The structure of F-actin and of actin filaments isolated from muscle

J. Mol. Biol. (1963) 6, 46-60

The Structure ofF-Actin and of Actin Filaments Isolated from Muscle JEAN HANSON AND J. LoWY

Medical Research Council Biophysics Research Unit King's College, Strand, London, W.C.2, England (Received 12 July 1962, and in revised form 3 October 1962) The filaments of the contractile apparatus have been isolated from a wide variety of smooth and striated muscles and examined in negatively-stained preparations in the electron microscope. In all cases there are thin filaments which are indistinguishable from the filaments in F -actin preparations. The filament consists of two helically-wound strands composed of subunits which appear to be alike and approximately spherical. The arrangement of the subunits corresponds to that of the scattering centres in one of the possible structures for actin deduced by Selby & Bear (1956) from the moderate-angle X-ray diffraction pattern of intact dried muscle. The number of globular subunits per turn of the helix (if integral) is 13 (cf. either ia or 15 in the models proposed from diffraction data). The spacing of the subunits along each strand is 56·5 A (of. 55 A in the models proposed from diffraction data). The cross-over points of the two twisted strands are spaced at intervals of 349 A along the filament (cf. either 351 A or 406 A in the models proposed from diffraction data). The overall diameter of the filament is about 80 A. It is shown in the case of rabbit skeletal muscle that this result is consistent with the quantity of actin in that muscle. There is good evidence that each of the globular subunits seen in the electron microscope represents one actin monomer. The structure of actin alone does not account for (i) the approximately 400 A axial periodicity observed in the I-substance of fibrils in the electron microscope, or (ii) the reflection at about 400 A observed in the axial diffraction pattern of intact muscle. It is suggested that both (i) and (ii) could be due to the combination of actin with other material, possibly tropomyosin B.

1. Introduction Bear (1945) discovered that the X-ray diffraction pattern of every type of muscle he examined included the same series of moderate-angle reflections on or near the meridian. These reflections were subsequentlyshown to be due to the presence of actin (Astbury & Spark, 1947; Astbury, 1949; Selby &, Bear, 1956; Cohen & Hanson, 1956). Meanwhile, Huxley (1953a,b) had found that in vertebrate skeletal muscle there are two kinds of filaments, and Hanson & Huxley (1953, 1955) had obtained evidence, mainly from extraction and recombination experiments, that one kind of filament contains actin, the other myosin. Their conclusion, that in vertebrate skeletal muscle actin is located in the thinner of the two kinds of filaments, is now firmly established (review by Huxley, 1960). In most of the other muscles which Bear had studied, thin filaments, similar in size and location to those of vertebrate skeletal muscle, have now been seen by electron microscopy in sectioned material 46

THE STRUCTURE OF ACTIN

47

(reviews by Hanson & Lowy, 1960; Lowy & Hanson, 1962), but whether or not they contain actin has not been conclusively established. The electron microscope is capable of resolving structural details such as those which give rise to the moderate-angle diffraction pattern of actin, but in sectioned fibres the thin filaments appear structureless. In certain respects the older practice of using intact isolated fibrils or filaments had given more promising results (Rozsa, SzentGyorgyi & Wyckoff, 1949), but neither shadowed nor positively-stained preparations had shown the characteristic structure that might have been expected from the diffraction pattern. This was disappointing because it is difficult to decide from diffraction results (i) whether the structure of actin is helical or planar, (ii) whether or not each scattering centre represents one monomer, (iii) how many centres there are in the non-primitive unit cell, and (iv) what is the precise diameter of the filament (Selby & Bear, 1956; Worthington, 1959). Elucidation of all these problems is needed for an understanding of the mechanism by which interaction between active sites spaced along actin and myosin filaments causes tension production and shortening in muscle. Other problems might be solved by using the electron microscope to study the structure .of actin filaments. In vertebrate skeletal myofibrils the I-substance, where the thin filaments are located, probably contains not only actin but also tropomyosin B (Perry & Corsi, 1958; Corsi & Perry, 1958; Huxley, 1960); and in the electron microscope the Lsubstance shows transverse striations spaced at intervals of approximately 400 A (Draper & Hodge, 1949; Carlsen, Knappeis & Buchthal, 1961). Is tropomyosin B situated in the same filaments as actin1 Are either or both of these proteins, or some other material, responsible for the approximately 400 A axial periodioity! And what is the origin of the reflection observed at about 400 A in the axial diffraction pattern of both vertebrate skeletal muscle and other kinds of muscle (Huxley, 1953a; Selby & Bear, 1956; Worthington, 1959)1 The present results were obtained by applying the method of negative staining to preparations of filaments isolated from a wide variety of muscles, and to preparations of F-actin. In this paper we describe the structure of the filaments and discuss the size and shape of the monomers, the diameter of the filaments, the location of tropomyosin B, and the origin of the approximately 400 A axial periodicity. Elsewhere (Lowy & Hanson, 1962) we discuss the implications of our finding that it is possible to define the location of actin in muscles other than the striated muscles of vertebrates. A preliminary account of the present results was given at a meeting of the British Biophysical Society in December, 1961. A summary has been published elsewhere (Hanson & Lowy, 1962).

2. Materials and Methods Muscles. Filaments isolated from the following muscles have been examined: psoas of rabbit; indirect flight muscles of Dytiscus and Oalliphora; striated adductor of Pecten; translucent and opaque parts of adductor of (Irassostrea ; anterior byssus retractor of Mytilus; mantle of Loligo; longitudinal muscle layer of body wall of Lumbricus; taenia coli of guinea-pig. Isolation of filaments. The method used for isolating filaments (Huxley, 1961) is based on the principle that cross-linkages between actin and myosin filaments in glycerolextracted fibres will be detached when the system is "plasticized" with ATP-Mg in the presence of EDTA (Watanabe & Sleator, 1957); a suspension of separated filaments suitable for negative staining can then be made by disintegrating the muscle in a blendor

J. HANSON AND J. LOWY

(Huxley, 1961). The procedure used was as follows. The water-glycerol mixture in which the muscle had been extracted was replaced with a solution containing 0·15 M-KCI, 4 mM-MgCI2 , 50 mer-veronal-acetate buffer, pH 7·0. The muscle was homogenized and centrifuged, and then resuspended in the same solution which now contained, in addition, 4 mM-EDTA. After the muscle had been homogenized again, 4 mM-ATP was added and the muscle homogenized once more. A suspension of filaments was obtained and, after centrifugation to remove large fragments, the supernatant solution was prepared for microscopy. Solutions and homogenizer were kept cold throughout these operations, the duration of each period of homogenization being as short as possible, for example 10 sec at high speed in a 5 ml. Virtis homogenizer. In the case of several molluscan muscles we have found that filaments can very easily be isolated from glycerol-extracted material without plasticizing it, and also that living muscles can readily be separated into filaments. Preparation of actin. F -actin was prepared from rabbit skeletal muscle by the method of Szent-Gyorgyi (1951), purified by one centrifugation (Mommaerts, 1951), and resuspended in 0'1 M-KCl. Negatively-stained preparations were made by methods based on those described by Huxley & Zubay (1960). (i) Copper grids were covered with carbon films which had been prepared on freshly cleaved mica (Spencer, 1959), or with thin films of collodion or formvar lightly coated with carbon. Films with small holes were also used. (ii) A drop of a suspension of filaments was placed on a grid and almost immediately withdrawn by touching the edge of the grid with the smooth edge of a filter paper. A drop of stain was immediately added and then withdrawn after 2 to 3 sec. (iii) The stains were aqueous solutions of uranyl acetate (0,3%, unbuffered) or potassium phosphotungstate. The latter was made by titrating a 1 % solution of phosphotungstic acid with strong KOH to p;EI 5'8; solutions of pH 5·3 and pH 7·0 gave the same results. (The use of uranyl acetate as a negative stain was mentioned by Huxley & Zubay, 1960, and has been further recommended on the grounds of the excellent contrast produced and the absence of reaction with most substances under investigation (Huxley, personal communication).) (iv) When uranyl acetate was to be used, the preparation was washed with a few drops of 0·1 M-KCI just before applying the stain. If the preparation was not washed, a precipitate formed. Washing was not necessary when potassium phosphotungstate was used, but some preparations were washed before applying this stain to check that washing did not affect results. When preparations had been made on films with small holes, the holes were found to be covered with films of stain (Huxley & Zubay, 1960) containing numerous filaments, even if the preparations had been washed before being stained. (v) After the drop of filament suspension had been placed on the grid, the filaments were sometimes fixed in osmium tetroxide vapour before the drop was withdrawn and the stain applied. No differences were observed when the actin filaments in such preparations were compared with others that had not been fixed. Microscopy. A Siemens Elmiskop I with 30 p, objective apertures was used. Pictures were taken at magnifications of 40,000 or 80,000, using an accelerating voltage of 80 kv. Calibration of magnification. Paramyosin filaments as well as actin filaments are present in many electron micrographs of preparations made from molluscan muscles. We have used the very regular axial periodicity which is visible in the paramyosin filaments in order to determine the magnification of these micrographs and of others taken during the same week and at the same microscope settings. The magnification of other micrographs was determined by using for calibration a carbon replica of a diffraction grating; the spacing of the lines in this replica was measured by light microscopy. According to this calibration the periodicity of paramyosin in our electron micrographs measures 145 A. According to X-ray diffraction results, this periodicity measures 144 or 145 A in living muscles, and also in muscles which have been glycerol-extracted, dried, fixed, or positively stained with phosphotungstic acid after fixation (Worthington, 1959; Elliott, 1960; Elliott & Lowy, 1961). Examination of micrographs. All observations and measurements were made on the original micrographs examined in dissecting microscopes magnifying 10 or 22 times.

THE STRUCTURE OF ACTIN

49

3. Results All the results apply to certain filaments isolated from every.type of muscle so far investigated, as well as to the filaments in preparations of F-actin. The filament is composed of two strands which are wound round each other. The positions where the two strands are seen to cross over one another are regularly spaced along the filament (Plate II). Frequently the two strands differ in contrast near the cross-over positions, one appearing to be denser than the other. The strand with the greater contrast appears to lie above the other and the helix gives the impression of being left-handed. This applies both to filaments lying on the supporting film and to filaments situated in stain-filled holes (i.e. in the films of stain which form over holes in the supporting film), and it applies to both uranyl acetate and potassium phosphotungstate preparations. However, because we do not know how the stain is distributed in the immediate vicinity of the strands, this appearance cannot, without further information, be interpreted to show the sense of the helix. If it could be demonstrated that when a filament is lying on the supporting film the upper strand, but not the lower one, has stain underneath it, then it could be argued that the strand which has the greater contrast in the micrograph is the lower strand; in that case the helix would be right-handed. Each strand consists of a single series of regularly spaced subunits which appear to be alike and approximately spherical. The subunits are seen most clearly in the filaments which are situated in stain-filled holes (Plates I to III). They are also visible in the filaments which lie on the supporting film (Plate IV) but in order to obtain sufficient contrast it is necessary in this case to take under-focus micrographs; in these the apparent structure of the background is coarse. In near-focus micrographs of stain-filled holes the subunits are seen much more clearly in uranyl acetate preparations than in potassium phosphotungstate preparations. On the other hand, under-focus micrographs of filaments lying on the supporting film show the subunits and the helical structure of the filaments rather more clearly if potassium phosphotungstate has been used for staining. At the ends of filaments, and in the regions between cross-over positions, the two strands are sometimes seen to lie separate from one another. This suggests that the attachments between subunits in a single strand differ from those between subunits in adjacent strands. Usually, however, the two strands lie very close to one another, and in the regions between cross-over positions one can see that the subunits of the two strands alternate, i.e. a given subunit in one strand lies alongside the position between two subunits in the other strand. Displaced subunits are occasionally observed, suggesting that attachments between subunits in the strand have broken. By counting the globular subunits it has been found that the number per turn of the helix described by each strand is either very nearly 13 or exactly 13 (Plate III(b)). Most of the observations which led to this result were made on filaments situated in stain-filled holes. Few of the filaments in anyone preparation were found to be suitable for counting subunits, and even in those filaments where long flawless sequences of subunits could be observed, it was not often possible to count along more than two complete turns of the helix described by either strand. For this reason we have not been able to determine if the helix is integral. Although filaments in stain-filled holes are more suitable for counting subunits than are those lying on the supporting film, they are less suitable for measurement because 4

50

J. HANSON AND J. LOWY

there is evidence that shrinkage, or sometimes stretching, has occurred. As shown later (p. 51), the subunits correspond to the scattering centres which are responsible for the moderate-angle X-ray diffraction pattern of F-actin. According to diffraction results, the spacing of the subunits along each strand should be about 55 A. Values considerably smaller or sometimes larger than 55 A were often obtained when the spacing was measured in filaments in stain-filled holes, but values close to 55 A were always obtained when measurements were made on filaments lying on the supporting film. The following results were all obtained by measuring filaments lying on the supporting film in preparations stained with potassium phosphotungstate. The spacing of the subunits in each strand, measured along the axis of the strand, was found to be 56·5 A. This value is the mean of 132 measurements which range from 52 to 58 A; the standard deviation of the mean is 0·25 A. This result was obtained by measuring a length of strand and dividing that length by the number of subunit periods in it. It does not take into account the fact that the strands are slightly LEGENDS OF PLATES I TO IV PLATE I. Electron micrograph of filaments isolated from the translucent part of the adductor of the oyster Orassoetrea angulata. The muscle had been extracted with water-glycerol and "plasticized" with ATP-Mg in the presence of EDTA. The preparation was negatively stained with uranyl acetate. This photograph has been printed with reversed contrast, i.e. black areas are unstained and white areas stained (cf. Plate IV). All these filaments are situated in a thin film of stain covering a hole in the carbon supporting film. There are numerous actin filaments and some much thinner filaments (arrow 1) which are frayed-out paramyosin subfilaments. In the actin filaments note the globular subunits (arrows 2) which, in this near-focus micrograph, cannot be confused with the apparent structure of the stain in the background. The helical configuration of the actin filaments is visible in many places, but is more clearly seen in Plate II. Magnification 430,000 x . PLATE II. Electron micrograph of a preparation of F-actin, negatively stained with uranyl acetate. The area shown here is part of a stain-filled hole in the supporting film. Note the helical structure of the filaments. The subunits are also seen clearly, but are demonstrated more convincingly in near-focus micrographs (Plates I and III(a)) where the apparent structure of the background is not as coarse as it is here in this under-focus micrograph. Reversed-contrast print. Magnification 455,000 x . PLATE III. (a) Electron micrograph of actin filaments isolated from the translucent part of the adductor of Orassostrea angulata. The muscle had been extracted with water-glycerol and "plasticized" with ATP-Mg in the presence of EDTA. The filaments were negatively stained with uranyl acetate and are situated in a hole in the supporting film. They are somewhat distorted but show the globular subunits very clearly. Near-focus micrograph. Reversed-contrast print. Magnification 425,000 x . (b) Electron micrograph of a preparation of F-actin, negatively stained with uranyl acetate. The area shown here is part of a stain-filled hole in the supporting film. In several places (one is marked) it is possible to count the number of globular subunits per turn of the helix described by each of the two strands of subunits which compose the filament. The arrows indicate positions where the two strands cross over one another. Under-focus micrograph. Reversed-contrast print. Magnification 525,000 x . PLATE IV. Electron micrograph of filaments isolated from the translucent part of the adductor of Crassostrea angulata. The muscle had been extracted with water-glycerol and "plasticized" with ATP-Mg in the presence of EDTA. The preparation was fixed in osmium tetroxide vapour and was negatively stained with potassium phosphotungstate (pH 5'8): unstained filaments (white) are seen against stained background (black). The filaments lie on a collodion-plus-carbon supporting film. In addition to the thin actin filaments there are two much thicker paramyosin filaments: The projections on the paramyosin filaments are believed to correspond to the bridges which in sectioned fibres appear to cross-connect actin filaments to paramyosin filaments (Hanson & Lowy, 1961). In the actin filaments note the globular subunits (arrow 1), the helical configuration (arrow 2), and the side-by-side packing (arrow 3). Under-focus micrograph. Magnification 250,000 x ,

PLATE

I

SOO .&. PLATE

II

(0 )

soo .&.

soo.&. PLATE

III

SOOA PLATE

IV

THE STRUCTURE OF ACTIN

51

curved. Thus the true spacing will be greater than the one we have quoted, but only by a very small amount (less than 1 A). (Measurements made on preparations stained with uranyl acetate also fell within the range of 52 to 58 A.) The spacing along the filament of the positions where the two strands cross over one another was found to be 349 A. This value is the mean of 156 measurements which range from 330 to 363 A; the standard deviation of the mean is 1·1 A. The total width of the two strands, measured where they lie alongside one another in the regions between cross-over points, was found to be approximately 80 A (the mean of 36 measurements which range from 70 to 95 A). This measurement cannot be a precise one because the boundary between the background and the globular subunits is not sharply defined, nor does it necessarily represent the true position of the edge of the filament. However, the same result was obtained when the lateral separation of two or more filaments lying alongside one another (Plate IV) was measured. The mean of 59 measurements, ranging from 73 to 86 A, is 80 A (standard deviation of the mean, 0·6 A). Finally, except for variations in length, no differences have been observed between actin filaments isolated from different muscles or between these filaments and those present in F-actin preparations. Very little material besides filaments of the type described here has been observed in the protein preparations.

4. Discussion (a) Comparison with X-ray diffraction results

From the X-ray diffraction pattern of an intact dried muscle (the translucent part of the adductor of Venus) Selby & Bear (1956) deduced certain features of the structure of the actin filaments (Fig. 1) but could not decide whether the scattering centres lie on a planar net or on a helix. Comparison of Plates I to IV and Figs. 1 and 2 shows that the arrangement of the globular subunits in the filament as observed in the electron microscope corresponds to that of the scattering centres in the helical model based on the diffraction results. According to Selby & Bear, the spacing of the scattering centres along the connections marked by dotted lines in Fig. 1 is about 55 A (cf. the figure of 56·5 A obtained for the spacing of the subunits along each strand as observed in the microscope). The diameter of the cylindrical shell drawn through the scattering centres in the helical model based on diffraction results is approximately 50 A (cf. the figure of approximately 40 A obtained for one half of the over-all diameter of the filament as observed in the microscope). Selby & Bear (1956) were also unable to decide between two slightly different arrangements of the seattering centres in the filament. They suggested that the nonprimitive unit cell either had 13 centres, in which case its length was 351 A, or had 15 centres and a length of 406 A (Fig. 1 (i) and (ii); Fig. 2 (i) and (ii)). In living relaxed vertebrate skeletal muscle Huxley (1953a) observed a reflection at about 400 A, but as he used a slit camera, only the layer-line position is known. Probably this reflection corresponds to the one found by Worthington (1959) in glycerol-extracted preparations of the same type of muscle. However, in this case the reflection is on the meridian and therefore it cannot relate to the pitch of the actin helix. Selby & Bear (1956) also found a reflection at 400 A in their material (a dried molluscan muscle), but for various reasons they considered that this reflection does not necessarily indicate the length of the unit cell.

J. HANSON AND J. LOWY

52

Worthington (1959), however, decided from a study of the diffraction pattern of intact dried Helix pharynx retractor muscle that the 15-centre cell was more nearly correct than the 13-centre cell. Selby & Bear (1956) had pointed out that, according to their results, the "genetic" helix, which is indicated by contiuuous lines in Fig. 1, (i )

{iil

u

FIG. 1. Two possible alternative arrangements of equivalent scattering centres in the nonprimitive unit cell of actin in intact dried muscle as proposed by Selby & Bear (1956) from a study of the moderate-angle X-ray diffraction pattern. Each diagram shows two unit cells of length U. The scattering centres are for convenience shown as if they were arranged on a planar net, but in fact they are helically arranged. If the net is rolled so that its two long sides (which are parallel to the filament axis and to the length of the unit cell) coincide, then the scattering centres will lie on the circumference (0) of a cylinder, the diameter of which is 50 A (cf. Fig. 2). The dotted lines represent one possible way of connecting the scattering centres; in one unit cell there are two such , strands of scattering centres, the spacing of which along each strand (S) is about 55 A. The pitch of the helix described by each strand is equal to the length of two unit cells. In model (i) there are 13 scattering centres per turn of this helix; in model (ii) there are 15 centres per turn. The continuous lines represent another possible way of connecting scattering centres into a helix (a "genetic" helix). In model (i) there are 13 centres in 6 complete turns of this "genetic" helix; in model (ii) there are 15 centres in 7 complete turns of this helix. The pitch of this helix (l--about 59 A) corresponds to a layer-line periodicity observed in the diffraction pattern. The spacing of scattering centres along the filament axis (k-about 27 A) corresponds to another layer-line periodicity in the diffraction pattern. The ratio of these two periodicities is slightly different in models (i) and (ii) and provides the only way at present available 'for distinguishing between them by diffraction methods (Worthington, 1959).

could have either 15 centres in 7 turns, or 13 centres in 6 turns. Worthington noted that the ratio 7 : 15 = 0'467, and the ratio 6 : 13 = 0·462, and he realized that it should be possible to decide between the two alternatives by making sufficiently accurate measurements of two layer-line periodicities in the diffraction pattern, one which corresponds to the spacing of the scattering centres along the filament axis, and another which corresponds to the pitch of the "genetic" helix (Fig. 1). Worthington reported that according to his measurements the ratio of these two periodicities is 0·467.

THE STRUCTURE OF ACTIN

53

No information about the pitch of the helix can be derived from the diffraction patterns as yet obtained from preparations of F-actin (Astbury & Spark, 1947; Astbury, 1949; Cohen & Hanson, 1956) because the reflections at the smaller angles are much more diffuse than in the patterns from whole muscle. Therefore measurements similar to those made by Worthington (1959) cannot be attempted at present. (i)

(ii)

FIG. 2. Two possible alternative arrangements of the globular subunits in an actin filament. The subunits are drawn as spheres of diameter 55 A. The centres of the subunits are helically arranged on the circumference of a cylinder. The positions of these centres in planar projections of the helical structure are shown in Fig. 1. There are two twisted strands of subunits; the course of these strands is shown by the dotted lines in Fig. 1. In Fig. 2 model (i) the pitch of the helix is 2 x 351 A; this is twice the distance between the two arrows as measured along the filament axis; the arrows indicate the cross-over points of the two strands. There are 13 subunits per tUTIl ofthe helix. In model (ii) there are 15 subunits per tUTIl ofthe helix, the pitch of which is 2 x 406 A. Model (i) is consistent with the results obtained by examining isolated filaments in the microscope, model (ii) is consistent with the results obtained by Worthington (1959) who studied the X-ray diffraction pattern of intact dried muscle.

The results obtained by electron microscopy show that in negatively-stained preparations of F-actin and of the actin filaments isolated from muscle, the number of subunits per turn of each strand is 13, or very nearly 13, and not 15. It is not known if the helix is integral. This conclusion follows not only from counting the subunits, but also from considering measurements of periodicities in the filaments. Thus: (i) the axial separation of the positions where the two strands cross over one another was measured in filaments lying on the supporting film. In such filaments the spacing of the subunits along the strand was always about 55 A, as in the intact muscle, and had not been changed by any shrinkage ofthe filament during preparation for microscopy. The pitch of the helix was found to be 2 x 349 A (range 2 x 330 to 2 x 363 A). This value is essentially the same as that deduced for the 13-centre model from diffraction results, namely 2 x 351 A (Selby & Bear, 1956). None of the measurements made in the microscope approached 2 x 406 A, the value deduced for the 15-centre model;

54

J. HANSON AND J. LOWY

(ii) the results obtained by measuring periodicities in filaments situated in stainfilled holes are variable, but it can be shown that they are consistent with our finding that in the same filaments the number of subunits that can be counted per turn of the helix is 13, or very nearly 13. Considering separately the measurements made on each particular region of a filament, it was found that values closer to 13 than to 15 were obtained when the value for the length of the strand along one turn of the helix (calculated from measurements of the pitch and diameter of the helix) was divided by the measurement of the separation of subunits along the strand. Worthington's (1959) conclusion that in intact dried muscle the number of centres per turn of the helix is more likely to be 15 than 13 is based on certain measurements which are difficult to make accurately enough to decide this point. If these measurements are confirmed, it will have to be concluded that in the process of isolating the filaments from muscle and preparing them for electron microscopy, the degree of twist of the two strands of subunits has altered. It should be emphasized that a very small change in the relative positions of subunits could lead to a large change in the pitch of the helix. One of the reasons why it is important to know the precise structure of actin as it occurs in the intact muscle is that contraction is due to interaction between specific sites on adjacent actin and myosin filaments. In the filament lattice of the A-bands of vertebrate skeletal muscle, each actin filament is "shared" by three myosin filaments (Huxley, 1953a,b), and as Huxley (1957) pointed out, the arrangement of projections on the myosin filaments is such that a 15-centre model for the unit cell of actin might allow all the projections on the three myosin filaments simultaneously to lie opposite one-fifth of the centres on the adjacent part of the actin filament. The 13-centre model does not, of course, allow any such "fit". But neither the 15centre nor the 13-centre model would seem to "fit" in the case of certain other kinds of muscle, where each actin filament is "shared" by only two myosin filaments (e.g, insect flight muscle-Huxley & Hanson, 1957a; oyster adductor muscleHanson & Lowy, 1961). Moreover, it has recently been found that the axial periodicity in the myosin filaments of vertebrate skeletal muscle measures 435 A (Elliott, 1960; Worthington, 1959, 1961); this is considerably more than the length of the unit cell in the 15-centre model for the actin filament. (b) Identification of globular subunits as monomers

F-actin is a polymer formed from units of molecular weight about 60,000 to 70,000. The subunits observed in F-actin filaments in the microscope are approximately spherical and about 55 A in diameter. A spherical molecule of this diameter and of density 1·3 would have a molecular weight of 68,400. There is also other evidence that the globular subunits can be identified as monomers . Huxley (1960) calculated from structural data that the quantity of actin in rabbit skeletal muscle will be either 16·5 or 20·25% of the total protein if the molecular weight of the unit corresponding to each scattering centre in the actin structure (as described by Selby & Bear, 1956) is taken to be either 57,000 or 70,000 respectively, these being two extreme values chosen from the range of published values for the molecular weight of monomeric G-actin. Huxley (1960) pointed out that his estimates are reasonably close to biochemical estimates for the quantity of actin in rabbit skeletal muscle. However, his calculation was made on the basis that the whole of the muscle is occupied by fibrils (a postulate he made deliberately for purposes not

THE STRUCTURE OF ACTIN

55

relevant to the present discussion). If one assumes that the fibrils in rabbit skeletal muscle account for 80% of the total volume of the muscle, the amount of actin as estimated from structural data (see Table 1) is either 13'3% (if the molecular weight ofG-actin is 57,000--Mommaerts, 1951) or 16'3% (if the molecular weight is 70,000Tsao, 1953). These values are in excellent agreement with all the biochemical estimates, namely 13 to 15% (Hasselbach & Schneider, 1951; Perry, 1952; Szentkiralyi, 1961). If two globular subunits represented one monomer (or if one subunitrepresented a dimer) the biochemical estimates would be too high (or too low) by a factor of two. This is unlikely to be the case, considering that all three biochemical estimates, though made independently and by different methods, are in close agreement. TABLE

1

Oomposition of sarcomere in rabbit skeletal muscle Protein

Absolute quantitymg/ml, of living fibril

Relative quantity% of total fibrillar structural protein

Actin

38·2

Tropomyosin B Myosin

27·3 98·4

21} 36 15 54

Unknown

18·2

10

Location Filaments of I-substance I-substance Thick filaments of A-band Z-, M-, N-lines and other stroma

182·1 This Table is based on an estimate of the absolute quantity of actin in the living fibril of the psoas muscle, calculated from the following data. As Huxley (1960) has shown, the number of actin filaments is 101S/ml. The length of one filament is taken to be 1 p.. The number of subunits in a filament of this length is 370. The molecular weight of each subunit is taken to be 62,000, a value which is consistent with recent determinations of the molecular weight of G-actin by Kay (1960) and by Ulbrecht, Grubhofer, Jaisle & Walter (1960). From the estimate of the absolute quantity of actin we have calculated the percentage of actin in the total fibrillar structural protein, on the following assumptions: (i) that 80% of the muscle volume is occupied by fibrils; (ii) that 70% of the total muscle protein is fibrillar structural protein (because myosin accounts for 54% of the fibrillar structural protein-Hanson & Huxley, 1957and for 38% of the total muscle protein-Hasselbach & Schneider, 1951). The total quantity of protein in whole muscle is taken to be 20% of the weight of the muscle. From the estimate of the percentage of actin, the relative and absolute quantities of the other constituents of the fibril are calculated, using information about relative quantities based on interference microscope measurements (Hanson & Huxley, 1957; Huxley & Hanson, 1957b). It is assumed, on good evidence (see p. 57), that all of the tropomyosin B is located in the I-substance. It is also assumed that tropomyosin B and actin account for the whole of the I-substance; this is consistent with Perry's estimate that tropomyosin B represents 10 to 15% of the total fibrillar protein (Perry, 1960).

Selby & Bear (1956) also considered the possibility that each scattering centre in the crystallographic structure represents a single "relatively globular" G-actin monomer, but they were unable to decide between this model and another one which took into account Tsao's estimate that the molecules of G-actin in solution have an axial ratio of about 12 (Tsao, 1953). In the model which is consistent with Tsao's estimate, four long monomers lie side by side in each of the two "rods", the axes of which are marked by dotted lines in Fig. 1, and the scattering centres are special regions within monomers.

56

J. HANSON AND J. LOWY

Electron micrographs of negatively-stained preparations show that each of the two strands in the filament consists of globular subunits which are very conspicuous and look alike, and do not appear to be local swellings along a continuous strand. There is no indication in these micrographs that each strand consists of several long molecules lying side by side. Moreover, certain preparations of actin, examined at a stage intermediate between G·actin and F-actin, show globular subunits which appear to lie in random positions with respect to one another (Hanson & Lowy, unpublished results), and according to recent studies on the shape of G-actin monomers in solution, the axial ratio could be unity (D. R. Kominz, personal communication). Rozsa et al. (1949), who studied shadowed preparations of Fvactin in the electron microscope, concluded that the filament is built of a series of "ellipsoidal rodlets" about 300 A long. They also isolated filaments of similar appearance from muscle. The rodlets were too large to be monomers, and the suggestion was made that each might be a unit formed at an early stage in polymerization by a group of G·actin molecules. A comparison of their electron micrographs with ours indicates that the rodlet corresponds to the portion of the filament situated between two successive cross-over points of the two twisted strands of subunits.

(c) The diameter of the filament

In sections of muscle the diameter of the actin filaments commonly appears to be about 50 A (Huxley, 1957), though recently Knappeis & Carlsen (1962) have observed a diameter of about 70 A in a certain type of preparation. The weight of actin in each filament (Table 1) is such that, assuming a density of 1·3, the filament would have a diameter of 60 to 65 A (depending on the molecular weight of the monomer) if it were a solid cylinder. If it were not a solid cylinder the diameter would be more than 60 A. It is clear, therefore, that a filament consisting of two twisted strands of globular subunits must have an over-all diameter (i.e. the total width of the two strands) which is considerably greater than the 50 A suggested by most electron micrographs of sectioned material. The total width of the two strands measures about 80 A in electron micrographs of negatively-stained preparations. But this result may not be reliable because the filaments may have flattened on the supporting film and may have become thinner on drying (though we have shown-i-p. 53-that they do not shrink significantly in the longitudinal direction). Figure 3 represents a model of the filament which is consistent with X-ray diffraction data: the spacing of scattering centres along the filament axis is 27 A, and the pitch of the "genetic" helix (continuous lines in Fig. 1) is 59 A. It is assumed in this model that the subunits are spheres of diameter 55 A, and that the scattering centres which give rise to the diffraction pattern lie at the centres of the spheres. The over-all diameter of this model is 98 A, i.e. the diameter of the cylindrical shell drawn through the centres of the spherical subunits (43 A-a value which is consistent with diffraction results) plus the radii of two subunits. The two strands of subunits in this model are a certain distance apart. (There would of course be cross-connections between the two strands.) Therefore one can consider another model which is also consistent with the diffraction data, but in which more material lies inside the cylindrical shell drawn through the scattering centres than lies outside this shell; consequently the two strands are nearer together than in the previous model. Such a filament would have an outside diameter of less than 98 A, and this could be reduced further if the density of Fvactin were greater than 1·3 (the value so far assumed), e.g. 1·4 as

THE STRUCTURE OF ACTIN

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found for G-actin (Kay, 1960). The over-all diameter of the filament could even approach 50 A if the monomers were very asymmetrical, fitting together to form a solid filament. But, as we have already pointed out, this is inconsistent with the appearance of the filaments in the microscope.

'-r--------'

C

FIG. 3. A model of an actin filament drawn as a planar projection of the helical structure (see legend to Fig. I). The model is consistent with X-ray diffraction data (Selby & Bear, 1956). The subunits are assumed to be spheres, of diameter 55 A, whose centres coincide with the scattering centres of the crystallographic structure. C is the circumference of the cylinder drawn through the scattering centres; the diameter of this cylinder is 43 A.

From all these considerations we conclude that the measurement of over-all diameter made on isolated filaments (80 A) is more reliable than that made on most sectioned material (50 A). An equatorial reflection at 55 A has been observed in X-ray diffraction patterns of dried F-actin preparations (Cohen & Hanson, 1956). The significance of this observation is at present not clear, because one does not know how the filaments are packed and whether or not this reflection is the first order of a series. (d) The location of tropomyosin B

There is good evidence that tropomyosin B is closely associated with actin in the fibrils of rabbit skeletal muscle and is situated in the I-substance (Perry & Corsi, 1958; Corsi & Perry, 1958; Huxley, 1960). All the filaments seen in sections of the I-substance (Huxley, 1953b, 1957), or deduced from X-ray diffraction data to be present in the I-substance of living unstimulated muscle (Elliott, Lowy & Worthington, 1962, manuscript in preparation) or glycerol-extracted muscle (Huxley, 1953a), must contain actin, because the quantity of actin as estimated biochemically (p. 55) is such that if the diameter of the filaments is not much more than about 80 A each of them must contain actin. Indeed, all the filaments we have observed in the I-bands of negatively stained glycerol-extracted rabbit psoas fibrils show actin structure. Therefore, one has to consider the possibility that tropomyosin B may be present in the same filaments as actin, perhaps, as Huxley (1960) suggested, forming a

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"backbone" which could maintain the integrity of the filament if the monomers at any time became detached from one another. However, in negatively-stained preparations, the filaments isolated from all the muscles so far examined, including rabbit psoas, appear to be identical to the filaments in F-actin preparations. (The quantity of tropomyosin contaminating our actin preparations, which had been centrifuged, is probably very small.) In order to assess the significance of this finding, it is necessary to consider how much tropomyosin might be present together with actin in the muscle filaments. The quantity of tropomyosin B in the fibril of rabbit skeletal muscle, as shown in Table 1, is about 27 mgjml. If all this tropomyosin were in the actin-containing filaments, each of them (Ill- long) would contain about 27 x 10-15 mg (there are 1015 filaments per ml. of fibril-Huxley, 1960). Let us suppose that the tropomyosin is situated inside the filament as a core around which are wound the two strands of actin monomers. Such a core, if a solid cylinder of density 1·3, would have a diameter of about 50 A. No such core has been observed. Moreover, if a core of this size were present in the actin-containing filaments isolated from muscle, it would be difficult to explain why these filaments do not differ from those in F-actin preparations either in diameter or in the arrangement of subunits. If a tropomyosin core had been removed during the preparation of isolated muscle filaments, then it would be difficult to explain why the arrangement of subunits, as seen in the electron microscope, corresponds closely to that of the scattering centres in the crystallographic structure (Selby & Bear, 1956) which is based on diffraction patterns obtained from intact dried muscle where, presumably, the tropomyosin core would be present. There is another model of a composite actin-tropomyosin filament which is not open to these objections. In this model there are two strands of tropomyosin which follow the actin helix and lie on the outside of the actin filament in the two grooves between the strands of actin monomers (Fig. 2). Such tropomyosin strands might easily be removed while preparing material for the microscope. There is no evidence available at present either for or against this model. It may be noted that in vertebrate skeletal muscle each I-band filament branches into four strands at the Z-line (Knappeis & Carlsen, 1962). This observation could be accounted for by the model just described, in which the diameter of each of the two tropomyosin strands is not much less than that of a single actin strand. (e) The 400 A axial periodicity

The I-substance of vertebrate skeletal myofibrils examined in the electron microscope shows a fine transverse striation apparently due to material concentrated at intervals of approximately 400A (review by Huxley, 1960; Carlsen et al., 1961); also, the axial diffraction pattern of intact vertebrate skeletal muscle includes a reflection at about 400 A (Huxley, 1953a; Worthington, 1959). Neither of these findings is accounted for by the structure of actin itself. The X-ray reflection, as seen in glycerol-extracted material (Worthington, 1959), lies on the meridian and therefore does not relate to the pitch of the actin helix. The subunits seen in the microscope all look alike. If they are monomers, as is very probable (see p. 54, section (b», then there seems to be no reason why particular subunits, spaced at intervals of about 400 A, should differ from other subunits by, for instance, possessing special chemical groups to which other material could be attached.

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59

Similarly, it is difficult to explain the localization of inorganic material at intervals of about 400 A in the I-bands of incinerated fibrils examined in the microscope (Draper & Hodge, 1950). Huxley (1960) has suggested that the striation observed in the fibrils which have not been incinerated might be accounted for if the cross-over points in all the filaments in the I-substance were to lie in transverse register. But it seems more likely that this striation has the same origin as that seen in incinerated material. Whether or not the meridional X-ray reflection also has the same origin cannot be decided. The results obtained by Worthington (1959) suggest that this reflection does not arise from the myosin filaments. If this is so, then both this reflection and the periodicity seen in the microscope might be due to a combination of actin with other material having a repeat of approximately 400 A, conceivably tropomyosin B. In any event, the structure of actin alone is unlikely to be responsible. We are very grateful to Dr. H. E. Huxley for introducing us to techniques of preparing negatively-stained isolated filaments, and to Dr. Huxley, Dr. M. Spencer and Dr. M. H. F. Wilkins for helpful discussion and useful suggestions. We thank Professor Sir John Randall for encouraging this research. REFERENCES Astbury, W. T. (1949). Exp. Cell Res. Suppl. I, 234. Astbury, W. T. & Spark, L. C. (1947). Biochim. biophys. Acta, 1, 388. Bear, R. S. (1945). J. Amer. Chern, Soc. 67, 1625. Carlsen, F., Knappeis, G. G. & Buchthal, F. (1961). J. Biophys. Biochem, Cytol. 11,95. Cohen, C. & Hanson, J. (1956). Biochim. biophys. Acta, 21, 177. Corsi, A. & Perry, S. V. (1958). Biochem. J. 68, 12. Draper, M. H. & Hodge, A. J. (1949). Aust. J. Exp. Biol. Med. Sci. 27, 465. Draper, M. H. & Hodge, A. J. (1950). Aust. J. Exp. Biol. Moo. Sci. 28, 549. Elliott, G. F. (1960). Ph. D. Thesis. University of London. Elliott, G. F. & Lowy, J. (1961). J. Mol. Biol. 3, 41. Hanson, J. & Huxley, H. E. (1953). Nature, 172, 530. Hanson, J. & Huxley, H. E. (1955). Symp. Soc. Exp. Biol. 9, 228. Hanson, J. & Huxley, H. E. (1957). Biochim. biophys. Acta, 23, 250. Hanson, J. & Lowy, J. (1960). In Structure and Function of Muscle, ed. by G. H. Bourne, vol. I, p. 265. New York: Academic Press. Hanson, J. & Lowy, J. (1961). Proc, Roy. Soc. B, 154, 173. Hanson, J. & Lowy, J. (1962). Proc, 5th Int. Congress for Electron Microscopy, vol. 2, abstract 09. New York: Academic Press. Hasselbach, W. & Schneider, G. (1951). Biochem; Z. 321, 461. Huxley, H. E. (1953a). Proc, Roy. Soc. B, 141, 59. Huxley, H. E. (1953b). Biochim. biophys. Acta, 12, 387. Huxley, H. E. (1957). J. Biophys. Biochem, Cytol. 3, 631. Huxley, H. E. (1960). In The Cell, ed. by J. Brachet & A. E. Mirsky, vol. 4, p. 365. New York: Academic Press. Huxley, H. E. (1961). Circulation, 24, 328. Huxley, H. E. & Hanson, J. (1957a). Electron Microscopy, p. 202. Stockholm: Almqvist & Wiksell. Huxley, H. E. & Hanson, J. (1957b). Biochim. biophys. Acta, 23, 229. Huxley, H. E. & Zubay, G. (1960). J. Mol. Biol. 2, 10. Kay, C. M. (1960). Biochim. biophys. Acta, 43, 259. Knappeis, G. G. & Carlsen, F. (1962). J. Cell Biol. 13, 323. Lowy, J. & Hanson, J. (1962). Physiol. Rev. 42, Suppl. 5, p. 34. Mommaerts, W. F. H. M. (1951). J. Biol. Chem. 188, 559. Perry, S. V. (1952). Bioehem, J. 51, 495. Perry, S. V. (1960). Annu. Rep. Chem. Soc. 56, 343.

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Perry, S. V. & Corsi, A. (1958) . Biochem. J. 68, 5. Rozsa, G., Szent-Gyorgyi, A. & Wyckoff, R. W. G. (1949) . Biochim. biophy8. Acta, 3, 561. Selby, C. C. & Bear, R. S. (1956) . J. Biophy8. Biochem. Oytol. 2, 71. Spencer, M. (1959). J. Biophqe. Biochem, Oytol. 6, 125. Szent-Gyorgyi, A. G. (1951). J. Biol. Chem., 192, 361. Szentkiralyi, E. M. (1961). Exp. o-u ReB. 22, 18. Tsao, T·C. (1953). Biochim. biophys. Acta, 11, 227. Ulbrecht, M., Grubhofer, N., -Iaisle, F. & Walter, S. (1960). B iochim. biophys. Acta, 45,443. Watanabe, S. & Sleator, W. (1957) . Arch. Biochem, Biophst« , 68 , 81. Worthington, C. R. (1959). J . Mol. Biol. 1, 398. Worthington, C. R. (1961). J. Mol. Biol. 3, 618.