated charge distribution shown in the lower part of Fig. 44b is generally consistent with the Patterson function, it can be regarded as a simplified model of the rod lamellar membrane and can also be considered for further Fourier syntheses. I n this connection it is of interest that Downer and Englander (1975) demonstrated a coupling not only between rhodopsin and lipids but also between rhodopsin and water; at least 60% of rhodopsin hydrogen is exchanged with all of that of water. The results of x-ray diffraction effected by vertically arranged rods, and the interpretation of such experiments, can be summarized as follows. Fourier syntheses of the electron density distribution agree surprisingly well with the descriptive results from electron microscope investigations of rod longitudinal sections. This is surprising because (1)the only, and insufficient, data published on reflection intensities indicate considerable fluctuation, and (2) the Lorentz factor in this case cannot yet be satisfactorily theoretically determined. The rebuttal made by Worthington (1973) concerning the Lorentz factor, L = C(h)-’ = h-l, is not convincing because, among other factors, C ( h ) = 1 only leads to “reasonable” electron densities in the membrane with estimated electron densities of 0.45 and 0.35 electron/& for protein and lipid, respectively; these values do not exactly agree with those we have calculated (Table XIV). Furthermore, Worthington’s argument is contradicted by the fact that the Fourier synthesis carried out by Corless (1972) does not reach the maximum pz = 0.560 electrodk, as determined by Worthington for C ( h ) = h; from Fig. Id in Corless (1972) ps = 0.465 e l e c t r o d k can be estimated. Finally, the experimental basis for determination of the phases of the reflection amplitudes is not fully reliable; an exception in this respect is the elegant solution found by Worthington, which is, however, valid only as long as the interlamellar space is assumed not to contain measurable quantities of protein. Regarding the position and the shape of the rhodopsin, apparently no one doubts the existence of spherical rhodopsin attached to, or implanted in, only one side of the membrane which is regarded as a more-or-less continuous double layer. We suggest an interpretation for the only slightly asymmetric charge distribution by assuming dumbbell-shaped rhodopsin aggregates (Fig. 44b, left) surrounded by a lipid bilayer. Both parts of the dumbbell are built similarly, apart from the retinylidene which is attached to the interlamellar part (see Fig. 28c). Evidence for this latter assumption is given in Section V,B,2,a. Indications of this rod lamellar membrane model are found, in our opinion, in many reports described in the preceding sections, as well as in the following description of the lamellar membrane.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

105

b. The Lamellar Membrane Viewed Directly. As shown in Table X, Blaurock and Wilkins (1969), as well as Chabre (1975b), found a diffuse reflection in completely intact rods at (55 A)-', corresponding to h = 0.0182 A-l. Blasie et al. (1969) recorded this, and a further reflection at (83 A)-', at different temperatures in centrifuged, moisturized lamellae and have discussed these reflections mathematically. These reflections, which yield idormation concerning the order of material in the lamellar membrane, should be carefully considered taking the following information into account. Chabre (1975b) again investigated rods in a much improved experimental situation, namely, intact rods in Ringer's solution, orientatian in a magnetic field, and only 5-10 minutes' irradiation time. Under these optimal conditions he could neither confirm that the (83 A)-l reflection exists nor that the protein distribution in the membrane changes significantly with the temperature. A plausible explanation for the occurrence of a second reflection in the small-angle range (83 A)-1 in the case of more-or-less compressed membranes is found in the hypothesis suggested by Guinier and Fournet (1955), that is, that the more compact the scattering centers of a particle are, the more pronounced a secondary maximum can arise due to a discontinuous course of the scattering amplitudes F(h).On the basis of their experimental conditions, at the time considered optimal, Blasie and Worthington (1969) put fonvard the hypothesis that rhodopsin molecules are arranged like a twodimensional liquid. In view of the experimental complexity of the problem many working hypotheses cannot be verified. This case also applies when investigating the arrangement of rhodopsin molecules. Unfortunately the important term liquidlike is not defined by the whrkers who use it. The formulas, and especially the use of the radial density function, however, imply that fluid in this case means an aggregate of material (rhodopsin molecules) which scatters x-rays according to the conventional Zernicke-Prins intensity function I (h). This intensity function, however, describes only a primitive fluid outside the range of smallangle diffraction (Hosemann and Bagchi, 1962). A primitive fluid consists either of only one kind of molecule (like mercury) or of molecules with an equal probability of orientation in all directions, that is, molecules that are at least rotationally symmetric. The latter is certainly not the case for rhodopsin, but exactly how the order of the rhodopsin molecules can be described remains a problem. Chabre (197513) claims that the proteins are randomly distributed in the membrane. If this were the case, the (55A)-l reflection could not have been found. The fact that only one reflection exists indicates a certain degree of order. According to Hosemann and Bagchi, this can be stated more exactly: Let the distance between every two

106

P R G E N ROSENKRANZ

particles in the membrane be ai,the average be i,and the fluctuation in at be Aa; if then 0.18 < Ad6 I 0.35, the order of the particles will be such that they produce just one interference; see also Section IV,D,l,a,i and D,l,b,i. Further conclusions, however, cannot be drawn from these facts alone. Whether the order of the particles in the membrane is amorphous or paracrystalline remains uncertain. If the order were paracrystalline, the vectors af corresponding to the absolute values af would form quadrilateral lattices or elementary cells which would be enumerable. If the structure were amorphous, this would not be the case. In our opinion, the material content of the (55A)-l reflection can only be the rhodopsin molecules of each hexagonal particle (rhodopsin aggregate), the centers of gravity of which are a distance of about 55 A from each other (Rosenkranz, 1976b). This assumption is also supported by experimental results reported in the following section in which the small-angle diffraction range is enlarged by a factor of 10 in the direction toward the primary beam. We have worked with synchrotron radiation, available at the Deutsches Elektronensynchrotron in Hamburg, to observe x-ray small-angle diffraction effected by the lamellar membrane. Three pieces of a retina were oriented one behind the other with their planes perpendicular to the beam and consequently the rod axes parallel to it; the test chamber was filled with Ringer's solution cooled to +4"C and had a thickness of 2 mm; the retinas were irradiated for 2 hours, the distance between preparation and film being 80 cm. Evaluation of the small-angle diffraction (Fig. 40) yields a single marked reflection at

h'

=

2yA-I = (0.38 & 0.04) x

lop2A-'

(13)

where 2'y = scattering angle. A significant difference between lightand dark-adapted retinas was not detected. A radius of gyration R, was calculated from the continuous region near h' = 0.0025 A-1 from scattering curves like Fig. 46c (Rosenkranz, 1976a). If a square lattice of side length p, is assumed, it follows from Eq. (13)that 240 5 p, 5 280 A. In Section IV,D,l,a,i and D,l,b,i the distances of neighboring hexagonal particles were measured as mean distances puL= 236 72 A and p G F = 192 65 A of a paracrystalline lattice; these results were therefore confirmed by an x-ray diffraction experiment on unfixed retinas. In the membrane, centers of mass 200-250 A apart are arranged as in a paracrystal; the relative fluctuation of the distances is 0.18 < Aplp 5 0.35. At least in the fixed material a hexagonal short order of these centers of mass is indicated; the

*

-

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

107

centers of mass must essentially contain rhodopsin, as this substance is (see Table XV) the one with by far the highest electron density in the lamella. The radius of gyration R, determined by Guinier and Foumet (1955)is the average distance of the atoms from the center of gravity of the whole particle; in principle it is calculated like the statistical error; in addition, however, each distance is weighted according to the atomic number of the respective atom. The radius of gyration originally derived for globular particles is also valid for nonglobular particles (Hosemann and Bagchi, 1962) if it is calculated from the inat h’ + 0 (Z = intensity of the small-angle crease in the curve In Z(II’~) diffraction). This was carried out within the limits of experimental possibilities: h’ = 0.0025 A-l. As is known, the radius of gyration R , can be used to calculate the dimensions of certain geometric bodies such as spheres, ellipsoids, and prisms in order to confirm or exclude a body hypothetically assumed from other experiments. On the basis of the previous observations we are convinced that the particles shown by this small-angle diffraction are aggregates of six rhodopsin molecules each forming a dumbbell-shaped particle. The peptide chain of each rhodopsin molecule is coiled up at both ends, leaving a distance of about 50 A between the two tangles. Six such small, dumbbellshaped particles form a larger, dumbbell-shaped particle without being closely linked one to the other. In freeze-etched cross-fractures one part of this dumbbell appears as a distinct hexagonal, truncated pyramid (Fig. 32) and likewise as a marked hump in longitudinal fractures, while the other part is much smaller (Fig. 38); only longitudinal sections show an almost symmetric pattern (Fig. 24). If one imagines this aggregate to be approximated by two cylinders of corresponding size in such a way that they are coaxially arranged with their centers of mass 55 A apart from each other (dumbbell), this particle has the following main moment of inertia (mass = 1):

The radius of gyration of this particle, because of Eqs. (14)and (15),has the theoretical value

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N R G E N ROSENKRANZ

where Oi = the ith main moment of inertia of the dumbbell-shaped particle, r = 65 A = Y2juL = %j&, the radius of the cylinders, and h, = 21 A, the height of each cylinder. Experimentally one finds from the ascent of the scattering curve (Fig. 46c), after Guinier and Fournet (1955), to be

Both values agree fairly well. This means that a necessary condition for the assumption of dumbbell-shaped rhodopsin aggregates is fulfilled. The analysis has been carried out assuming that (1) the scattering curve is caused by the kind of particles that represent the largest-if not the only-group of monodisperse particles of this size in the retina, and (2) the increase in the curve In Z(hr2)is constant for h’ c 0.0025 A - I . The description of the lamellar membrane surface is still more incomplete than that of the cross-sectioned membrane. The number of experiments with intact rods has so far been small. This may be caused by the fact that the order is less marked as compared to that in sections parallel to the rod longitudinal axis, so that the experiments are perhaps less attractive. We suggest an interpretation of the experimental data after careful consideration and taking into account the ideas described in Section IV,D,l and D,2. A rhodopsin molecule peptide chain is coiled up into two tangles of not quite equal size; these tangles are located on top of each other in the lamellar membrane which is totally penetrated by them. The two ends of the peptide chain emerge either freely into the interlamellar space or are directed toward the respective ends of neighboring rhodopsin molecules. The rhodopsin molecule may also be considered a small dumbbell. Six such small dumbbells each form a dumbbell-shaped rhodopsin aggregate of 75 A height and 140 diameter. They in turn form a hexagonal short-range order which is sufficiently marked for light diffraction but is otherwise not easily detectable. Further confirmation of x-ray diffraction effected by the membrane surface is desirable (and, it is hoped will perhaps be stimulated by this article).

-

4. Light Optical Znvestigations in Rods

Information referring to the size and position of the chromophores in the rod, that is, the retinylidene groups, is obtained from measure-

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ments of the absorption spectra by means of linearly polarized light; since decisive information appears only for the dark-adapted rod, this is dealt with in Section V. It remains only to be said that the interpretation of the absorption spectrum of unpolarized light is not yet complete. As is known, illuminated rhodopsin shows three absorption maxima at 380, 275,* and 226* nm (Liebman, 1962, 1972). Retinal is indicated by 380 nm, and the protein band at 280 nm points to tyrosine and tryptophan, but there is still no conclusive explanation for the 226-nm maximum, although it is suggested that because it lies near 233 nm it may be caused by the conjugated dienes of the unsaturated phospholipids (Klein, 1970). Measurements of the absorption spectrum by means of circularly polarized light are interesting only when they are performed on dark-adapted material. Circular dichroism AE; = EL - ER Z 0 (EL = absorption of left circularly polarized, ER = absorption of right circularly polarized light) is known to occur when molecules are asymmetric such that the mirror image cannot be brought into register with its original. Digitonin extracts of light-adapted rods do not show circular dichroism in the visible range. In the range near 220 nm, Crescitelliet al. (1966), however, found circular dichroism; the relative circular dichroism (for R . pipiens and R. catesbeiana) was determined to be

-

where Eunp(A,,,) = absorption of unpolarized light at A, = 500 nm; this value was taken to be equal to unity. Crescitelli et al. (1969) repeated the measurements of the circular dichroism with a rod suspension from R. temporaria. The wavelength range investigated again lay between -200 and .250 nm and 340 and 600 nm. The difference spectra of the light- and dark-adapted states were equal to those of the digitonin extractions, except for the surroundings of 340 nm. The absolute values of the circular dichroism were greater than the corresponding data for the digitonin extracts by more than an order of magnitude in the case of AE, > 0; in the case of AeZ < 0 they were lower by a factor of 1.5. Unlike the digitonin extracts at 5 400 nm the suspended rods did not show decrease in AeZ when bleached. According to Mommaerts (1969) AeZ (A = 220 nm) = -24 X lC3, which means that -65% of the opsin is in the form of a right-handed a helix. This conclusion must be made with caution, since the helix interpretation is based on measurements made in 1966; these measurements do not provide all the necessary information one needs to

110

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ROSENKRANZ

support the above statement, because the range of measurement did not include the ultraviolet value down to 190 nm.

-

5. lmrnunological Experiments An obvious approach to the problem of rhodopsin localization in the lamellar membrane is use of the antigen-antibody method, for example, a fluorescent antirhodopsin could attach to the rhodopsin and reveal its position by fluorescence. If one assumes only the three simplest possibilities in the attachment of rhodopsin-to the cell membrane, to the interlamellar side of the lamellar membrane, or to the intralamellar side-one may expect the following. If the rhodopsin is marked only in the cell membrane, the fluorescence should be almost equal along the whole rod surface no matter whether the rod is observed sideways or parallel to its longitudinal axis (from above). Marking of the interlamellar membrane side would imply that the lamellae are isolated and that the intact rod has pores in its cell membrane and interlayer, which are large enough to let a cylinder at least 32 k 2 A in diameter and 240 k 10 A in length pass through; this description is assumed by Blasie and Worthington (1969) for the antirhodopsin molecule on the basis of x-ray small-angle diffraction experiments. In this case the fluorescence should be much more intense when the rods are observed from above. The same would apply if the antirhodopsin were coupled to the intralamellar side; in this case one would also have to assume that the intralamellar space is accessible to the large antirhodopsin molecules. The latter is not expected (see Section IV,D,l,a). One cannot therefore be sure that the antirhodopsin molecules have free access to all the rhodopsin molecules in the membrane. Dewey et al. (1969) performed experiments with Formalinfixed retinas of R. pipiens; they produced antirhodopsin serum in the rabbit and treated retina sections first with the antiserum and then with fluorescein-labeled sheep antirabbit y-globulin. Among other structures, all receptors of the retina were labeled by the fluorescein in such a way that they were much more fluorescent when seen from above. Dewey et al. suspected that intact rods could be penetrated by such large molecules as antirhodopsin, an assumption they wished to confirm by analogous experiments using ferritin or peroxidase as a marker substance. We believe that these rods were no longer intact; this opinion is based on the results of experiments by Yoshikami et al. (1974) with fluorescent N,N '-didansylcystine which cannot penetrate intact cell membranes, and on the attempts of Jones (1974) to fix rods with formaldehyde; these rods showed atypical swelling and damaged lamellar rims. The experiments of Dewey et al. (1969) indicate

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only that the cell membrane itself contains visual pigment but do not convincingly demonstrate that rhodopsin is situated only or generally on the interlamellar membrane side. 6. Hypotheses on the Structure of the Lamellar Membrane This section refers to light- and dark-adapted rods. During the 1970s six suggestions have been made concerning the structure of the lamellar membrane of the rod. The essential differences lie in the shape of the rhodopsin molecule and its location in the membrane. Three models have been ublished in which rhodopsin was assumed to be a sphere about 40 in diameter. If the sphere is assumed to lie on the intralamellar side of the lamellar membrane, the term intrasphere model is used; analogously, intersphere model or amphosphere model is used if the rhodopsin is assumed to lie on the interlamellar side or on both sides of the membrane. In three further models the rhodopsin molecule spans the whole lamellar membrane but has a different shape, described as a toy balloon, a dumbbell, or a dumbbell aggregate (to a first approximation). a. The Zntrasphere Model. This model assumes the rhodopsin molecules to be spheres, in the dark-adapted state on the intralamellar side of the lamellar membrane, and embedded in a phospholipid layer. After illumination the rhodopsin sinks either 7 A, according to Worthington (1974, based on Blasie’s work) or totally into the membrane, according to Abrahamson and Fager (1973).Both groups regard this model as only one of at least two possibilities, both of which are rather probable. This is implied in Worthington’s article (1974) by phrases such as “. . . the possible location of rhodopsin, if spherical, in the disc membranes of frog retina is shown . . .”. Abrahamson and Fager do not exclude uncertainties in the identification of the freeze-fractured surfaces on which, however, their interpretation decisively depends. In our opinion the rhodopsin cannot be located on only one side of the lamellar membrane, as it represents at least 80% of the electrondense material in the rod; the electron density distribution is to a first approximation symmetric around the center of the membrane but may also be slightly asymmetric with its center a little to the interlamellar side. The intrasphere model is further contradicted by a comparison of the charge density distributions before and after illumination of the rod; only on the interlamellar side were minor charges observed after illumination. Shifting of the highly electron-dense rhodopsin toward the center of the membrane should have affected the charge density distribution in its central region.

w

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flRGEN ROSENKRANZ

b. The Zntersphere Model. This model, generally assumed for vertebrates, is described by Daemen (1973),who suggests that “a continuous phospholipid bilayer most likely forms the backbone of the disk membrane” into which from the interlamellar side, the spherical rhodopsin molecules are embedded as reported by Blasie (1972); this means that in the dark-adapted state three-quarters of the rhodopsin molecules emerge into the interlamellar space, while in*the lightadapted state the rhodopsin sphere sinks into the lipid layer to a depth of half its diameter. Daemen himself states that these details concerning the position of the rhodopsin molecules cannot be observed in experiments with rods of intact retinas. Furthermore, it should be noted that the almost symmetric electron density distribution is in sharp contrast to the rhodopsin distribution on one side. Finally, careful estimation of the lipid content of the rod shows that, especially in the frog, a continuous lipid bilayer cannot exist because of the lack of sufficient material (Section III,C,5). c. The Amphosphere Model. Vanderkooi and Sundaralingam (1970) were the first who tried to explain the almost symmetric electron density distribution in the lamellar membrane by assuming that the rhodopsin was equally distributed on both sides of the membrane and that the interspaces between the spherical rhodopsin molecules were filled with a lipid bilayer. An ordered structure of the globular proteins appearing, for example, in electron micrographs was interpreted by these workers as being artificial; they stated that these molecules could be assumed to be ordered like a plane liquid. This model would be acceptable except that, especially in the case of the frog, the arrangement would require a rhodopsin concentration that is too high by a factor of at least 2. Furthermore, x-ray diffraction indicates a weak mass shift after illumination toward the interlamellar side only, as a rhodopsin distribution acwell as a diffuse reflection at (55 cording to Vanderkooi and Sundaralingam would result in a reflection at about (85 A)-1 in uiuo. The same argument applies to the model introduced by Borovjagin et al. (1971),which is essentially similar to the one described above. d. The Toll Balloon Mode2. Besides the traditional rhodopsin sphere model, Worthington (1974) also considers the possibility of a extended rhodopsin shape. In this case, the marked asymmetric arrangement on the intralamellar side of the membrane remains as previously described, but the somewhat shrunken rhodopsin molecule has a thin extension directed toward the interlamellar side, resembling a toy balloon. Kreutz (1972) has introduced a similar although mirror-inverted model for the vertebrate rod. The base mem-

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brane in this model consists of two layers: lattice proteins on the intralamellar side of the membrane, and one lipid layer on the interlamellar side. The rhodopsin molecules are attached to the base membrane on the interlamellar side in such a way that the spheres partly emerge into the interlamellar space and project thin extensions toward the lattice protein. There is, however, one problem with this membrane model, especially when it is applied to the frog retina: The lattice protein, if it is considered equal to the nonrhodopsin protein, amounts to only onefifth of the total protein in the rod, and this distribution would, especially in the frog, again lead to a marked asymmetric electron density distribution. e. The Dumbbell Model. After careful consideration of all essential results concerning frog rod ultrastructure, we postulate the lamellar membrane model shown in Figs. 28 and 29 (Rosenkranz, 1976a). The single rhodopsin molecule, shaped like a small dumbbell, is distributed approximately evenly on the inter- and intdamellar sides of the membrane. The two halves (tangles) are connected to one another by a peptide chain. The two peptide chain ends emerging from the tangles can form either networks or lattices (thus partly taking on the function of the lattice proteins described by Kreutz). The lattice of rhodopsin molecule aggregates is b y no means ordered as exactly as shown in Fig. 29b over large membrane areas, but sometimes over smaller ones. The rhodopsin aggregate of six hexagonally arranged single molecules forms a pore (Fig. 28c) which, because of the isomerization capacity of retinylidene, can open and close the connection to the interlamellar space. The chromophore is assumed to be found in the interlamellar tangle of opsin solely on the basis of the electron density distribution observed in the light- and dark-adapted states; other experiments do not appear to be conclusive. Since, however, the opsin distribution is almost symmetric about the center of the membrane, this hypothetical dumbbell aggregate model could still be valid if the retinylidene group were located on the intralamellar side. As in the model of Vanderkooi and Sundaralingam, the lipids are considered to fill the parts of the membrane not occupied by the hydrated protein. The relatively small proportion of nonrhodopsin proteins could either be part of the interlayer or could act as lattice proteins, as described by Kreutz; in both cases they reinforce the skeleton of the lamellar body. The dumbbell model, which was discussed by several investigators by Po0 and Cone (1973) and which was described in Section 111,B74, must also be considered. I n summary, it appears that at the present

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time there is a trend toward the idea that rhodopsin exists not as a sphere but rather as a dumbbell spanning the whole lamellar membrane.

E. THE RIMS

OF THE

LAMELLAE

1. The Rims as Observed in an Ultrathin Section The rim surrounds (see Figs. 3 and 15) the whole margin of the lamella and accompanies all its incisures. It connects one lamellar membrane with the other and thus builds the lamella into a body with a closed surface. Owing to deep incisures the length of a rim is approximately five times as long (110 pm) as it would be without the incisures. I n cross section (Figs. 24 and 27) the shape of a rim resembles a circle lacking a third or fourth of its periphery; the resulting ends are fused with the membranes of a lamella. The diameter of the remaining circle (250-300 A) is similar to that of tubules occasionally found in the rod. Tubules and rims also seem to be structurally similar as far as can be determined in electron micrographs; Fig. 21a and d can be compared with Fig. 41. In both cases neighboring rings or pieces of rings constitute an essential part of the structure. The tubules are found either embedded parallel to the lamellae in the lamellar body without disturbing the regular order of the stacked lamellae or, less frequently, parallel to the rod longitudinal axis, sometimes at a distance of about 150 from the cell membrane (Fig. 21a), or in the lamellar body (Rosenkranz and Hauser, 1972). No success has been obtained in determining where the tubules end in the cytoplasm or on the cell membrane; it is, however, believed that the lamellae do not taper off as tubules in the ciliary matrix as is observed in the gecko. Figures 16 and 17 show conclusively that the rims of successive lamellae are connected to each other at the end of their incisures by anastomoses in the form of short tubules. If one assumed that the lamellae at the end of each common incisure are connected to one another, this system of rims and tubules, which can also be referred to as a quasi-tubule system, would have a volume of 6% of the total rod volume; it may be of importance when considering function to realize that the lamellar body would then be traversed by a maximum of 20 to 30 longitudinal channels. The tubules and their contents were found to be similar to the lamellar structure proper, according to an earlier fixation method employing oso4or KMn04. The tubule walls appeared to represent the curved part of the lamellar membrane. In these experiments the intratubular space behaved exactly like the intralamellar space. Falk and

a

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Fatt (1969) were the first to observe that the rim system was chemically totally different from the lamellae proper. Subsequently several other observations showed that the rims behaved differently from lamellae when many fixation and staining agents were used. Falk and Fatt found that only the rims remained intact (i.e., could be stained) when they treated rods first with a phosphate-buffered 40 mM OsOl solution, followed by 10 mM tris at pH 7.2-7.5. These investigators suggest that tris was bound to the osmium in the tissue, thus producing a water-soluble compound which dissolved the lamellar body and resulted in rod ghosts. We do not agree with this opinion, considering the extremely well-preserved location of the rim system after Os04-tris treatment; it seems more probable that only the part of the lamellar body responsible for staining was dissolved. Another essential part remained and kept the rims in their original positions. How the lamellar membrane differs chemically from the rims remains uncertain. These experiments demonstrate clearly, however, that there is a difference. This is supported also by observations on staining behavior. There was a marked difference (Fig. 27) in staining behavior when glutaraldehyde-fixed rods were postfixed with PtC1, and embedded in Epon. Nir and Pease (1973) demonstrated an increase in the contrast of the rim wall due to Os04 postfixation and staining with uranyl acetate and lead citrate (Fig. 25). When osmium postfixation is omitted, the contents of the rims are markedly stained (Jones, 1974; Fig. 26). The results are, however, not sufficient to obtain a reliable picture of the chemical composition of the rim system.

2. Freeze-Fractured Lamellar Rims; Rims Seen in the Scanning Electron Microscope The few reports on the structure of the quasi-tubule system based

on results obtained from freeze-etching and from the scanning electron microscope confirm the view outlined in the preceding section. Figure 47 shows the structure of a rim built of circular segments, and Fig. 49 (or, even more distinctly, Fig. 48) demonstrates the great mechanical strength of the rim system, which is known to be achieved in the simplest way-by a tubelike structure built of ring segments.

3. A Synoptical Znterpretation of the Results Concerning Structure and Arrangement of the Lamellar Rims The small volume and the practically linear arrangement of the quasi-tubule system of the rod, among other factors, make investigations technically very difficult Because of this there is still no clear description of this part of the rod. We cannot say with certainty that all

116

flRGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

117

lamellae are connected at the ends of the incisures by tubules, or that this system is normally connected to the cell membrane via a narrow cleft One can only state with some certainty that not the tubules but parts of some rims are connected to the ciliary matrix. Furthermore, the rims of neighboring lamellae approach each other up to a distance of 30-40 A. Thus far there is no evidence concerning the chemical composition of the wall and lumen of the rim system. It is certain, however, that it differs from the rim and its continuation in the lamella (i.e., in the lamellar body proper), and that an essential structural unit of the tubule and rim is a ring-shaped segment (shown by ultrathin sections and freeze-etching). These results, together with the apparent morphological similarity between tubules and rims, could lead to the supposition that one originates from the other.

F. ROD CYTOPLASM 1. The Rod Cytoplasm as Observed in Ultrathin Sections

The observations considered in Section IV,D,l,a suggest that the ground cytoplasm is located only in the space between the cell membrane and the quasi-tubule system, and that the surfaces of these two organelles almost exclusively limit the cytoplasm. Thus cytoplasm is found only on the intracellular side of the cell membrane and in the sometimes deep incisures of the lamellae. If the whole lamellar body and the quasi-tubule system were dissected from the rod, there would remain a long cylinder (wall thickness 100-150 A) and about 25 walls of the same length and a thickness of 60 30 8, pointing to the center of the cylinder; these walls would reach different distances into the cylinder and would not always be parallel to the outer cylinder wall because of slight undulations. Besides the cell membrane the cytoplasm can also be clearly demonstrated to run along the whole rod parallel to the longitudinal axis; in contrast to the cell membrane,

*

~~

FIG.47. Part of a cross-fractured,freeze-etchedrod. qv, Fibrils crossing an incisure (es) and linking adjacent lamellar rims. Pretreated with 10% glycerol-Ringer’s solution, platinum-carbon replica. FIG.48. Scanningeledron micrograph of extreme resolution of a rod surice. The fibrils (qv), crossing incisures (es), and the lamellar rims (rw) are distinctly seen. OsO, vapor fixation in a hanging droplet; distance between rod and fixing agent 7.5 cm, time of fixation 1.5 hours, shadowed with gold. Autoscan scanning electron microscope, beam voltage 20 kV. FIG.49. Rod with indentations (eb). Preparation as in Fig. 48. Stereoscan S4 scanning electron microscope; U = 20 kV. Bar, 1 pm.

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JURGEN ROSENKRANZ

however, the lamellar membrane is at no point separated from the cytoplasm by more than 0.5-0.8 pm. The volume of the cytoplasm is about 26 pm3, that is, 2% of the total rod volume assuming that the interlayers described in Section IV,D,l,a,iii are not part of the cytoplasm. This assumption has to be made, as previously mentioned, because of the morphological separation of both spaces by the quasitubule system, the results of the lanthanum nitrate experiment performed by Cohen (Section IV,D,l,a,iii), and the total destruction of the cytoplasmic contents after pronase or hyaluronidase treatment, which Borovjagin et al. (1973) supposed to be caused by the destruction of glycoproteins and/or mucopolysaccharides. Differential staining of only this cytoplasm region in the rod has not been reported, except for the staining observed after treatment with barium sulfate, which is assumed to occur when the cell membrane is damaged. It is difficult to explain why the rates of diffusion of the two atoms barium and lanthanum in the rod are different when the two chemicals react similarly in other biological experiments. 2. Freeze-Etching of the Rod Cytoplasm; The Cytoplasmic Space Observed in the Scanning Electron Microscope The description of the cytoplasmic space obtained from ultrathin sections is completed by experiments with freeze-etched rods, which indicate that the deep incisures contain not only cytoplasm but also fibrillar cross-connections (Fig. 47). These cross-connections presumably have diameters of 40-50 A and connect neighboring segments of one lamella over distances of 160-260 A. To be more exact, they connect adjacent rims (Fig. 47). They seem to originate from the ringshaped segment of one rim and to fuse with the opposite segment. By extrapolating from Fig. 47, it appears that every fourth ring-shaped segment of a rim possesses such a cross-connection. The cytoplasmic space in the rod seems to be enlarged after freeze-etching; the distance from the lamellae to the cell membrane, as well as the width of the incisures, is about 200 A. This suggests a cytoplasmic volume of about 60 pm3, corresponding to 4%of the total rod volume. If one looks at a rod in the scanning electron microscope (Fig. 48), the cell membrane appears to touch the core so closely that its contours are clearly visible. The rims of the stacked lamellae and the connections between them are shown. This observation adds to the evidence for the existence of such connections, independent of that obtained from freeze-etched preparations.

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3. A Synoptical Interpretation of the Structure and Distribution of the Cytoplasm It is possible to differentiate two compartments in the traditional cytoplasmic matrix in which the lamellae are assumed to “float,” namely, the interlayers limited by neighboring lamellae and parts of their rims, and the ground cytoplasm between rims and cell membrane, which probably amounts to 4% of the rod volume. Both regions are morphologically connected only by the 30- to 40-kwide clefts between neighboring rims. As a cleft is smaller than a membrane, the quasi-tubule system must appear as a coarsely porous membrane. The observation of a second cytoplasmic compartment (by Cohen, 1968) indicates that either a coarsely porous membrane exists, through which certain ions (e.g., La3+)are allowed to pass and others (e.g., Ba2+)are not, or that the interlayers have a composition different from, for example, the cytoplasm next to the cell membrane, or that both cases exist. It is, however, certain that a cytoplasmic sheet with a thickness of 200 A lines the inner surface of the cell membrane and protrudes radially into the lamellar body. In the incisures this sheet is penetrated by about 2000 fibrillar cross-connections per lamella. These cross-connections are not clearly visible in most ultrathin sections because of their small size and the summation effect of staining. The cross-connections lead to a mechanical stiffening of the lamellar segments; they also display a large area of contact with the cytoplasm. The short cross-bridges and the parts of the rim directed toward the cytoplasm together create a surface area more than eight times that of the intracellular membrane surface.

V. The Dark-Adapted Rod Since much of the information given in the preceding section is common to both light- and dark-adapted rods, only structural details characterizing the state of dark adaptation are dealt with here. They could help to identify the location of the chromophore in the rod.

DETAILSO F DARK-ADAPTED RODS AND LOCATIONOF FUSCIN Although the fuscin in the microvillous projections of the pigment epithelial cell does not actually belong to the rod, its obviously light-dependent position immediately adjacent to the rod should be considered; the melanin bodies containing the fuscin migrate vitread A.

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m R G E N ROSENKRANZ

along the rods within minutes after the beginning of illumination (Murray and Dubin, 1975). After the termination of illumination they return to the pigment epithelial cell body. P. Fatt (unpublished, cited after Falk and Fatt, 1972) did not find light-optical differences in length between light- and dark-adapted rods (species not mentioned) in a Ringer's solution of pH 7.0; his measurements were such that a 2 2 %change in length and a 25% change in diameter should have been noticeable. The wavelength of light used on the dark-adapted rods was between 725 and 765 nm, and the bleached rods were exposed to light for 1 second. In a refined experiment, Enoch et al. (1973)report a 2-4% increase in diameter after illumination of frog rods in aqueous humor from the eye of a goldfish; the reference value was obtained in infrared light (A = 826 nm) from five carefully selected intact rods. The value of 2% is in good agreement with the volume increase in rhodopsin after illumination, which Heller determined to be 36% (Section V,C) and which we determined to be 44%(Section V,B,l,c). In both cases, however, the rhodopsin was not embedded in the lamellar membrane. Enoch et al. (1973) report that the optical path length 6 (A = 826 nm) = 0.45* is increased by 0.9*%after illumination. According to Liebman and Entine (1968)6 (A = 502 nm) = 0.09.

B. ULTRASTRUCTURE

OF THE

DARK-ADAPTED ROD

1. Electron Microscope Observations a. Ultrathin Sections of the Lamellar Body. Electron microscopy of ultrathin sections of rods is not the best method to determine changes in rod ultrastructure after illumination. As the retinal in its all-trans form is about 17 8, long, the changes, if any, will be of the order of magnitude of 10 A. Although this does not represent the limit of the reciprocal resolving power of a modern electron microscope, it is the thickness of the section that introduces the limitations. It is therefore to be expected that Falk .and Fatt (1972), Nir and Pease (1973), and Rosenkranz (1976b) did not find significant differences between ultrathin sections of light- and dark-adapted rods. In contrast to these investigators, P. Rohlich (1967, cited after Rohlich, 1971) found a smaller periodicity or lattice constant in dark-adapted, embedded rods than in light-adapted ones. b. Freeze-Fractured Lamellar Body. In R . esculenta, Rohlich (1971)found increased periodicity in the axial direction after soaking the retina in 20% glycerol in 0.1 M cacodylate buffer; in the darkadapted rod he found a.periodicity of 290 8, (average of seven rods)

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and 360 A in the light-adapted rod; the latter value, however, has not yet been confirmed by other workers. Mason et al. (1974) claim to have found great differences between dark- and light-adapted lamellar membranes of R. catesbeiana after freeze-etching (using isolated rods and rods in the retina); in the dark-adpated state all membranes show a smooth, hydrophilic, interlamellar surface (PS face), a particle-free, rippled, hydrophobic fracture face (PF and E F face), and an intralamellar surface with many particles (ES face); the particles have a diameter of 50 & and are therefore different from the 125- to 175-A particles of the light-adapted membranes. With the use of sonication experiments before and after illumination these investigators hypothetically identified these particles as rhodopsin. After illumination the rhodopsin migrates into the rippled, hydrophobic membrane layer, forming particles of 125- to 175-A diameter which emerge 20-30 8, from the cleavage surface and which seem to consist of four to eight rhodopsin molecules. These investigators do not conclude that the evidence is unequivocal either in demonstrating the described order of the rhodopsin molecules or migration due to illumination. Pedler and Tilly (1967)showed (although in X . Zaeuis) that rods cannot be decomposed into their lamellae by sonication without prior OsOl fixation. c. Rods after Spreading. Spreading experiments with dark-adapted rods showed hexagonal particles with diameters of f N F d= 145 & 18 A (Rosenkranz, 1976b). Within the limits of error the ratio Vpy between the volumes of the truncated pyramids of light (h)- and dark (d> adapted rods is the same as the ratio VSTbetween the rod volumes Vh and V , (Section V,A); in one case

and in the other (after considering Sections II1,A and IV,D,l,c)

as the height of the truncated pyramids after illumination remains unchanged at about 50 A.

2. X-Ray Diffraction Effected by the Lameltar Body a. Rods Viewed Longitudinally. According to Corless (1972) there is only one significant difference in the electron density distribution of light- and dark-adapted rods, namely, a minor increase in

122

flRGEN ROSENKRANZ

electron-dense material adjacent to the interlamellar membrane side in the dark-adapter state with a maximum at x = 80 A (see Fig. 46b to compare the position of the x axis). The reasons for the electron density change are small but significant reflection changes with order numbers h = 2,3, and 4, Corless does not draw any definite conclusions from this result but leaves the position of the rhodopsin in the membrane open to question. Chabre (1975b), in an extensive investigation of R. esculenta, also found an increase in electron-dense material after illumination only at about 82* A; he does not confirm rhodopsin shifting as described by Blasie et al. (1969) and Blasie (1972). Chabre made his measurements 5 minutes after total bleaching of the retina; unlike Corless, he recorded higher intensities in the light-adapted state for the reflections h = 2 and 3 than in the dark-adapted state; the intensities of the remaining eight reflections, however, lay below their initial values. After 100 minutes the diffraction pattern became normal in that the intensities of all the reflections were only slightly below their initial values. With R. pipiens Worthington (1973) found that the state of adaptation had no influence on the lattice constant a but affected the reflection intensities; the intralamellar space seemed to become narrower after illumination. This result is in contrast to the findings of Corless and Chabre. Worthington stresses, however, that any interpretation of the reflection intensities from his experiment are only preliminary, owing to the complicated experimental situation. If it is assumed that Worthington found a shift in intensities similar to those observed by Corless and Chabre, the following conclusion can be drawn. The difference in the change in electron density distribution after illumination found by Corless and Chabre in one case and Worthington in the other cannot be due to the different phase set (Table XIII), as the influence of the respective reflections h = 2 and 11is much too small; the difference can essentially be due only to the assumption of a different Lorentz factor to correct the reflection intensities (Section IV,D,3,a. After considering the information in Section IV it seems more probable that the retinylidene group of the rhodopsin is located on the interlamellar side of the lamellar membrane. b. Rods Viewed in Cross Section. According to Chabre (1975b), the illumination of rods in the retina leads to a negligible change in the broad (55 A)-1 reflection. If a rhodopsin aggregate is assumed, this means that the outer dimensions of the aggregate remain constant in the membrane. The average distance p , between neighboring rhodopsin aggregates also does not change during illumination, as pre-

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

123

viously mentioned in Section IV,D,3,b. These findings contradict the light-optical measurements made by Enoch et al. (Section V,A).

3. Light-Optical Investigations a. Absorption of Linearly Polarized Light. In linearly polarized light, dark-adapted rods do not show an absorption band at 380 nm (Section IV,D,4) but a weak p band at 350* nm (Kropf, 1972) and a very strong a band at approximately 502 nm. The p or cis band is due to induced electron oscillations along the bent polyene chain of the ll-cis-retinal (Dartnall, 1972). The maximum of the a band was measured in single rods of the same species (R. pipiens) by Liebman and Entine (1968) under linearly polarized light at A,, = 502 f 1nm and (under unpolarized light?) by Liebman (1962) at 506 nm in the majority of cases with fluctuations between 500 and 511 nm; Liebman regards Amax = 510-511 nm as the most probable in situ. The shifting of the maximum of ll-cis, 12s-cis-retinal from 380 to 510 nm in the retinylidene opsin can be explained b y the protonated Schiff base of the rhodopsin (Ebrey and Honig, 1975). An important factor in the elucidation of rod ultrastructure is rod dichroism; linearly polarized light with its electrical vector oscillating perpendicularly to the longitudinal axis of the rod (absorption constant e ~ is) absorbed 4.5* to 6 times more strongly at,,A than parallelly polarized light (el,); the first value was measured by Wald et al. (1963), and the second by Liebman (1962). The 280- and 235nm bands are not dichroic (Liebmann, 1972). Assuming that all retinylidene groups form the same angle with the lamellar surface u, Liebman calculated this angle from the dichroic ratio, with V, = /ell = 6, arctan u = (l/2V,)1/2= 16"

+

respectively 18.5" for V, = 4.5. Since ell 0, one cannot, however, necessarily conclude that all chromophores form an angle u with the membrane plane; the chromophore itself is already aplanar, and how much this factor contributes to the dirchroic ratio is still unknown. b. Absorption of Circularly Polarized Light. In analogy with the linear dichroism (Section B,3,a) there is also circular dichroism; this phenomenon can be used to detect asymmetries in the molecule and its surroundings. Crescitelli et al. (1966) determined the circular dichroism in digitonin extracts of dark-adapted R . catesbeiana and R. pipiens; they found two circularly dichroic extinction maxima of approximately

124

P R G E N ROSENKRANZ

equal intensity: he, (A = 490* nm) = 0.52 x lo+* and Aez (A = 336* nm) = 0.58 x both values were taken from the respective figures and were calculated as described in Section IV,D,4. The fact that the a and /3 bands of rhodopsin, but not of pure retinal, show a strong extinction suggests an asymmetry in the coupling between retinylidene and opsin; the shift of the absorption spectrum toward shorter wavelengths by -10 however, remains unexplained, as does the reason for the asymmetry, that is, whether it is due to the asymmetric chromophore itself or to the surroundings. Similar questions arise concerning the dichroism of rhodopsin in the ultraviolet range (Ebrey and Honig, 1975); Crescitelli et a2. (1966) found a decrease in the circular dichroism of -4 x l W * after illumination (see Section IV,D,4) and attributed this decrease to a loss in the proportion of helically formed proteins in the opsin [Mommaerts (1969)assumes a reduction of 10% to 65%], but whether or not these measurements allow such an interpretation must be carefully considered (Section IV,D,4). Shichi et a2. (1969)extracted rhodopsin from bovine rods with nonionic detergents and determined an “apparent” helical protein proportion of about 60% in the unbleached rhodopsin; after “irreversible illumination” this proportion was reduced to 48%. However, when the circular dichroism in suspended rods was measured, a light-dependent reduction was not observed. While the investigation methods of circular dichroism and the equivalent optical rotation dispersion are technically of importance, conclusions about the complicated structure of rhodopsin in the rod can be made only with greater theoretical knowledge. The statement of Velluz et a2. (1965) remains valid: “One can hardly expect at the moment to be able to calculate a priori the optical activity of a given asymmetric molecule, even a fairly simple one.”

c.

RESULTS OBTAINED FROM ISOLATED RHODOPSIN On the basis of chromatographic behavior, Hall et a2. (1969),as well as Heller (1969), concluded that in R. pipiens there was an increase in rhodopsin volume after illumination. Heller showed that the Stoke molecule radius r d = 23 A for unbleached rhodopsin and r h = 25.5 A for illuminated rhodopsin, assuming rhodopsin to have a spherical shape. The results indicate a change in the secondary structure of the rhodopsin, but they do not provide evidence about the real shape of the molecule. It should be noted that from Stokes’ work the following proportionality applies for a force K acting on a sphere of radius r i n a solvent of viscosity rl0:

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

125

In addition,

applies for diluted newtonian solutions of, for example, rotational ellipsoids as particles, where r ) = viscosity of the solution, V = viscosity factor for rotational ellipsoids, after Simha, and @ = total volume of the solid phase in the unit volume of the solvent, and therefore also

where r' = apparent radius (Netter, 1959). Equation (18)shows that the Stokes' radius cannot yield information about the shape of the rhodopsin as long as V is not known.

VI. Changes in Rod Ultrastructure with Time A. DEVELOPMENT INTO

A

MATURE ROD

1. Electron Microscope Observations At the present time there exist essentially two hypotheses concerning the development of the rods. Sjostrand assumes that the lamellae are produced by invaginations of the cell membrane at the vitreal end of the rod; and this idea has been adopted by Nilsson (1964b) for R. pipiens; de Robertis, however, assumes (also de Robertis and co-workers, 1970)that the lamellae originate from vesicles in the cytoplasmic region of the rod. He suggests that they are only partly produced by invagination and; if so, only at the lateral cell membrane. Both groups of investigators agree about the first stage of rod formation, that is, that one ofthe two centrioles in the rod inner segment migrates to a position x of the cell membrane, where it lines itself up with its longitudinal axis parallel to the membrane and to the longitudinal axis of the inner segment. According to Nilsson, who has observed rod development carefully and in great detail, this procedure occurs about 5.5 days after fertilization of the frog egg. Some time later the cell membtane begins to arch at this place (x), and as early as the sixth day a badloonlike bulge can be seen. Inside this bulge, filamentous struc~ures(microtubules?) and the first lamellae develop, but no vesicles, contrary to the view of d e Robertis et al. (Fig. 52). Ac-

126

J~~RGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

127

cording to Nilsson (1964b), from the cell membrane conical invaginations at the base of the growing outer segment the lamellae develop; the invagination widens in the interior of the rod to become at first sacculelike and finally disklike; between one-half and two-thirds of the disk circumference is connected to the cell membrane at this stage. Nilsson states that the invaginations migrate sclerad, gradually separating from the cell membrane. Two days after the first appearance of the lamellae the fine structure of the rods is the same as that found in adult frogs; the deep incisures are formed, and all lamellae, except for one or two, are isolated from each other and from the cell membrane. Nilsson (1964b) claims to be able to differentiate between rods and cones in the first stages, because only rods have lamellae with an intralamellar space of z 50*-A width. This is almost always the case, however, on about the eighth day most rod lamellae, similar to those of cones, do not appear to have intralamellar spaces. Some remaining rod lamellae with intralamellar spaces can also be observed and at this time still make identification possible. The postulation that all lamellae develop as invaginations of the cell membrane can be confirmed only when the experiments of Nilsson (196413)are supported by autoradiographic investigations of developing rods similar to those carried out by Young and Droz (1968)on rnature rods. Until then, the results of de Robertis et al. (1970) and Rosenkranz and Hauser (1972) should be equally considered, even though they were obtained in a less systematic way. Figure 51 shows a longitudinal section of a rod from an adult frog. This type of rod was observed on several occasions and was always situated among other normal, well-structured rods. The well-defined, centrically structured ciliary matrix indicates that it is a rod in a stage of early development and not of decomposition; de Robertis considers that the numerous tubules and vesicles also favor this assumption. The fact that the frog was an adult i s not necessarily inconsistent with FIG.50. Part of a longitudinal section of a developing rod. The lamellae (la) appear to be formed some distance away from the cell membrane. Hexagonal particles (hx) a p pear to aggregate in ribbons (bd),reminiscent of the observations in the spread experiments of Blasie et al. (1965); these ribbons are obviously incorporated into already existing fragments of lamellae. hxl, Hexagonal particles seen in section. Preparation as in Fig. 13. (Rosenkranz and Hauser, 1972.) FIG. 51. Longitudinal section of a developingrod. The core of the rod is filled with many tubules (tu) and fragments of lamellae (la), sometimes without any detectable order, sometimes appearing to be attached to the ciliary matrix (cx). This electron micrograph agrees with the conception of de Robertis and contradicts that of Nilsson, as discussed in the text. Preparation as in Fig. 13. (Rosenkranz and Hauser, 1972).

128

JURGEN ROSENKRANZ

this phenomenon, as rod development starting at the posterior pole is known to be asynchronous. Owing to the manifold results (see Figs. 50 and 51) it is difficult at present to present a detailed alternative to Nilsson’s view on lamellar development or to confirm it. The clearly shown invaginations of the cell membrane and the lack of vesicles and tubules in the tadpole rods shown by Nilsson (1964b) differ from the tubulelike invaginations of the cell membrane in Fig. 51 and the numerous structural building blocks of all stages of aggregation up to the completed lamella shown in Figs. 50 and 51. These differences between the well-known micrographs of Nilsson and Figs. 50 and 51, of R . esculenta, may be due to the method of fixation. Nilsson used 1% OsOl in Verona1 acetate buffer, and we used 2.5%glutaraldehyde in collidine buffer followed by 1%O s 0 4 in collidine buffer. As previously mentioned, further experiments are necessary to clarify the problem of the development of the lamellae.

2. Light Microscope Observations Liebman and Entine (1968) found, in R . pipiens, that the visual pigments of the receptors drastically change during the metamorphosis from tadpole to adult frog. They determined different visual pigments

in three developmental stages: in the tadpole (no matter whether legless or with hindlegs and almost fully developed forelegs), in the frogpole (the stage of almost completed metamorphosis), and finally in the adult frog (Section V,B,3,a). In the tadpole stage they found red and green rods in the same quantity and of the same morphology as in the adult frog. The red rods contained only one visual pigment, P.527,. As indicated by the notation the absorption maximum of this pigment is at h = 527 -+ 1 nm, and as a chromophore this pigment contains 3-dehydroretinal or retinal, (derived from vitamin A,) which differs from retinal by an additional double bond in the ring. This visual pigment, which is also called porphyropsin has a linear dichroic ratio v , (P527*)= 4, which is similar to that of rhodopsin. After illumination, P527, shifts its absorption maximum to 400-405 nm, which is the same as that for retinal,. The green rods contained a visual pigment, P438, indicating Amax = 438 2 1 nm; the chromophore is assumed by these workers to be retinal,; the dichroic ratio is 0, (P438,) = 3.5. In a certain frogpole stage these in= 513 nm. They vestigators found an absorption maximum at,,A showed that this was a mixture of the two visual pigments P527, and P5021. Furthermore, they ascertained that this change from porphyropsin to rhodopsin takes place synchronously and to an equal extent in all rods.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

B. CONSTANTRENEWAL OF

A

ROD

IN THE

129

ADULT FROG

In 1967 Young, using autoradiography and light microscopy on rods of R . pipiens, showed that tritiated methionine is built into the rods. In 1968 Young and Droz traced the pathways of several radioactively labeled amino acids in R . esculentu rods, using electron microscopy. From these experiments it was concluded that the amino acids migrate through the connecting cilium partly into the ciliary matrix but mainly into the vitreal part of the rod and form a band equally distributed across the whole section. The whole band moves sclerad with the same velocity parallel to the longitudinal axis of the rod (see Fig. 53), until it separates from the rod at its sclerad end. It is then engulfed by the adjacent pigment epithelial cell (Fig. 54). The velocity of the shifting band is 36 lamellae per day in R . esculenta at 22.5"C. According to Young, the continual renewal of lamellae at the vitreal end of the rod takes place in a way similar to the rod development observed in tadpoles b y Nilsson. Since Matsubara et al. (1968) and Hall et al. (1969) showed that radioactively labeled amino acids such as methionine, phenylalanine, and leucine are also assembled into the opsin, the band mentioned above must be mainly made up of lamellar membranes containing rhodopsin. If these are formed, as Fig. 52 suggests, by invagination of the cell membrane, one has to consider why the opsin, instead of passing through the connecting cilium, is not initially present in the cell membrane in the sclerad part of the inner segment. Perhaps invagination of the cell membrane is not the decisive procedure in the formation of lamellae. It should also be noted that the opsin is not renewed, that is, the band does not migrate sclerad when the frog is kept at a temperature of +4"C, but the velocity is doubled with every 10°C temperature increase (Young, 1967).

c.

DIFFUSIONOF RHODOPSIN

Experiments of Brown (1972), Cone (1972), Liebman and Entine (1974), and Po0 and Cone (1974) have shown that rhodopsin mole-

cules diffuse in the lamellar membrane. This diffusion is thought to be caused by Brownian movement. The assumption can be made as long as no contradictory direct measurements of the viscosity constant 77 are made, since the two interesting diffusion constants are indirectly proportional to 7; this value has been determined theoretically under the condition that the geometry of the rhodopsin molecule is known. According to Einstein (1906) the two directly measured diffusion constants of rotation Dr, and of translation Dt, are related to the viscosity

130

J~~RGEN ROSENKRANZ

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

131

constant 7 . The shape of the particle, however, is assumed to be elliptical and not spherical (as Einstein assumed), since we regard this as the more probable. Several assumptions can be made for the following calculations. The rhodopsin molecule may have the form of a prolonged rotational ellipsoid which is located with its longest diameter 2al perpendicular to the lamellar membrane and which rotates around a,. According to Section III,B,4, 2al = 75 A, and the diameter at right angles to 2al is 2uz = 30 A. Alternatively it can be assumed that six rhodopsin molecules each form a rhodopsin aggregate and that this can be approximated by an oblate ellipsoid with 2a, = 75 A and 2uz = 140 A (Section IV,D,3,b); again the rotational axis is a,. The translational movement to which, in both cases, the lateral diffusion constant is related is always observed in a direction through az. 1. The Rotational Diffusion of the Rhodopsin The rotational diffusion constant D,(in sec-’) is defined by Eq. (19) according to Fick‘s second law which describes nonstationary diffusion processes:

anlat

= D,(azn/ae2)

(19)

where t = time, 8 = angle between the chromophore axis and the reference direction, for example, the direction of the polarization vector of the light used for the observation, and n = number of rhodopsin molecules (or aggregates) per unit area lying between 6 and ae during FIG.52. Longitudinal section of a rod of a 6-day-old tadpole. Note that there are almost no cytoplasmic regions, except for the ciliary matrix (cx), which are not occupied by lamellae. All 16 lamellae are invaginations of the cell membrane. Rana pipiens. Fixed in 1% OsO, solution buffered with Verona1 acetate (pH 7.2-7.4), dehydrated with acetone, embedded in Vestopal W. Reproduction from Nilsson (196413). FIG. 53. Longitudinal section of a fully developed rod. The autoradiographic labeling is due to the following tritiated amino acids: histidine, methionine, leucine, and phenylalanine; all were injected 1 week before fixation of the rod. Note the equally fast migration ofthe labeled region. Rana escuknta. Fixed with a 4% methanol-free formaldehyde solution (phosphate-buffered)and postfixed with 2% 0 s . in phosphate buffer (pH 7.1). Reproduction from Young and Droz (1968). FIG.54. “A large phagosome (p), containing approximately 42 rod outer segment discs, has just been engulfed by the pigment epithelium. A cytoplasmic extension of the pigment epithelial cell (c) has flowed around the phagocytized discs to occupy the space formerly filled by the discs , . . (m) melanin granulae.” Rhesus monkey rod. Fixed in 0.8% glutaraldehyde. The same phagocytosis presumably exists with frog rods but an illustration of this is not available. From Young, 1971.

132

JURGEN

ROSENKRANZ

the time at. Following Cone (1972)a solution of this differential equation is n=l+fexp(-4Df)cos%

(20)

f describes the depolarization of the optical system of the microscope;

it has been determined experimentally, on the basis of the maximal dichroic ratio in the lamellar plane, V,,, = 3, to be f = 0.7, With Eq. (20) the linear dichroic ratio is x p - 4D,t v, = 22 +-ffeexp - 4D,t

From the plot V, = V,(t)determined experimentally by Cone one obtains, with Eq. (21) and with V,(T,)= 1 + e-l, the relaxation time 7, of the molecule: t = 7, = (4DJ-l = 27* psec and

D, = 0.9 x 104 sec-I According to Einstein (1906) but using the present terminology

D,

=

2kTB

(22)

where k = 1.38 x W sec/degree, T = absolute temperature, and B = mechanical mobility of the rhodopsin molecule (W-l sec-'). B depends on the shape of the molecule; according to Einstein, for a sphere with radius T it is

According to Edwardes (1893) and Perrin (1934), B is for a prolonged rotational ellipsoid rotating very slowly around its long diameter 2ul:

and for an oblate rotational ellipsoid rotating around its short diameter 2u,:

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

133

If one assumes the rhodopsin to be spherical, as Cone does, with a radius r 21 A, and to have a relaxation time 7, = 20 psec at 2WC, it follows from Eqs. (22) and (23) that J

A rotational ellipsoid with al = 37.5 8, and az = 15 A (Section III,B,4) has the same volume as a sphere with radius r = 21 A; because of Eqs. (22) and (24) this leads to

If one finally assumes that a rhodopsin aggregate rotates as a whole and, as described in Section IV,D,S,b, that one diameter a, = 37.5 A but a2 = 70 A,the approximation for a hexagonal particle [taking Eqs. (22) and (25) into account] is

These estimations show that, on the basis of the data given by Cone, a viscosity coefficient q is calculated to be about 10 times higher than that suggested by Cone (1972). The value of r) is almost as high when the rhodopsin molecule is assumed to be an ellipsoid rotating as a single molecule. It is interesting that our assumption that a rhodopsin aggregate exists that is able to rotate only as a whole leads to a value of q that can be compared with that of olive oil, that is, it can be regarded as a realistic value. This estimation shows further how necessary it is to make an independent measurement of the viscosity constant q of the lamellar membrane. 2. The Lateral Diffusion of Rhodopsin Analogous to D,, the diffusion constant of the lateral or transversal diffusion Dt (mz sec-l) is defined as anlat = Dt (an2/aX2)

(29)

In this case, n indicates the number of nonilluminated rhodopsin molecules (or aggregates) per unit area that move forward a distance a x in a certain direction in time at. Equation (29) describes a one-

134

flRGEN ROSENKRANZ

dimensional diffusion process; this simplification of the problem is allowable because of the experimental procedure, since one-half of the rod lamella was always illuminated. Diffusion could therefore start vertically along a whole lamellar diameter d = 2r, and not only from the surface center. If we regard primarily the diffusion along the central vertical line on the diameter d , that is, 0 5 x 5 rl, a general solution to the differential Eq. (29) is n(x, t) = N(sin kx

+ const. x cos kx) exp - DA2t

(30)

where ij7 = number of nonilluminated rhodopsin molecules (or aggregates) per unit area in the nonilluminated lamella, and x = space coordinate through the center of the lamella vertical to the diameter that separates the nonilluminated half of the lamella from the illuminated half, where x = 0. Considering the initial condition n(x, 0) = N, Eq. (30) leads to

and produces the special solution n(x, t) = Nexp

-

(7iDtt/4x2)

(31)

If one considers the half-time tln during which half the rhodopsin molecules at the point x = r M are again not illuminated, the lateral diffusion constant from Eq. (31) becomes

Dt =

(In 2)4rM2 +tlB

Po0 and Cone (1974) put r M = r1 but measured t l n at r M = o.5*rl. Liebman and Entine (1974) reported r M = 0.6*r1= 2.5 pm, tln = 4.0 & 0.5 seconds. This leads to Dt = 4.4 x lo+’ cm2/sec,provided the lamella is regarded as a circular area without incisures. Based on the suggestions made by Cone, Liebmann, and Entine, incisures in the lamellar membrane mean an increase in the diffusion constant by a factor of 1.7 to 6, determined from analogous experiments, and therefore

0.7 x 1W8 5 Dt 5 2.6

X

1W8 cm2/sec

Dt = 10W2m2/sec (33)

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

135

Thus the measurement of Liebman and Entine (1974) analyzed with Eq. (32)lead to the same diffusion constant as that obtained by these workers in other ways. This is not the case with the half-time measured by Po0 and Cone, and Eq. (32). The diffusion constant of translation Dt can also be used to obtain information on the viscosity constant 7;in analogy with Eq. (22)the following applies:

Dt = 2kTB

(34)

For a sphere (radius r ) B is, according to Einstein (1906),

For a prolonged rotational ellipsoid moving perpendicular to the long diameter 2a1 (Perrin, 1934) it follows that

This relation was deduced by Oberbeck (1876) under the condition that the rotating ellipsoid was surrounded by an unlimited liquid; in the case of the almost two-dimensional lamellar membrane with many neighboring ellipsoids it can only be regarded as an approximation. For an oblate rotational ellipsoid moving vertically to the short diameter 2ul:

With the same values for r, al, a2,and T as in the case of rotational diffusion, and with Dt = 1W8 cmz/sec the lateral diffusion of a spherical rhodopsin molecule, taking Eqs. (34) and (35) into account, leads to

For a single elliptical rhodopsin molecule, from Eqs. (34)and (36),it follows that

136

JORGEN ROSENKRANZ

And finally for a rhodopsin molecule aggregate, from Eqs. (34) and (37), as an approximation for a hexagonal particle,

While the viscosities derived from the rotational and translational diffusion constants differ by a factor of more than 10 when all rhodopsin molecules are assumed to be isolated from each other, qbx [Eq. (28)l agrees relatively well with qkx [Eq. (40)] (Rosenkranz, 1976a). It should be noted that the viscosity of olive oil is 0.84 P. This kinematic study of the rhodopsin molecule produces even more results indicating that the rhodopsin in the lamellar membrane forms aggregates of six molecules.

VII. The Green Rod A.

CHARACTERISTICS

I n this section only the deviations of the green rod from the red rod are described. The matrix in which rods are implanted can be assumed to be identical to that of the red rods, and the description therefore is not repeated. The number of the green rods in the frog retina is listed in Table I, and the length and diameter in Table 11. A general description is given in Section I.

B. ULTRASTRUCTURE 1. Electron Microscope Observations According to Nilsson (1965),the green and red rods seem to be mor-

phologically identical, except for the features mentioned in Section VI1,A. Similarly, significant differences cannot be shown between the lamellar membranes. Jones (1974) found that the optimal phosphate buffer concentration for isotonic fixation of green rods is 64 mM and not 50 mM as for red rods.

2. Light Optical lnvestigations For 26 dark-adapted, intact green rods, observed at right angles to their longitudinal axis, Liebman and Entine (1968) found a single absorption maximum at ,,A,, = 432 nm; furthermore, they found a dichroic ratio v ~for,A, ~ of the same magnitude as that for red rods;

from the slightly incomplete spectra in their Fig. 4 we have estimated

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

137

vem = 4*. If the absorption coefficient for the pigment P432 [i.e., the

pigment having its absorption maximum at 432 nm, which is referred to as chloanopsin by Gaupp (1904)l is equal to that of the red rod, Liebman and Entine calculate the concentration of the chloanopsin in the green rod to be 2.5 mM. From extracts of green rods of Rana cancriuora, Dartnall(l967) isolated a visual pigment based on retinal, (of vitamin A,) and found an absorption maximum between 320 and 620 nm at Amam; his best estimation for this value was A,, = 433 2 2 nm. Crescitelli (1972) could not find an explanation for the fact that a green rod is blue-sensitive. In spite of many observations of rods in the retina (R. esculenta) parallel to their long axis we did not observe green rods, provided the microscope light had a color temperature comparable to that of daylight. Green rods appeared gray-yellow, which lies near brown-yellow, the color complementary to blueviolet. C. RENEWAL In addition to the information contained in Section VI,B it should be noted that the lamellae of the green rod do not move as fast as those of the red rod sclerad along the rod longitudinal direction. Young and Droz (1968)report a renewal of 25 lamellae per day at 22.5"C for R. esculenta, which is only 70% of the red rod rate.

VIII. Summary This article attempts to give not only a summary but also a critical survey of present knowledge concerning the ultrastructure of frog rod outer segments. Attempts have been made to close the gaps in this knowledge wherever it was technically possible. Our attempts to bring together the majority of experimental results concerning the structure of the frog rod outer segment, taking into account the differences in opinion and techniques of observation, leads to the following conclusions. The rod outer segment is directly connected to the rod inner segment by a cytoplasmic bridge of 0.07-pm2 cross-sectional area; a further, indirect, connection exists between the cell membranes of the inner and outer segment through about 25 inner-segment apical microvillous processes. In this way membrane configurations are produced which, on a smaller scale and between two different cells, would be referred to as zonulae adherentes. The total membrane area per rod structured in this way is equal to about 500 zonulae adherentes. Fil-

138

JOURGEN ROSENKRANZ

aments pass through the 10-pm-long microvillous processes as well as through the connecting cilium; in the first case there are about 20 microfibrils with a diameter of 80 f 40 A per microvillous process passing vitread beyond the elliposid; in the second structure 9 microtubule doublets extend from the basal body through the connecting cilium 0.1 pm into the rod. Depending on the preparation method employed, the cell membrane appears to be either of normal thickness or of thickness comparable to that of a lamella. Which of the observations represents the real structure is uncertain. The situation is still more complicated because differences in the fine structure of various areas of the cell membrane have not been observed, for example, between the vitreal and scleral area, and between the intra- and extracellular sides of the cell membrane. A different structure of the cell membrane along the rod longitudinal axis can be expected, considering the observation that an invagination of the cell membrane at the vitreal end leads to the generation of lamellae (according to many investigators), while the same process at the scleral end merely serves to shed a pile of old lamellae. The ground cytoplasm in the rod outer segment exists essentially as a 200-A-thick layer; as such it lines the intracellular side of the cell membrane and the deep incisures of the lamellar body. Thus the ground cytoplasm represents only 4% of the total rod volume; the distance between the cytoplasm and any point of a lamellar membrane is on the average not more than 0.5 pm owing to its special distribution. Because the deep incisures are traversed by many short fibrils, the contact area between the cytoplasm and lamellar body is eight times as large as the total intracellular cell membrane surface. The system of the lamellar rims is chemically different from the lamellar body proper, and consequently from the system of the lamellar membranes. If the lamellae are really generated by invagination of the cell membrane, the cell membrane must be transformed in the hairpin region into a tubulelike segment; another possibility is that tubules are the sites of development of the lamellar membranes; the rims would then be derived from tubules. This hypothesis is consistent with the observation that at the ends of some incisures the rims of neighboring lamellae are connected by tubules. Rims of lamellae lying one on top of the other approaoh each other up to a distance of -30 A, while the respective lamellae in vivo remain 150 apart; therefore the lamellar period which, according to x-ray diffraction is 300 A i n oiuo, consists of half of a lamella and half of an interlayer; the interlayer contains not only cell water (or cytoplasm) but also fibrils of 45 f 5 A diameter, the exact course of which has not

-

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

139

yet been detected within this layer. Investigations suggest that the lamellae do not float in the cell water as though they were isolated from each other, but seem to be connected through the interlayer. The lamella proper consists of three layers: two lamellar membranes surround an intralamellar space with a height of about 10 A. This should be regarded as a third layer rather than a space, since osmotic shocks do not suggest a primarily aqueous content. The lamellar membranes themselves again consist of three layers which are mirror-inverted about the intralamellar layer. The results of chemical, light and electron optical experiments, and neutron and x-ray diffraction techniques, can be best interpreted by making the following assumptions. First, the secondary structure of a rhodopsin molecule is dumbbell-like; the dumbbell has a length of 75 A, that is, it spans the whole lamellar membrane, and the mass of the rhodopsin polypeptide chain is distributed in two tangles of approximately equal size. Second, not single rhodopsin molecules but rhodopsin aggregates of six single rhodopsin molecules are surrounded in two dimensions by a bilayer of lipid molecules. Each rhodopsin aggregate represents a hexagonal particle of 140-A diameter, contains six prosthetic groups on the interlamellar side, and can function as a pore with a diameter affected by illumination. Appendix 1: Extreme External Influences and Their Consequences A.

CHANGES IN ROD STRUCTURE AFFECTEDBY OSMOTIC SHOCKS

Investigations on rod behavior toward different osmotic pressures as a function of illumination, and toward cell poisons, are beyond the scope of this article. Such experiments are mentioned only when they contain important information about ultrastructure. In Table XVI some of the results of osmotic shock experiments are compiled. These results have been obtained mainly by induced osmotic changes and only in one case (Zuckerman) by electrophysiological measurements. Perhaps one of the reasons for the contradictory results shown in this table lies in the method of rod isolation or even fragmentation often applied. Experiments performed with J. Schelten, with thermal neutrons at the DIDO reactor in the Kernforschungsanalge Julich, Germany, suggest that isolated rods of R. escuZentu, even after 7 hours in D20-Ringer's solution, did not take up DzO; the Ringer's solution was prepared only with heavy water. This

140

flRGEN ROSENKRANZ

interpretation follows from a consideration of the differential cross section d8/dSZ for coherently scattered thermal neutrons:

d8- Z(h) exp(Z‘D) dSZ DN+’E ‘P(h)

n

where Z(h) = neutrons scattered by the lamellae into the solid angle A i l , 8 ’ = total cross section for neutron scattering and absorption, D = thickness of sample, N = number of lamellae contained in sample, +’ = neutron flux, E ’ = irradiated sample area, and P(h) = probability of meeting two lamellae of different rods a certain distance away from each other. Our experiments led to the value dZ/dSZ = 1.4 cm-’ for rods in H 2 0 as well as in D20-Ringer’s solution, although from Fig. 44b one would theoretically expect

dCH/dfl= 1 cm-’

for H20-Ringer’s solution

dZD/dSZ= 50 cm-’

for D20-Ringer’s solution

and

Perhaps the cell membrane was transformed into an unphysiological state by the separation of the rods from the energy source in the rod inner segment. Obviously this is not the case when rods in the retina are irradiated by thermal neutrons, as the experiments of Chabre (1975a) with the same species have shown. Useful information on the permeabilities of different ion species can therefore only be expected from such experiments performed on the entire, intact rod cell. Another possibility mentioned, among others, by Cohen and Chabre is that even in the intact retina many rods may experience leakages which make exact measurements of osmotic behavior difficult as long as these possibilities of inaccuracy cannot be excluded. These statements are, however, not completely sufficient to explain all discrepancies. For instance, it remains open to question why Korenbrot et al. (1973) are the only investigators who found sodium permeability to be light-dependent. The results shown in Table XVI for the intralamellar space indicate that this is not a space filled mainly with water, but rather a layer filled with unhydrated material. The results of Heller et a2. (1971) seem to contradict this opinion, although their investigations were performed on isolated lamellae separated from the rod and retina. For this reason the appearance of artifacts cannot be totally excluded.

TABLE XVI PERMEABILITIES OF SOME IONS AND NONELECTROLYTES FOR THE WHOLE ROD, THE CELL MEMBRANE, AND THE LAMELLAR MEMBRANE Hod

Isolated" hvb Permeable Impermeable

-

+

-

+ -

+

+ + + + 'I

NaCI, KCI NaCI, KCI

Cell membrane Permeable

Impermeable

Permeable Impermeable

Ammonium acetate

NaCl

KCl NaCl

Experiment involved isolated rods (+) or rods in the retina (-). (+) or dark-adapted (-) state. Rod or lamella behaved (approximately) like an osmometer (+) or did not (-).

* Observation of the light-adapted

Rod

- Rod + Rod

r

+

Osmometef

+

KCl, melezitose, saccharose NaCl NaCl K+ more than CH,SO,-, Na+, C1-, CO,*CH,COONa+. C1-

%

C1-, NO,-, K+, SO,L-, glycerol, melezitose acetate, ammonium + NaCl - Na+, C1-

Lamellar membrane

Mg2+, C1-, saccharose, Na+, K+, Cas+

Reference Chabre and Cavaggioni (1975) Cohen (1971)

+ +

Zuckerman (1973) Rod Cobbs and Rod Hagins (1974) Lamellae Heller et al. (1971)

+

Rod

Korenbrot and Cone (1972)

+ +

Rod

Korenbrot and Cone (1972) Korenbrot et al. (1973)

Rod

142

P R G E N ROSENKRANZ

B. THE BEHAVIORO F THE RODS fN A MAGNETIC FIELD Chalazonitis et al. (1970) exposed rods in Ringer's solution to a homogeneous, constant magnetic field with a flux density B between 3 and 20 kG (in the Gaussian system 1G 1 Oe, the unity of the magnetic field intensity H). A vector m can be assigned to the rod longitudinal axis so that it points from the vitreal to the scleral end. Chalazonitis et al. found that m is parallel as well as antiparallel to B with the same probability, and that ca. 3* kG and 2 minutes are sufficient for m to rotate from its approximately perpendicular initial position to a position parallel to the field lines; they found further that at 7.6* kG 70%*of the rods and at 20* kG 90*%aligned themselves parallel to the field within this time. The rotation is independent of the state of adaptation. At 3 kG, where the rotation just starts, an increase in the rotation angle was observed following illumination of the rods; if the angle between m and B is (Pd = Q (m, B)d in the dark-adapted state, after illumination

Furthermore, it was observed that the rod lost its rotation capacity when the lamellar order was disturbed. In an attempt to interpret the behavior of the rods when subjected to a magnetic field, Hong et al. (1971) claim that it is unlikely that paramagnetic molecules in the rod are sufficiently concentrated to mask the diamagnetism. They do not support their claim but confine their interpretation to the case of diamagnetic anisotropy of the rods and assume an anisotropy such that the magnetic susceptibility xa is greater in the axial direction than xr, that in the radial direction. Hong et al. (1971) also tried to derive the time dependence of the rod rotation in the magnetic field. They considered the moment of inertia 8 of the rod, its magnetic energy E, and a friction forcef'd which counteracts the rod rotation by the angle cp in Ringer's solution with viscosity qm. The rod here is approximated to be a prolonged rotational ellipsoid. They derive the differential equation

8+ + f'+ + (dE /do) = 0

(41)

wheref' = 4.6 x gm cm2/sec dEldcp = 4.5 x 10-lo sin Q * gm cm2/sec2,cp = Q (m, B) = Q (m, H), and 8 = 3.4 x gm ern2. Equation (41) thereby becomes

+ + 1.35

X

lCr+

+ 1.32 x

1Cr sin 2q = 0

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

143

An approximate solution to this nonlinear, inhomogeneous differential equation is obtained by the linear approximation of sin 2cp = 2q, which leads to a rotation time T for cp = 89" to cp = 1" of T = 4 seconds. As this approximation is valid only for small cp ( 55") but not for the present case (cp = go"), a more exact solution is proposed on the basis of more recent experiments (J. Rosenkranz, unpublished results, 1975). Experiments in our laboratory investigated whether the rods were para- or diamagnetic; they were found to be diamagnetic. This conclusion is based on the observation that rods in an inhomogeneous field move parallel to the magnetic field lines away from the higher magnetic field line density; this applies when the rods are suspended in aqueous 0.4 M FeCl2.4H20solution, as well as in distilled water, Ringer's solution, or saturated aqueous Ca(N03)2 4 H 2 0 solution. These solutions had the following magnetic susceptibilities xm(measured by the method of Quincke): FeC12. 4H20 solution: Xr = + 57.4 x Water: xw = -9.0 x l W 6 (calibration medium) Ringer's solution: Xh = -6.7 x Ca(N0J2 4 H 2 0 solution: Xk = - 10.1 x 10-6

For an unknown molecule in the rod the magnetic dipole moment induced by the magnetic field strength H is determined in the case of diamagnetism by m = V'poxH

where V ' = volume occupied by the considered molecule in the rod (in the case of rhodopsin or lipid, V ' = 0.1V = 150 pm), p o = 47r lo-' Vs(Am)-', x = diamagnetic susceptibility of the rod; as the force AK' on m in the gradient awaz of the magnetic field is determined by

it follows, because

that -\XI <

-Xm

=

Xk

=

-lo-'

The exact numerical value of x could not be determined from A K = 0, as no suspension liquid could be found that left the rods intact and

144

@RGEN ROSENKRANZ

had a lower diamagnetic susceptibility than that of a saturated calcium nitrate solution. As shown by Fig. 55, the orientation of the rod longitudinal axis in a homogeneous as well as in a inhomogeneous field immediately indicates that the magnetic susceptibility in the axial direction xa is much higher than that in the radial direction xr; this follows, considering the existing diamagnetism and that the energy E = -mH tends to become a minimum. If lxal > lxrl were not true, the rod longitudinal axis and the magnetic field lines would not remain parallel, but a deviation is not observed. There are two pieces of information which help to identify the molecules responsible for the diamagnetism of the rod. The orbits of the electrons causing the diamagnetism must lie mainly in the lamellar plane (Fig. 55), as the dipole moment of a rotating electron is given by

where e = electron charge, u' = oscillation frequency of the electron, r = orbit radius of the electron, and n = unit vector normal to the

orbit. As the vitreal and scleral ends of the rod respond equally in the magnetic field, exactly the same distribution in or on both membranes of a lamella must exist. These characteristics are shown by the rho-

FIG.55. Part of a cross-sectional view of a lamella (la) showing two alternatives: the electrons causing, besides H, the diamagnetism spin in the lamellar membrane plane (km)or at right angles to it (km');see Eq. (42). m Lis the dipole moment caused by the Larmor frequency. M = m,, m, or mi,mi,respectively, is the resulting dipole moment on which H acts to give the moment of rotation D = M x H. As D, = (m + mL)x H and Dz= (-m + mL)x H,the resulting moment of rotation is AD = D, - D2= 2m x H.

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

145

dopsin molecules which therefore can be assumed to cause the diamagnetism of the rod (apart from H). To derive the equation of motion of the rod in the magnetic field calculations must be based on the time-dependent change in the angular momentum L of the rod in a homogeneous magnetic field; this is equal to the moment of rotation N diminished by the frictional resistance R to which the rod is exposed in Ringer’s solution during the rotation:

L=N-R

(43)

The diamagnetic anisotropy in the rod leads to a magnetic moment m so that

The frictional resistance is

The change in the angular momentum is finally

Thus Eq. (43)becomes

Vp, VL,pp, and pL With the values taken from Section 111 for 1, d , VHZ0, the mass m of the rod is m = 1.46 x lo-’, kg, and its moment of inertia around an axis vertical to m is

e = e,,, = - [):(

+

$1

=

2.5 x

kgm2

(45)

The same moment of inertia

also applies for a rotational ellipsoid with the semiaxis b, = 29 p m parallel to 1, and b, = 3 p m at right angles to 1; these values were

146

P R G E N ROSENKRANZ

used for the following calculation of the frictional coefficient. Furthermore,

V ‘ = 1.52 x m3 IXa - Xrl IXalz H = 3.5 x 1(Y A/m 4.4 kOe = (HI The frictional coefficient c2 is calculated according to Edwardes (1893)and Perrin (1934) for very slow movement of a rotational ellipsoid with a long diameter 2b, = 58 p m and a short diameter 2b2 = 6 pm and with qm= qRinger*s = 0.0112 P and 9 = 1.25; the fact that not only one rod rotates but that many neighboring rods move at the same time in a flattened liquid droplet leads to mutual interference which can be described by the coefficient 9 of qm;9 = 1.25 has been estimated by model experiments, keeping the Reynold’s number constant. c2

=

16+: - b t ) h m 3([(2b2, - b2,)/(b:- b2,)’I2] In {[b,+ (b2,- b2,)112]/b2} - b,) (46) =

1.2 x

kgm2/sec

With these particular values Eq. (44) becomes Q+5x

lo5++ 1 0 6 s i n c p = 0

(47)

A solution of this nonlinear, inhomogeneous, second-order differential equation is possible when the short time range is separated from the range of longer times (we thank V. Ram for this suggestion); Eq. (47) is then solved separately for each range: solutions cpK and (pL. For short times t’ Eq. (47) becomes, because cp (t = 0) = d 2 ,

with a = 5 x lo5 and b = lo6. If the substitution t’ = a-’ t is introduced, dt’ = u-’ dt; in this way Eq. (48) becomes Q

+ (F, + b/a2 = 0

(49)

Equation (49) can be solved under the initial conditions ~ ( t=’ 0) = d 2 and dt’ = 0 ) = 0:

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

147

For long times t ' Eq. (47) becomes, with the substitution t ' = ab-'t,

+ + + sin cp = 0

@/a2)+

For small b/a2= 5, if one expands cp = cpL comes, in the zeroth approximation,

(51)

+ cp15 + . . . , Eq. (51) be-

&, + sin cpL = 0

(52)

Regarding cp = cpL ( t ' = 0 ) = d 2 , an integral of Eq. (52) is (PL =

2 arctan (exp - Z(b/u)t')

(53)

Observations of rods in a homogeneous magnetic field in our laboratory showed that the majority of the rods were oriented almost parallel to the magnetic field within somewhat less than 10 seconds. If one regards the data used in Eq. (47) as being of the right order of magnitude, Eq. (53) indicates a response time of 1 second for the change from a position exactly vertical to the field lines to one deviating by only about 2" from the direction of the field lines. Appendix 2: On the Limitations of the Experimental Techniques Used A. ELECTRONMICROSCOPE PREPARATION TECHNIQUES 1. Ultruthin Section Technique

In recent years the following standard method for the preparation of rod ultrathin sections has been developed. A retina, often darkadapted and with the pigment epithelium attached, is fixed in glutaraldehyde solution, often postfixed in osmium solution, dehydrated in ethyl alcohol, and embedded via propylene oxide-Epon mixtures in pure Epon. The glutaraldehyde fixative consists of 1% glutaraldehyde in sodium phosphate or sodium cacodylate buffer, the osmolarity of the buffer alone being 2 120 mosM. The osmium solution is prepared from 1% Os04 in the same buffer. After sectioning parallel to the rod longitudinal axis the sections are generally double-stained with uranyl acetate and lead citrate. Unless otherwise specified, the use of this standard method is assumed.

148

flRGEN ROSENKRANZ

The interpretation of an ultrathin section of a rod is difficult in many respects, as the overlapping effects of artifacts and a staining pattern which cannot be clearly differentiated have to be considered. The denaturating and, for example, dissolving effects of Os04and ethyl alcohol are known for a multitude of specimens, but these experiments also show that it is not always possible to apply the results observed in rat liver or pancreas tissue or cattle serum albumin to other biological material such as rods. A complete biochemical investigation of the fixation, dehydration, and embedding agents used for rod preparation is necessary to make a more exact interpretation of ultrathin sections. As such comprehensive investigations have not yet been carried out for the rod, it is necessary to refer to the respective investigations on other biological objects, always bearing in mind that the structure preservation and staining effects in the rod may be different. Structural changes are caused b y loss of tissue material and are related to osmotic pressure changes and changes in enzymic activity. While it is generally assumed that glutaraldehyde alone or with OsOl as a postfixative does not denature proteins, fixation by oso4alone is known to cause denaturation; thus about 80%of the soluble protein of rat zymogene granules is dissolved in the fixative after fixation with 1% oso4(Amsterdam and Schramm, 1966). It should be noted that the isolation of the lamellas by sonication (Section IV,D,l,a,iii) is possible after Os04 fixation but not in the unfixed state or after fixation with glutaraldehyde. Even after glutaraldehyde fixation the activity of some enzymes is reduced; while the cholinesterase activity is still as high as 75%, that of Mg-activated ATPase is reduced to 15% (Hopwood, 1973).As far as the phospholipids are concerned they are all extracted from the tissue (rat hypothalamus) following glutaraldehyde fixation except for phosphatidylserine and ethanolamine (Roozemond, 1969). At present there is still some uncertainty concerning the effects of the osmolarity of the buffer in the fixation solution; the osmolarity of the fixative itself does not seem to be decisive for structure preservation. Inspired perhaps by investigations carried out by Bone and Denton (1971), Jones (1974) showed that only a buffer with -50% of the osmolarity of Ringer's solution effects minimal changes in rod volume (observed as regularity of the lamellar order). Volume changes are, however, only one criterion of structure preservation. Others include changes in membrane permeabilities, membrane potential, and enzymic activities. Another important influence on structure preservation, but not necessarily on the final staining pattern, is exerted by the ethyl alcohol

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

149

used for dehydration. I n Acanthamoeba, with a lipid composition probably different from that of the rod, 84% of the phospholipids and all the neutral lipids are extracted b y ethyl alcohol, no matter whether their fatty acid residues are saturated or unsaturated. The situation changes when Acanthamoeba is postfixed with 0 ~ 0 4 Then, . all neutral lipids are again missing in the section, but only 13%of the phospholipids are extracted, mainly the saturated ones (Korn and Weisman, 1966). The cross-linking mechanism brought about by glutaraldehyde and affecting mainly proteins is frequently as suggested by Richards and Knowless (1968): \

CHI HC(COH)-CH~- (COH) CH-HC’ NH I

R

I

HN

R

I

The most reactive group is the e N H 3 group of the lysine which has a reaction constant that increases proportionally with pH value. The cross-linking brought about by OsO, in phospholipids is based on diester formation at sites of former double bonds in the fatty acid residues, according to Korn (1967):

The esterfied osmium is stably bound; the OsO2 is not bound; perhaps it deposits at the interface between lipoprotein and water. According to Bahr (1954), the cross-linking of proteins is partly brought about by their amino and sulfhydryl groups and the disulfide bond, but mainly by their content of tryptophan, cysteine, and histidine. The good fixation and staining properties of carotenes, precursors of retinal, are again due to the presence of double bonds. The staining of sections must also be judged with caution. Not all structural details that have “survived” fixation, dehydration, and embedding are stained, except in the case of OsO, fixation. After glutaraldehyde fixation, radioactively labeled amino acids like leucine, but not tyrosine (Hodson and Marshall, 1967) or reduced OsO2 (Korn, 1967), deposit in areas theoretically assumed not to be related. Finally, the reactions of different stains in embedded rods have not yet been

150

JORGEN ROSENKRANZ

chemically investigated. Thus, when the stain uranyl acetate is used, one can only suppose that uranyl ions form complexes with hydroxyl, carboxyl, and phosphate groups (Rothstein and Meier, 1951). Lead citrate, which in our experiments contributes only a little to staining intensity and nothing to staining modifications in the rod, appears to attach to cysteine, phosphate groups, and glucose polymers of the biological material (Reynolds, 1963). In conclusion, some results should be mentioned which partly simplify and partly complicate an interpretation of ultrathin-sectioned rods. Practically no difference in structure preservation was found when cacodylate, collidine, or phosphate buffer was used, although the first seems to be the most suitable (Rosenkranz, 197613). It also appears to be unimportant whether monovalent ethyl alcohol or bivalent alcohols like glycol or hexylene glycol are used for dehydration, at least as far as the rod core is concerned. Sjostrand and Barajas (1968), however, working with mitochondria, suggest that ethylene glycol diminishes conformational changes in the cell membrane. Generally, it has not been found that water-soluble embedding agents like Durcupan or glutaraldehyde-urea resin, which do not require alcohol as a dehydrating agent, produce sections showing more details than, for example, Epon (see Figs. 5 and 23). When this is taken into account, it is difficult to understand why postfixation with platinum destroys large parts of the lamellae of rods embedded in Epon, whereas they remain intact after Durcupan embedding (Figs. 27 and 23). 2. Freexe-Etching The standard procedure is often as follows. A small piece of tissue is soaked for about 1 hour in 20% glycerol, generally in Ringer's solution, deep-frozen first by Freon 22 and then by liquid nitrogen, fractured under vacuum (usually torr), etched at about - 100°C for from 1to 5 minutes (i.e., the cell water sublimes at a cooler face), and then shadowed by a platinum-carbon mixture. This contrasting metal layer is mechanically reinforced by a carbon layer; the replica is finally cleaned from remaining biological tissue by, for example, 70% chromosulfuric acid. This standard method has at least two disadvantages. First, the treatment with glycerol may alter the structure of the biological tissue; whether it does, and if so to what extent must be determined for every specimen by omitting the glycerol treatment in a control experiment. Second, the deep-frozen biological specimen is warmed up during the transfer to the vacuum recipient and during shadowing (Rosenkranz, 1975b); this could also lead to unpredictable structural changes. The deep freeze-fracture method tries to avoid

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

151

these two disadvantages; here the temperature of the preparation never increases above - 134°C from the first deep-freezing until the production of the replica. This demands, after very fast freezing, a low temperature throughout the whole procedure, and shadowing equipment that does not negatively influence the production of the metal layer by electron or ion currents. It should be noted that the possibility of obtaining double replicas exists, which makes it easier to recognize artifacts produced by fracturing and shadowing; this technique has not yet been applied to rods. Finally, it should be stated that freeze-etched replicas allow exact measurement only of spacial periodicities; the size of all other structural details can only be estimated.

3. Spreading Spreading of biological material occurs in trough as well as in drop preparations. The present experiments with rods were performed mainly with drop preparations (with a diameter of several millimeters); the curvature of the drop is negligible compared to the rod diameter, so that the surface tension is considered only when observing whether the fragment floats on the surface or sinks into the drop. In the first case the surface tension can be neglected as a minor force directed tangentially to the surface. If the fragment sinks, it is expected that these forces will tear the biological fragment apart and push it downward in an unpredictable manner in order to minimize the drop surface. Calculations show that the latter is the case for rods on a drop with a diameter of several millimeters. As is true of many other stains, the chemical reactions of PTA or silicotungstic acid with material in the rod have not yet been investigated. It is also not clear to what extent PTA cross-links or fills gaps in the biological material. The information gained by the drop technique is therefore valuable only when combined with information obtained from other investigations.

B. DIFFRACTION METHODS

1. Light Diffraction Light waves, like all other waves, are diffracted b y bodies which differ in density from their surroundings, in this case in the optical density of the material. If the distances between neighboring bodies (or contrast spots on the negative) are equal to or greater than the wavelength A of the diffracted coherent waves, these waves interfere and thus produce an intensity distribution of light radiation in the space behind the bodies (spots). This intensity distribution depends,

152

JURGEN ROSENKRANZ

apart from A , only on the shape of the bodies and on their mutual arrangement when several bodies are hit b y the light beam at the same time. While the shape of a body can be determined from the arrangement of the intensity differences of a diffraction pattern, the number and position of the reflections or diffraction maxima (light areas in the diffraction pattern) yield information on the type and regularity of these bodies. The usual difficulty in the interpretation of diffraction patterns lies in the fact that the effects due to shape, type, and arrangement of the bodies, overlap. A missing reflection in a certain area can be due to three reasons: the special shape of the body, the special lattice it produces together with other similar bodies, or too low a degree of order. For the diffraction patterns presented here it should be stated, without dealing more thoroughly with structural analysis, that a hexagonal lattice as a diffraction pattern with a distance h between two reflections clearly indicates a hexagonal order of spots on the negative, and thereby particles on the membrane. The distance p between the spots on the negative is correlated with h in the following way: hp

=

(2Id3) X const.

where the constant is an apparatus constant pertinent to the particular diffraction apparatus. 2. X-Ray Small-Angle Diffraction The reflection intensities of an x-ray diffraction pattern divided by the Thompson, Lorentz, and polarization factors form the intensity function Z(h); h is the spatial coordinate in the reciprocal space or Fourier space and is related to the spatial coordinate x in the physical space. Generally the electron density distribution p x ( x )is the convolution root of the inverse Fourier transform of the intensity I, in sh01-t:~

1

h space

Z (h)exp(2?rihx)do,, = p , ( x )

* px( - x) = W ' Z

(h)

p x ( x ) = @Z(h)

Under the conditions outlined in Section IV,D,3,a, and with the notation described there, the following applies for the rod: 9-l

Z (h) = Patt(x)

a relation normally only applying exactly to infinite structures, and In general, the convolution square U = g(x) * g(-x) = Jg(y)g(x + y)du,. A solution of this quadratic integral y-space equation is called convolution square root fi= g(x).

ULTRASTRUCTURE OF FROG ROD OUTER SEGMENTS

153

further, Patt(x) = Patt(x

Ia ) =

Qo = pX(x)* pX(-x)

Qo is also referred to as the autocorrelation function and depends only on the charge distribution of an elementary cell. The intensity of the x wave diffracted by the structure also depends on the atom form factor or scattering amplitude of the atom or ion. For x-rays, this scattering amplitude increases markedly with increasing atomic number and decreases markedly with increasing diffraction angle. A hydrogen atom therefore scatters only 0.028th of the quantity scattered by a carbon atom in the direction h = 0. X-rays therefore yield a distorted picture, especially of biological material, as they almost completely ignore the most frequently occurring element, hydrogen. In our case, however, compounds and not single atoms are of interest, and the electron density distribution px(x) determined by Fourier synthesis is

where x = spatial coordinate in the elementary cell (of length a = 300 A), L = 6.02 x lP3 molecules/mole, M = molecular weight in gm/mole, p = mass density in gm/cm3,and Zi = atomic number of the ith atom of the compound in question. From Eq. (54)one can see that x-rays cannot differentiate between two compounds when p x ( x ) is equal for both.

3. Neutron Small-A ngle Diffrac tion Neutron diffraction is in principle described in the same way as light or x-ray diffraction. The amplitude of the neutron wave resulting from diffraction by an atom is

JI

=

exp i(2mlA) - (b'/r)exp i(2.rrt-A)

The first term of this equation describes the neutron wave incidence parallel to x, with wavelength A (often A = 7 A); the second term describes the diffracted neutron wave observed at a distance r from the diffraction center. b ' is the scattering length of the respective atom and is of the order of cm; this magnitude makes neutron diffraction interesting; it is a real magnitude for the atoms dealt with in biology, but it depends among other factors on the kind of atom and isotope. The coherent part of the scattering length that alone contributes to the interference is referred to as b . For the isotopes in which we are cm) (Bacon, 1962: lH, interested, it has the following values (in

154

JORGEN ROSENKRANZ

-0.37;‘H, +0.65;“C, +0.66;I4N, +0.94; “0, +0.57;“P, +0.53;“S, +0.31. From these data and the data of Section I11 the scattering length densities

Pn =

scattering length cm-’ volume

were calculated for the lamellar membrane

where 6 , = coherent scattering length of the ith kind of atom, N = number of identical molecules in the volume V, V = volume directly occupied by N molecules. ACKNOWLEDGMENTS

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