The cytoskeleton of retinal pigment epithelial cells

CHAPTER 2

The Cytoskeleton of Retinal Pigment Epithelial Cells KATSUSHI OWARIBE

Department of Molecular Biology, School of Science, Nagoya University, Nagoya 464, Japan CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Organization of Microfilaments and Associated Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Two Types of Microfilament Bundles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Circumferential Microfilament Bundles (CMBs) and Zonula Adhaerens . . . . . . . . . . . . . . . . . . 2.3. Paracrystalline Microfilament Bundles (PMBs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Intermediate Filaments and Desmosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The Intermediate Filament (IF) System in RPE Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Desmosomes in RPE Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 3.3. Intermediate Filaments and Desmosomes in Cultured RPE Cells . . . . . . . . . . . . . . . . . . . . . . . .

35 35 36 38

4. Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. I N T R O D U C T I O N

The retinal pigment epithelium (RPE) is a onecell layer, ("simple") cuboidal epithelium located between the neural retina and the choroid of the eye. The RPE plays several major roles in eye physiology such as phagocytosis of outer segments of photoreceptor cells, absorption of light and trans-epithelial transport of nutrients (e.g. Burnside and Nagle, 1983; Young and Bok, 1969; Zinn and Marmor, 1979). The RPE cells display a pronounced polarity, and their cytoplasmic architecture and differentiation, including membrane specializations and organelle distribution, is closely related to their function (Nguyen-Legros, 1978). RPE cell polarity and the regional specializations of the cytoplasm involve the disposition of its filamentous cytoskeletal elements, i.e. microfilaments, intermediate filaments and microtubules which together represent a major portion of the RPE protein mass. Conversely, the cytoskeleton appears to be

Epithelial cells generally form single- or multilayered cell systems (epithelia) covering the surface of the body and lining the lumina of the internal organs. BasaIly, an extracellular matrix " l a m i n a " (basal lamina, basal membrane) is sandwiched between any particular epithelium and the underlying mesenchymally derived tissue. There are certain exceptions which do not fulfill these morphological criteria but show a similar pattern of gene expression and internal cell architecture. These are believed to be derived from true epithelia, with perhaps the most prominent example being the epithelial reticulum of the thymus. In addition to their function as protective layers, epithelia can serve a variety of other functions such as secretion, absorption, molecular transport and sensory reception. 23

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closely related to the function of the epithelium. The cytoskeleton of the RPE cells includes the junctional complexes which also show polarity of distribution. It is very convenient for studying the cytoskeleton of RPE cells that they have a very stable and regularly polygonal morphology and that, when grown in culture, they form an epithelium like monolayer similar to that in situ. In this article, I shall describe the cytoskeleton of avian and mammalian RPE cells, with focus on the molecular organization of major cytoskeletal filament systems.

2. O R G A N I Z A T I O N OF M I C R O F I L A M E N T S AND ASSOCIATED PROTEINS 2.1. Two Types of Microfilament Bundles

As in other eukaryotic cells, the actincontaining microfilaments are major components of the cytoskeleton and the contractile apparatus of the RPE cells. In avian RPE cells, two types of microfilament bundles can be distinguished which differ structurally and functionally. One is the circumferential microfilament bundle (CMB) associated with the zonula adhaerens region, i.e. a circular, plaque-bearing, subapical junctional belt typical of many polar epithelial cells (Farquhar and Palade, 1963). The filaments in this bundle, which are relatively loosely packed and display random polarity, contain actin, myosin and other actin-associated proteins (Drenckhahn and Wagner, 1985; Gordon and Essner, 1987; Turksen and Kalnins, 1987; Turksen et al., 1983). They have been shown to be contractile (Owaribe et al., 1981; Owaribe and Masuda, 1982). Similar CMBs have been described in other polar epithelial cells and seem to be important for the maintenance of the polarized epithelial structure in general (cf. Crawford et al., 1972; Farquhar and Palade, 1963; Hull and Staehelin, 1979; Owaribe et al., 1979). The second microfilament system comprises the paracrystalline microfilament bundles (PMBs) in which the filaments show the same polarity (Owaribe and Eguchi, 1985). PMBs appear in a later stage of development and are found in apical projections. They seem to

F~G. 1. Actin immunofluorescence of RPE cells from an l 1-day-old embryo. (a) Apical view. Note that bright bands are intersected by dark lines showing cell boundaries (b) Basal view. Bar, 10 ~ m

contribute to intercellular interaction and pigment migration (Burnside et al., 1983; Murray and Dubin, 1975; Snyder and Zadunaisky, 1976). In mammalian RPE cells CMBs but not paracrystalline bundles have been described.

2.2. Circumferential

Microfilament and Zonula Adhaerens

Bundles ( C M B s )

2.2.1. ISOLATION AND CONTRACTION OF C M B S

CMBs can be best demonstrated in avian and mammalian RPE cells by immunofluorescence microscopy. Figure 1 shows the RPE cells from an l 1-day-old chick embryo stained with actin antibodies in a top view of the cells in which the CMBs appear as broad polygons, in basal view, only a few thin actin bundles are found in several

RETINAL

PIGMENT

EPITHEL|AL

25

CELLS

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~ i' ~

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. . . . . . . .

~~

~

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FIG. 2. Isolation of CMBs from chicken RPE cells by homogenization. Phase-contrast micrographs of RPE cells before (a) and after (b) homogenization. Arrows show isolated typical CMBs. Note that these CMBs and the RPE cells have almost the same dimension. Bar, 20/am. cells in the epithelium. They are located at the cell periphery, and the cells tightly adhere at this level. Whole CMBs have been isolated from chicken RPE cells and the contractility of the isolated CMBs has been shown directly (Owaribe and Masuda, 1982). For this the pigment epithelia obtained from l 1-day-old chick embryos are treated with a 50% glycerol sotution containing 0.1 M KC1, 5 mM E D T A and 10 mM sodium phosphate buffer (pH 7.2) for 24 hr or more at 4°C. The glycerinated epithelia are then incubated in a " T r i t o n solution" (1% Triton X-100, 0.1 M KC1, 10 mM sodium phosphate buffer, p H 7.2) for l0 min at 0°C and cells are lysed by passing through a syringe needle (Fig. 2). In the resulting homogenate, CMBs and other cell components such as nuclei and pigment granules can be seen with a phase contrast microscope. The isolated CMBs and the RPE cells have nearly the same diameter. Contractility of isolated CMBs is demonstrated after perfusion with a MgE+-ATP solution under observation with a phase contrast microscope. The photographs shown in Fig. 3(a - d) have been taken successively during perfusion of Mg 2~-ATP solution. The CMB seen in these photographs on the left hand shows fast and extensive shortening

!

d"

"''

_.-

FIG. 3. Contraction of isolated CMBs. Phase-contrast mlcrographs were taken successivelyat 0 (a), 2 (b), 5 (c) and 10 min (d) after perfusion of Mg-ATP. Each CMB shows clear contraction. The contractile profile of the left one has been analyzed in Fig. 4. Bar, 10/am. but the overall polygonal shape is maintained. When the total length of CMBs is measured and plotted against the time of perfusion of Mg 2+A T P solution (Fig. 4a) the final length is about 40% of the original length, and the shortening rate during the first 2 min is about 8 / a m / m i n (at room temperature). Fragments of CMBs also show shortening in Mg2+-ATP solution. The rate and the extent of CMB shortening varies somewhat from specimen to specimen, depending on the interaction of the bundles with the substrate, usually a coverslip or a slide.

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A b

50

21.

~3o ._1

10 I t , , , i , , , , , , 0

5 Time(min)

10

FtG. 4. C h a r a c t e r i z a t i o n of c o n t r a c t i o n of the CMB. (a) M e a s u r e m e n t o f the rate a n d degree of s h o r t e n i n g of the C M B (the left o n e in Fig. 3). The t o t a l length o f the C M B d u r i n g the s h o r t e n i n g was traced a n d m e a s u r e d . The initial rate of the s h o r t e n i n g was a b o u t 1 0 / a m / m i n , a n d the s h o r t e n i n g degree was 60°70. (b) Effect of N E M SI on s h o r t e n i n g o f a C M B . No s h o r t e n i n g was observed.

N-et hylmaleimide-modified myosin subfragment 1 (NEM-SI) is a specific inhibitor of actin-myosin interaction (Cande, 1980). Incubation of the CMBs with 3 mg/ml NEM-S1 for 5 min at 0°C completely inhibits the shortening in the Mg2*-ATP solution (Fig. 4b), suggesting that the shortening of CMBs is due to actin-myosin interaction. The contraction is Ca2+-independent even in the Triton model of the sheets (Masuda et al., 1984).

2.2.2.

CONTRACTION OF C M B S IN THE CELLS AND ITS MORPHOLOGICAL SIGNIFICANCE

Chicken RPE cells grown in culture form an epithelia-like monolayer similar to that formed in situ. Well-differentiated cultured RPE cells reveal thick CMBs in their subapical region (Fig. 5) which probably are necessary to maintain the epithelial organization as destruction of the bundles by cytochalasin B causes breakdown of the epithelial structure (Crawford et al., 1972; Owaribe et al., 1979). When CMBs contract in such RPE cell layers, morphological changes are observed (Owaribe et al., 198!). For example, when well-differentiated pigment epithelial cell monolayers grown in culture are treated with glycerol as described above and then transferred to Mg-ATP solution at room temperature the CMBs contract immediately. As a result of such induced CMB

FIG. 5. i m m u n o f l u o r e s c e n c e m i c r o s c o p y o t cultured chicken R P E cells stained with actin a n t i b o d y (a) and with n o n i m m u n e l g G (b). The cells were treated with T r i t o n before staining. C M B s a p p e a r as b r i g h t p o l y g o n s at the a p i c a l region o f cell periphery. Bar, 10/am.

contractions the epithelial sheet is often cleaved into smaller colonies consisting of 2 0 - 3 0 cells, and the residual "colonies of cytoskeletons" change their shape to cup-like forms and finally detach from the substratum (Fig. 6). These shapes are much more readily seen by scanning electron microscopy (Fig. 7). Transmission electron microscopy of ultrathin sections through such contracted cytoskeletons show that the condensed filamentous CMB material is retained in the subapical region (Fig. 8). These observations suggest that certain shape changes in the living epithelium in situ are induced by contraction of the subapical CMB, for example

RETINAL PIGMENT EPITHELIAL CELLS

27

la FIG. 7. Scanning electron micrograph of contracted pieces of the sheet in the ATP solution. Bar, 20/~m.

C

Fie. 6. Phase-contrast micrographs showing the contraction of glycerinated RPE sheets. Photographs were taken successively at 0.25 (a), 2.5 (b) and 10 min (c) after transfer to the ATP solution. Bar, 50/an.

FIG. 8. Thin-sectin electron microscopy showing the apical region of the contracted RPE cells in the ATP solution. Bar, 1 lain.

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changes from a cuboidal to a more cone-like cell shape. Thus, the CMBs of the RPE cells may function in both ways, as a cytoskeletal part of the junctional complex maintaining the polar epithelial organization, and as a contractile system responsible for physiologically relevant changes of cell shapes in the epithelium. Crawford (1979, 1980) has reported focal contractions in monolayer cultures of chicken RPE cells during differentiation. Honda and Eguchi (1980) have suggested, from computer simulations, that the RPE in vivo can attain a stable regular hexagonal pattern by shortening the lateral wall length without a change in the surface area per cell (for applications to other polar epithelial cells see Honda, 1983). Therefore, CMBs might participate, as stable, locally fixed contractile machinery, in the formation of a stable cellular pattern in the RPE and in the maintenance of the formed pattern. Moreover, in a more general way, the formation of cup-like shapes of RPE cell colonies as a result of contraction of CMBs can be regarded as an example of three-dimensional morphogenesis from a one-layered epithelial cell sheet. The formation of tubular and cup-like tissue structures from epithelia is an essential process of organogenesis during normal development (Baker and Schroeder, 1967; Wessells et al., 1971).

Fl6. 9. SDS-PAGE patterns of RPE cells treated with glycerol and Triton (a) and the purified CMB fraction (b,c). Molecular weight standards; M (myosin heavy chain, 200 kDa), phosphrylase a 0 4 kDa), boring serum albumin (68 kDa), A (actin, 42 kDa).

2.2.3. STRUCTURAL COMPONENTS OF C M B s

When isolated CMB fractions from avian RPE. cells are purified by successive Percoll density gradient centrifugations and analyzed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) the resulting polypeptide pattern shows three major and several minor bands (Fig. 9). Molecular weights of the major components were estimated to be ~ 200, 55 and 42 kDa. By coelectrophoresis with authentic muscle proteins, the 200 and 42 kDa polypeptides have been identified as myosin heavy chain and actin, respectively, and the 55 kDa protein is the subunit of the intermediate filaments (IFs) detected by electron microscopy in association with these CMB fractions, i.e. vimentin (see below and Owaribe et al. , 1986).

In addition, several microfilament-associated proteins such as myosin, tropomyosin, ~-actinin and vinculin have been identified in CMBs by immunofluorescence microscopy (Drenckhahn and Wagner, 1985; Gordon and Essner, 1987; Opas and Kalnins, 1985; Turksen and Kalnins, 1987). These results show that the composition of CMBs of RPE cells is essentially similar to that of intestinal epithelial cells (e.g. Bretscher and Weber, 1978; Craig and Pardo, 1979; Geiger et al., 1981; Hirokawa et al., 1983; for review see Mooseker, 1985). Figure 10 shows the immunolocalization of two major components of the z o n u l a a d h a e r e n s plaque involved in CMB attachment, i.e. vinculin (in bovine RPE cells) and plakoglobin (in rat RPE cells and cultured chicken RPE cells). These proteins will be discussed in

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29

greater detail below. The localization pattern shows, in corresponding focal plaques including the subapical region, that both proteins are located along the cell peripheries but that the resulting fluorescent polygons are much thinner than those obtained with actin antibodies (compare Figs. la and 5a) and a-actinin (see Fig. 27d), which is in agreement with the notion that vinculin and plakoglobin are located in the plaque between the CMB and the junction membrane (see also Tokuyasu et al., 1981; and Cowin et al., 1986).

2.2.4. MORPHOLOGYOF CMBS

b

Most of the isolated CMBs appear as pentagons or hexagons or fragments thereof (Fig. l l a - d ) . In electron micrographs of low magnification, isolated CMBs appear as peripheral filament bundles integrated into a meshwork of more internal filaments (Fig. l le). It is not clear whether the internal meshwork filaments are directly connected with the peripheral CMB. At higher magnification, the CMB filaments are resolved as actin microfilaments of a diameter of about 6 nm (Fig. 12) whereas the filaments of the internal meshwork are found to be primarily, but not exclusively, IFs of a diameter of about 10 nm (Fig. 13). However, there are also some microfilaments detected within the internal meshwork. Overall view of the apical cytoskeleton of RPE ceils is well observed by the quick-freezeetching method (Fig. 14). Microfilaments in CMBs run parallel to the cell membrane, and some microfilaments are anchored to the membrane. At the inner periphery of CMBs, microfilaments are intermingled with IFs. Perhaps, some interaction between microfilaments and IFs may stabilize the whole structure.

2.3. Paracrystalline Microfilament Bundles (PMBs) 2.3.1. STRUCTURAL ORGANIZATION OF P M B S FIG. 10. Immunofluorescence microscopy showing localization of vinculin and plakoglobin in RPE cells. (a) Bovine cells in situ stained with monoclonal antibody to vinculin. (b) Rat cells in situ stained with monoclonal antibody to plakoglobin. (c) Cultured chicken cells stained as b. Bars, i0/am.

The organization of the filaments in avian PMBs can best be shown in isolated bundles. For this, RPE cells from newly hatched chicks are treated with glycerol and Triton buffer solution

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FIG. 11. Morphology of CMBs. (a-d). Phase-contrast micrographs of typical CMBs. Each CMB is contractile. Bar, 10 tam. (e). Low-magnificationelectron micrograph of negatively stained CMB showing thick bundles and central meshwork. Bar, 3 tam. (see above and Owaribe and Eguchi, 1985) and are ruptured by passing through syringe needles. After gentle rupture single cells are still seen (Fig. 15). Bundles, 2 0 - 3 0 / ~ m in length, are recognized with the specific length distribution depending on the shearing forces applied. By appropriate shearing, two types o f microfilament

bundles are released and found in the homogenate which are readily distinguished in the electron microscope. One type comprises the CMBs described above. The other type, which is more abundant, is characterized by more densely packed paracrystaUine-like bundles of microfilaments, i.e. the PMBs (Fig. 15,b,c). Figure 15b shows an example of a single PMB in which characteristic transverse striations are also resolved. In RPE cell homogenates from 15-dayold embryos, thin short bundles are observed but their paracrystalline structure is less apparent (see below). When PMBs are decorated with heavy meromyosin (HMM) and negatively stained, the bundles appear to taper into single filaments decorated with H M M and at the ends of the frayed out filaments the polarities of the actin filaments can be determined. In every decorated bundle, all actin filaments have the same polarity. Figure 15c presents an example. From the observation o f H M M decorations in whole cells or in cell lysates it appears that the typical HMM arrowheads all point toward the cell center. The appearance of the PMBs is not altered in the presence or absence of Mg2÷-ATP or Ca 2÷, indicating that they are not contractile themselves and also do not contain Ca2÷-dependent proteins which sever or bundle together actin filaments as described for villin in the core bundles of actin filaments in the brush borders of intestinal cells (Bretscher and Weber, 1979, 1980). PMBs have not been found in homogenates of RPE cells from cows, rabbits and guinea pigs. In these three species only fragments of CMBs are observed. However, it is not clear why mammalian RPE cells have a less ordered filament system that may be functionally different from the avian PMBs. The optical diffraction technique is particularly useful for analyzing periodic structures such as tightly packed bundles of actin filaments (e.g. DeRosier and Tilney, 1981; Klug and Berger, 1964). Figure 16 shows an optical diffraction pattern of a bundle similar to that shown in Fig. 15b. The first layer-line which occurs at about 1/36 nm- 1, and the sixth layer-line at about 1/5.9 nm -1, are typically present in diffraction patterns of actin bundles. Besides these layerlines, a meridional reflection has been observed at

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FXG. 12. Electron microagrphs of the bundle region of a CMB. In this CMB depleted of meshwork, the peripheral region of a thick bundle is loosened and microfilaments are well-recognized (a). Bar, I/~m. (b) High-magnification photograph of the peripheral region of the bundle bracketed in a. Each filament shows a beaded, double-stranded structure that is typical in actin filaments. Bar, 0.2 gm.

about 1/12 nm -1, suggestive of the presence of another component, probably cross-linking protein(s). Optical diffraction patterns of this image show ratios of the Z coordinates of layerlines of 1 : 2.92 : 6.05, indicating that the individual actin filaments in the bundle comprise 13 subunits in 6 turns of the genetic left-handed helix and that the average interval of the transverse striations is about 1/3 of the half-pitch of the actin double helix. From the row-lines in the diffraction pattern the spacing between actin filaments in the paracrystalline bundle is estimated to be about 8.8 nm. The optical diffraction pattern of the PMBs of chicken RPE cells suggests that they are composed of hexagonally packed actin filaments (DeRosier and Tilney, 1981). The transverse striations and filament spacing of the bundles are closely similar to that induced by fascin, a 58 kDa actin cross-

linking protein (Bryan and Kane, 1978; DeRosier et al., 1977; DeRosier and Tilney, 1981; Otto et al., 1980; Spudich and Amos, 1979). However,

SDS-PAGE analyses of chicken RPE (see Fig. 20A) cells show no distinct band corresponding to 58 kDa, and RPE-PMBs are stable in buffer solutions containing 0.25 M NaC1, conditions in which the fascin-containing bundles are disorganized (Edds, 1979). On the other hand, SDSPAGE of such fractions reveals several components of actin-accompanying polypeptides that could be candidates for cross-linker proteins (see Fig. 20A), especially one of polypeptides in 50-55 kDa range. As the highest degree of regular packing of actin filaments in PMBs is seen in the microvillus-like projections of the apical surface of earlier stages of chicken eye development (see below) the ultrastructure of these apical projections has been studied in intact and glycerinated RPE cells. Thin

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FIG. 13. Electron micrograph of meshwork region of a CMB. This region is abundant in intermediate filaments. Bar, 0.2 ~m.

section electron microscopy shows that intact RPE cells of a 1-day-old chicken have many long apical projections, about 30/am in length and 0.2/am in diameter (Fig. 17a,b), which extend into the extracellular space between the outer and inner segments of photoreceptor cells, often protruding as far as the outer limiting membrane. Pigment granules often found in these projections are closely associated with microfilaments. During glycerination of RPE cells of this stage the apical projections of many cells fused to produce a large, cone-like protrusion containing many microfilament bundles (Fig. 18). These apical projections frequently tend to fuse to one another as the RPE are separated from the neural retina even without glycerol treatment. The RPE cells from cows, rabbits and guinea pigs, which have more flexibly appearing, fingerlike apical processes, do not contain such large amounts of actin, nor do they show PMBs, in agreement with reports that the apical projections of mammalian RPEs do not possess "core filaments" (Burnside and Laties, 1976). In apical

projections of frog RPE ceils, Murray and Dubin (1975) have shown the presence of tightly packed bundles of actin filaments by HMM decoration but these structures have not yet been characterized in greater detail.

2.3.2.

INCREASE IN ACTIN CONTENTS AND FORMATION OF P M B S DURING DEVELOPMENT OF CHICKEN R P E

When RPE cells from day 11 to day 21 embryos or newly hatched chickens are analyzed by SDSPAGE, it is obvious that the cytoskeletal composition changes during the development. Figure 19 shows the electrophoretic patterns of cytoskeletal components of RPE cells at various stages of development. The relative content of actin increases markedly in embryonic RPE ceils between day 15 and day 21. This observation is also supported by immunofluorescence and electron microscopy (see below). When the SDSP A G E polypeptide patterns are traced by densitometry, and the amount of each component measured (Fig. 20A), the relative amounts of actin

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FIG. 14. Cross cut of glycerinated RPE cells from an 1l-day-old embryo showing CMBs and IFs. The specimen was quick frozen with a liquid helium-cooled apparatus, freeze-fractured and rotary-shadowed. MF: microfilaments in CMBs, IF: intermediate filaments in meshwork, P: pigment granules. Photograph courtesy of S. Tsukita and S. Tsukita. Bar, 1 /am.

to myosin heavy chain (Fig. 20B) in RPE cells from 21-day-old embryos has increased more than four times, compared with that o f l 1-day-old embryos, and the corresponding value from l-month-old chicken is even 2 - 3 times higher than that of 21-day-old embryos. A rough estimation from the densitometry data revealed that the actin accounted for about 60°70 of the cytoskeletal protein in the RPE cells of both stages. When chicken RPE cells o f various stages of embryogenesis are stained for immunofluorescence microscopy with actin antibodies the process o f formation and elongation of the new actin bundles different from CMBs can be visualized (Fig. 21). In early stages (13 - 15 days old) many short actin

bundles are seen at the apical side of the cells; then they grow and appear to protrude into the extracellular space, obviously reflecting the formation of apical microvillus-like projections (Fig. 21a - e). During this increase and elongation the forming PMBs seem to become rigid. In early stages of elongation of the actin bundles, the CMBs are still well recognized in the cell peripheries. However, with increased numbers of the new bundles and their elongation, the CMBs are hard to resolve and seem to be displaced from the apical to a more basal position (Fig. 21f). The elongation of the PMBs apparently is closely correlated with the increase in total cytoskeletal actin contents observed by SDS-PAGE.

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FiG. 16. Electron micrograph of a negatively stained actin bundle (a) and its optical diffraction pattern (b). Bar, 0.2/~m.

FIG. 15. Electron micrographs of negatively stained actin bundles from apical projections. (a) Overall view of isolated cells. Long apical projections are clearly observed. The projections of each cell are probably fused during glycerination. Bar, 10/~m. (b) Isolated bundles of microfilaments by homogenization. Bar, 0.2/~m. (c) Bundles decorated with HMM. Arrows show the polarity of the arrowheads. Bar, 0.2 ~m.

As the myosin content does not increase concomitantly, the PMBs seem to contain little, if any, myosin. However, in this respect conflicting immunolocalization results have been reported: According to Turksen and Kalnins (1987) myosin, tropomyosin, a-actinin and vinculin are absent in the apical projections; Drenckhahn and Wagner (1985) have reported similar results. On the other

FIG. 17. Electron micrographs of the intact RPE cells from a 1-day-old chick. (a) Low-magnification photograph showing long apical projections extending along outer and inner segments of photoreceptor cells. N; nucleus. Bar, 5 /am. (b) High-magnification photograph of apical projections, in which the bundles of microfilaments is clearly seen. Bar, 0.2 t~m.

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a b e d

e

f

M-

55KA-

FIG. 18. Electron micrograph of glycerinated RPE cells from a 1-day-old chick. There are many actin bundles in the large cone, which is formed by fusion of apical projections. Bar, 5/am (a); 2.5/am (b); 1 /am(c).

hand, Philp and Nachmias (1985) have reported the presence of myosin, vinculin, a-actinin and fodrin in PMBs.

3. INTERMEDIATE F I L A M E N T S A N D DESMOSOMES 3.1. The Intermediate Filament (IF) System in RPE

Cells 3.1.1. IDENTIFICATION OF I F PROTEINS IN CHICKEN AND BOVINE R P E CELLS

In chicken RPE cells, only one type of IF protein is seen by SDS-PAGE and immunoblotting which can be enriched in the residual cytoskeleton after treatment with high salt buffer (Fig. 22 and 23d; cf. Owaribe et al., 1986). This agrees with results obtained by immunofluorescence microscopy (Docherty et al., 1984; Philp and Nachmias, 1985). Figure 22 shows the specificity of the

Fro. 19. SDS-PAGE pattern showing the cytoskeletal components of chicken RPE cells at various stages of development. The cells from 11 (a), 13 (b), 15 (c), 17 (d), 19 (e) and 21-day-old embryos (f). M: myosin heavy chain, A: actin.

vimentin antibody reaction with the approximately 55 kDa polypeptide and the cross-reaction with human vimentin which has a slightly lower SDSPAGE mobility (cf. Franke et al., 1982b). The IF system of bovine RPE cells has been also examined immunochemically and biochemically (Owaribe et al., manuscript in preparation). The SDS-PAGE pattern of the Triton X-100 treated cells ("Triton-cytoskeleton") shows two major bands corresponding to 53 and 43 kDa and a faint band at about 58 kDa in the region of potential IF proteins (~ 4 0 - 7 0 kDa, except for the neurofilament polypeptides N F - M and N F - H) (Fig. 23). Two-dimensional gel electrophoresis (Fig. 23f) and immunoblot analysis (Fig. 23b' , b " ) has shown that the ~ 53 kDa band is cytokeratin A and the 43 kDa band contains both residual actin and cytokeratin D (cf. Franke et al., 1981a,b). These two cytokeratin polypeptides correspond to human cytokeratins 8 and 18, respectively (Moll et al., 1982; Schiller et al., 1982).

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A

U

i, I M

charge considering immunoblot analysis (cf. Moll et al., 1982) (Fig. 23f). These results show that RPE cells can profoundly differ in different species: for example, chicken RPE cells have only vimentin IFs whereas bovine RPE cells contain a large amount of cytokeratin IFs and a smaller proportion of vimentin IFs.

3.1.2. INTRACELLULAR DISTRIBUTION OF VIMENTIN AND CYTOKERAT1N

AIM 2C 15

10

11

13

15

17

19

Days

21

FIG. 20. (A) Densitometric tracing of Fig. 19; (a) the pattern of 19f, (b) the pattern of 19a. In addition to actin, the amount of several components including the 55 kDa protein increased in a as compared with b. (B) Increase in cytoskeletal actin content of RPE cells during development. Ordinate: relative values of actin to myosin heavy chain. Abscissa: incubation time of the eggs. The actin content increases more than 4 times between 15-dayold and hatching stages.

The IF components have been further characterized after enrichments in cytoskeletal preparations obtained after treatment with 1.5 M KCI in the presence of 1% Triton (cf. Achtst~itter et al., 1986). While the IF polypeptide pattern of chicken RPE cells reveals only one component of a ~ 55 kDa, i.e. vimentin (Fig. 23d), and no cytokeratin can be detected, bovine cytoskeletal proteins comprise four major bands corresponding to 58, 53, 43, 42 kDa (Fig. 23e). The ratio of the 43 kDa to the 53 kDa component is now similar due to the removal of most of the actin, compared with the "Triton cytoskeletons" (Fig. 23b). By two-dimensional gel electrophoresis, including co-electrophoresis with authentic bovine proteins and immunoblot analysis these components have been identified as vimentin (58 kDa), cytokeratin 8 (A, ~ 53 kDa) and 18 (D, ~ 43 kDa) by molecular mass and

IFS

In the central region of bovine retina, the RPE cells contain only few or no pigment granules (Berman et al., 1974), and such non-pigmented cells are particularly convenient for immunofluorescence microscopy. As expected from gel electrophoretic and immunoblot analyses, bovine RPE cells contain both cytokeratin and vimentin 1Fs, but their distribution patterns are quite different (Fig. 24). While keratin IFs form a dense meshwork extending throughout almost the entire cytoplasm, vimentin IFs are seen predominantly in the peripheral cytoplasm (Fig. 24c,d). Within a given RPE sheet, not all cells are positive for vimentin, indicating that two types of RPE cells can be distinguished, those rich in vimentin IFs and those expressing little or no vimentin. Rat RPE cells in situ are usually binucleated and this distinctive feature is particularly useful in immunofluorescence microscopy of ocular tissue or isolated RPE cell sheets of albino rats (Kuwabara, 1979; Stroeva and Mitashov, 1983). When examined in this way, rat RPE cells are abundant in cytokeratin IFs but negative for vimentin IFs (Fig. 25). The special arrays of cytokeratin IFs in these cells are best visualized in sheet preparations of RPE cells (Fig. 25c). Examination of RPE cells from other mammalian species such as mice, guinea pigs, rabbits and human embryos, by immunofluorescence microscopy also revealed cytokeratin IFs. Hiscott et al. (1984) have also reported positive cytokeratin staining of human RPE cells in epiretinal membranes in situ and in vitro. 3.2. D e s m o s o m e s in R P E Cells

The junctional complexes of RPE cells of chicken (Crawford et al., 1972; Hudspeth and

RETINAL PIGMENT EPITHELIAL CELLS

37

FIG. 21. Elongation of the actin bundles in chicken RPE cells. The cells were stained with actin antibody ( a - e ) or NBDPhallacidin (f). The cells from 15 (a), 17 (b,c) and 19-day-old embryo (d). (f) The cells from a l-day-old chick. (f) shows both sides of the epithlium in the same field because a part of the epithlium was folded and turned over. The CMBs that disappeared in the apical view are found on the basal side, but they are not found in the basal side of every epithelium. Bars, 10 tam ( a - e ) , 20 tam (f).

Yee, 1973) and a variety of vertebrates (Hudspeth and Yee, 1973) have been extensively examined ultrastructurally, and a typical junctional complex composed of gap junction, tight junction and the zonula adhaerens has been described in these cells. Unequivocal desmosomes which are characteristic for the junctional complexes of many other epithelia (Farquhar and Palade, 1963; Cowin et al., 1985; Franke et al., 1987) have not been described in chicken RPE cells, or notably, in other submammalian species. Only Miki et al. (1975) have reported in human and other RPE

cells very small desmosome- and hemidesmosomelike structures by thin-section electron microscopy. Docherty et al. (1984) have confirmed that chicken RPE cells have no desmosomes by immunofluorescence microscopy, also using antibodies to desmosomal proteins, and have suggested that the RPE is a special kind of epithelium like the endothelium (Franke et al., 1979; Blose and Meltzer, 1981) and the "lens epithelium" (Ramaekers et al., 1980) which contain vimentin and no keratin IFs and are devoid of desmosomes. However, recently, using

38

K. OWARIBE 3.2.2. IMMUNOLOCALIZATION OF DESMOSOMAI. PROTEINS

FIG. 22. Immunoblot analysis of the 55 kDa protein. SDS-PAGE gel of Triton-treated chick embryo RPE cells were transferred on a nitrocellulose sheet. The sheet was stained with amidoblack (a), 55 kDa antibody (b), vimentin antibody (c) and keratin antibody (d). Blots of cultured h u m a n fibroblasts (MRC cells) were stained with amidoblack (e), 55 kDa antibody (f) and vimentin antibody (g).

adequate and sensitive markers, desmosomes have been found in RPE cells of certain mammalian species (Owaribe et al., manuscript in preparation).

3.2.1. DETECTION OF DESMOSOMAL PROTEINS IN R P E CELLS

Figure 26 presents the results of gel electrophoretic and immunoblotting experiments aimed at the detection of desmosomal proteins in bovine and chicken RPE cells. Desmoglein, which is a 165 kDa glycoprotein spanning the desmosomal membrane and contributing to the desmosomal plaque (Schmelz et al., 1986a, b; Steinberg et al., 1986) is readily detected in IF cytoskeletons of bovine RPE but not in those of chicken (Fig. 2 6 a ' , b ' ) . Plakoglobin, which is a 83 kDa protein present in a variety of intercellular adhaerens junctions, including desmosomes and zonulae adhaerentes (Cowin et al., 1986), is detected in both bovine and chicken RPE cells (Fig. 26c',d'). These results indicate that, whereas bovine RPE cells are connected by true desmosomes, chicken cells lack these structures, although they do have a well developed zonula adhaerens junction.

Desmoplakin I which is a 250 kDa polypeptide generally present in desmosomal plaques (Mueller and Franke, 1983; Franke et al., 1982a; Cowin et al., 1985) can be identified, by immunofluorescence microscopy, in bovine RPE cells (Fig. 27). Remarkably, desmoplakin immunofluorescence was clearly observed in pigmented RPE cells but less clearly in non-pigmented cells, probably because of the abundance of autofluorescent granules in the cytoplasm of the latter. When bovine RPE cells are incubated in medium containing 2 mM EDTA, the intercellular junctions are split, and the individual cells separate from each other, as reported by Kartenbeck et al. (1982) and Volberg et al. (1986) for cultured bovine kidney epithelial (MDBK) cells. In these EDTA-treated cells, CMBs dissociate from the cell membrane and appear to contract somewhat (Fig. 27d). In early stages of EDTA-induced RPE cell separation desmosomes also split but the resulting vesiculated, endocytosed fragments are still located in the cell periphery (Fig. 27c). It is remarkable that after this induced "junction splitting" by EDTA treatment, desmosomal material and CMBs can be observed in different distributions in the cytoplasm. By thin-section electron microscopy, typical desmosomes with a distinct midline structure, a pair of dense cytoplasmic plaques and attached IF bundles are observed along lateral cell membranes of bovine RPE cells (Fig. 28a). Moreover, electron microscopic immunolocalization with desmoplakin antibodies identifies these junctions as true desmosomes (Fig. 28b,c).

3.3. Intermediate Filaments Cultured R P E Cells

and

Desmosomes

in

3.3.1. EXPRESSION OF VIMENTIN AND DESMOSOMAL PROTEINS 1N CULTURED BOVINE R P E CELLS

When bovine RPE cells are grown in culture, the synthesis of cytokeratins seems to stop whereas the cultured cells continue - - or begin - to synthesize vimentin soon after onset of the culture. After about two weeks of culture, almost all

39

RETINAL PIGMENT EPITHELIAL CELLS

V O

i,ii~!i!~ ~iiii? FIG. 23. Characterization of IF proteins of chicken and bovine RPE cells. (a - b"): (a) Molecular weight markers (from top to bottom), myosin heavy chain (200 kDa);/3-galactosidase (120 kDa); phosphorylase a (94 kDa); bovine serum albumin (68 kDa); actin (42 kDa); carbonic anhydrase (29 kDa). (b) SDS-PAGE pattern of Triton treated bovine RPE cells. (a' - b") Corresponding immunoblots stained with a monoclonal antibody to cytokeratin No. 18 (a' ,b' ) and a monoclonal antibody to cytokeratins, which shows more wide cross-reactivity to keratins (b"). (c,c') SDS-PAGE pattern of bovine fibroblasts (B1 cells) and corresponding immunoblot stained as b ' . (d,f) SDS-PAGE patterns of IF cytoskeletons of chicken (d) and bovine RPE cells (e). (f) 2D-PAGE of IF cytoskeleton of bovine RPE cells. NEPHGE: Nonequilibrium pH gradient electrophoresis, B = Bovine serum albumin, P = Phosphoglycerokinase.

cells display, by immunofluorescence microscopy, vimentin IF fibrils but not cytokeratin IFs (Fig. 29). In these cells, cytokeratin expression has not been detected, even after three months of culture. These data are in agreement with those of Haley et al. (1983) who have analyzed cytoskeletal proteins of cultured human RPE cells by twodimensional gel electrophoresis and have presented a polypeptide pattern in which large amounts of vimentin seem to be present. These results suggest that the cells express cytokeratins only in situ, perhaps due to the requirement of some interaction with the basal lamina or with other retinal cells. Otherwise, all cells produce only vimentin IFs (for changes of expression of IF proteins in other cultured ceils see Schmid et al., 1983; Venetianer et al., 1983). In contrast, desmosomal plaque proteins are continually expressed during later stages of growth in culture. Among the various desmosomal proteins examined, plakoglobin is the first to be detected at cell- cell boundaries, i.e. in junctions, probably reflecting its presence in nondesmosomal intercellular junction regions, e.g. fasciae and zonulae adhaerentes (Cowin et al., 1986) which are formed prior to the formation of

desmosomes. Subsequently, the cells form desmosomal junctions (Fig. 29a shows typical linear punctate arrays of desmoplakin localization). Antibodies to plakoglobin have been observed to react with junctions in all cells of RPE cell cultures. This is not the case for desmoplakin and desmoglein in all cells. Ceils with desmoplakin-positive desmosomes are mostly located in the central part of the growing monolayer colonies. In these bovine RPE cell cultures, the IFs anchoring at desmosomes are probably vimentin IFs, similar to the situation with human meninges and meningiomal cells (Kartenbeck et al., 1984).

3.3.2. DYNAMIC PROPERTIES OF VIMENTIN FILAMENTS IN CULTURED CHICKEN RPE CELLS

Chicken RPE cells in situ and in vitro show neither cytokeratin IFs, nor desmosomes, but they possess an apical meshwork consisting of vimentin filaments, which can be isolated together with the "polygonal plate" (see Fig. 11). This may suggest that there is some interaction between the CMB microfilament system and the vimentin IFs.

40

K. OWARIBE

FIG. 24. Immunofluorescence microscopy showing cytokeratin filaments in bovine RPE cells. Phase-contrast (a) and immunofluorescence(b) micrographs of frozen sections stained with a monoclonal antibody to cytokeratins. Only the RPE cell layer reacted with the antibody. Double immunofluorescencemicrographs of RPE sheets stained with a monoclonal cytokeratins (c) and polyclonal vimentin (d) antibodies. Bars, 20 om. During formation of an epithelial monolayer sheet in culture, RPE cells change their shape from single elongated cells to densely packed colonies of polygonal cells (Crawford et al., 1972; Owaribe et al., 1979; Turksen et al., 1983). In early stages, vimentin IFs are observed as typical wavy bundles of filaments extending throughout the cytoplasm (Fig. 30a). With ongoing development, the IF bundles disperse gradually into thinner bundles of filaments, and become distributed rather uniformly (Fig. 30b,c), in contrast to the enrichment of actin in the cell periphery (Owaribe et al., 1979). During further cell differentiation, actin bundles gradually increase in size and accumulate to develop typical CMBs (see Fig. 5). The gradual change of the localization of actin in stress fibers to its

appearance in CMBs seems related to a change from the cell-substratum adhesion alone to the establishment of continuous c e l l - c e l l contacts (Opas et al., 1985). When well developed RPE cells are treated with 5 ~M colcemid in culture medium for 2 4 - 36 hr at 37°C, their cell shapes become irregular, and cytoplasmic pigment granules aggregate. The arrangement of vimentin IFs is known to be affected in many cell types by colcemid (e.g. Bennett et al., 1978; Blose and Mehzer, 1981; Osborn et al., 1980) and, similarly, in RPE cells treated in this way, thick vimentin-positive, perinuclear fluorescent rings or coils are formed, as shown in focal planes at the cell bases (Fig. 31b). In subapical planes, however, extended vimentin IFs are still recognized although they

RETINAL PIGMENT EPITHELIAL CELLS

41

FIG. 25. Immunofluorescence microscopy showing cytokeratin filaments in albino rat RPE cells. Phase-contrast (a) and immunofluorescence (b) micrographs of frozen sections stained with a monoclonal antibody to cytokeratins. In the sheet preparation, cytokeratin filaments are well recognized (c). Bars, 20 gin.

b'

tl'

im e

m

~lllllmw

FIG. 26. lmmunoblot analysis of desmosomal plaque proteins. ( a - d ) Coomassie patterns of the IF cytoskeleton of bovine RPE cells (a,c) and the Triton cytoskeleton of chicken RPE cells (b,d), gels; 10% (a,b), 7.5% (c,d). (a' - d ' ) Corresponding immunoblots stained with a monoclonal desmoglein ( a ' , b ' ) and a monoclonal plakoglobin (c' ,d') antibodies.

tend to change from meshwork-like arrangements to coarser bundle-like structures (Fig. 31a). Electron microscopy of the perinuclear bundles of vimentin IFs (Fig. 32a,b) reveals their composition of many parallel IFs. Mitochondria are often observed in close alignment with the IF bundles, suggestive of some interaction with IFs (cf. Hynes and Destree, 1978). As the result of the formation of these thick IF bundles, pigment granules are excluded from the central, i.e. perinuclear region and are displaced to the peripheral cytoplasm. IFs and actin filaments of the subapical region are also affected by colcemid treatment (see above and Fig. 31c). When such subapical bundles of colcemid-treated cells are examined by electron microscopy, parallel, somewhat alternating arrangements of bundles of microfilaments and IFs are seen (Fig. 32c,d). These newly formed massive microfilaments also show bundles having distinct "dense bodies" similar to those known from smooth muscle cells. The contours of the plasma membrane parallel to the microfilament

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K. OWARIBE

FIG. 27. Immunofluorescence microscopy showing localization of desmoplakins and a-actinin. (a) Normal sheet preparation stained with a mixture of monoclonal antibodies to desmoplakins. (b-d) Phase-contrast (b) and double immunofluorescence micrographs of the sheets pre-treated with EDTA stained with monoclonal desmoplakins (c) and polyclonal a-actinin antibodies (d). Bar, 10 ~m.

and IF bundles appear relatively smooth, and the CMBs are similar to those of untreated cells, whereas the contours of the cell periphery perpendicular to the direction of the filament bundles is irregular which presumably is related to a disordered microfilaments alignment in the CMBs. Effects of colcemid treatment on IFs and microfilaments have been observed only in cultured RPE cells. RPE cells in situ, e.g. from an day 11-embryo, do not form extensive perinuclear IF bundles although the epithelium undergoes some general shape change during colcemid treatment.

4. M I C R O T U B U L E S

The organization of microtubules has been described in detail for chick RPE cells in culture

(Owaribe et al., 1979; Turksen et al., 1983). In uncoupled, elongated cells, the extended arrays of microtubules are well recognized: Most microtubules radiate from the perinuclear region, probably the centrosome, toward the cell periphery as observed in cultured cells of many other cell types. Concomitant with the formation of a continuous epithelium, the cells become thicker, pigmentation increases, and the individual microtubules are less well resolved, although the overall organization of microtubules does not change dramatically. When dense RPE cell monolayers are treated with colcemid, the cell shape is not considerably affected, at least over short periods of time; this is different from the effects of cytochalasin treatments. Hence, one might conclude that microtubules are not critically involved in the maintenance of cell shape of RPE cells. Irons and Kalnins (1984) have examined the localization of tubulin in cultured RPE cells of

RETINAL PIGMENT EPITHELIAL CELLS

FIG. 28. Conventional and immunoelectron micrographs of bovine RPE cells in situ. (a) Thin-section electron micrograph showing the apical junctional complex consisting of gap junction (G), tight junction (T) and zonula adherens (Z). Arrows show desmosome-like structures. Bar, 0.5/am. (b,c) Immunoelectron micrographs showing localization of desmoplakins in the desmosome-like plaques. The cells were stained with a mixture of monoclonal antibodies to desmoplakins and visualized by second antibodies coupled with 5 nm gold particles. Bars, 0.2 tam.

normal and dystrophic rats and have not found any differences in cell shape or in the distribution of microtubules. A similar result has also been reported for actin distribution (Chaitin and Hall, 1983). Bruenner and Burnside (1986) have described that the number o f microtubules in the apical projections of R P E cells of teleost fishes increases during pigment granule aggregation. They have suggested that teleost RPE cells behave like amphibian melanophores in pigment granule migration although the force-producing

43

FIG. 29. Immunofluorescence microscopy of cultured bovine RPE ceils stained with monoclonal desmoplakins (a) and polyclonal vimentin (b) antibodies (double staining). Bar, 10 lam.

mechanism is unclear. Klyne and Ali (1981) have examined the structural differences of RPE cells of light- and dark-adapted eyes of fishes by electron microscopy: In the light-adapted cells, microtubules are distributed throughout the cytoplasm in association with other organelles such as nuclei, mitochondria and pigment granules, whereas in the dark-adapted cells, microtubules appear to be absent. These results are in concert with the concept that, in RPE cells, microtubules are primarily involved in movements of intracellular organelles as in many other cells (e.g. Allen et aL, 1981; Burnside and Laties, 1979; Schliwa, 1984; Vale et al., 1985).

44

K. OWARIBE

FIG. 30. Immunofluorescence microscopy of cultured chicken RPE cells stained with antibodies to the 55 kDa protein (chicken vimentin) showing various stages of the epithelium formation. (a) S q u a m o u s stage; the cells are almost confluent but immature. (b) Intermediate stage; the cells are polygonal and lightly pigmented. (c) Mature cuboidal stage; the cells form a simple cuboidal epithelium and are heavily pigmented. Bar, 10 tam.

FIG. 31. lmmunofluorescence microscopy of cultured chicken RPE cells treated with colcemid. (a) Apical view of mature cells stained with vimentin antibody. Vimentin filaments change their distribution from non-polarized meshwork to polarized bundle-like structure (cf. Fig. 29c). (b) Basal view of the cells stained with vimentin antibody. Thick perinuclear bundles are formed in the cells. (c) Apical view of the cells stained with NBDphallacidin. Many actin bundles are formed besides CMBs. Bar, I0 tam.

RETINAL PIGMENT EPITHELIAL CELLS

FIG. 32. Elecron microscopy of cultured chicken RPE cells treated with colcemid. (a) Overall view of a cell showing a thick perinuclear bundle at low magnification. The rough endoplasmic reticulum lies near the nucleus in this cell. Mitochondria are aligned along the bundle, and pigment granules are excluded from central region of the cell. (b) High-magnification photograph of (a), each filament can be seen. (c) Low-magnification photograph of apical region of the cells. (d) Highmagnification photograph of apical region showing parallel alignment of IFs and microfilament bundles that have prominent dense bodies. Bars, 5 tam (a), 1 tam (b,d), 4/am (c).

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K. OWARIBE

46 5. CONCLUSIONS

Chicken RPE cells have two types of microfilament bundles, which are structurally and functionally different. CMBs are elements common to avian and mammalian RPE cells. They are associated with the zonula adhaerens and appear to be involved in the maintenance of the epithelial organization, as a major subapical cytoskeletal element. This i s similar to the situation in other polarized epithelial cells. On the other hand, PMBs are core filaments of apical projections which appear to be involved in the maintenance of the projections and probably also in pigment granule distribution. PMBs seem to be a cytoskeletal element specific for chicken RPE cells. Probably, the functions of the PMBs in chicken RPE cells are served by other structures in mammalian RPE cells, perhaps by the loose meshworks or bundles o f microfilaments described in corresponding positions of mammalian RPEs (Burnside, 1976; Burnside and Laties, 1976). During the development of RPE cells, the CMBs are formed much earlier than the PMBs. Apparently, the differentiation of RPE cells first requires the general cytoskeletal organization of a polarized epithelial cell. Only after formation of the epithelial sheet do the cells develop the RPE function-specific cytoskeletal element(s) as a subsequent program. This would also be consistent with the finding that, in culture, RPE cells form CMBs but not PMBs. Perhaps these cells need the intimate interaction with photoreceptor cells for differentiation of the full, cell-type specific cytoskeletal complement. The finding o f cytokeratin IFs and desmosomes in RPE cells of various mammalian species confirms the epithelial character of these cells. The reason for the absence of cytokeratin IFs and desmosomes in chicken RPE cells is not clear, neither is there any obvious explanation for the existence of a vimentin cytoskeleton in the avian RPE cells. The biological functions of IFs and IF proteins are generally unclear. It has been proposed that they play certain supportive mechanical roles (e.g. Lazarides, 1980), and examples indicating a special role in the

intracellular distribution of certain cell particles has been demonstrated, for example for vimentin IFs during adipose conversion of mesenchymal cells (Franke et al., 1987b). Clearly, however, IFs are not indispensible for fundamental cell functions as they are absent in a number of cell types (for references see Franke et al., 1987b). Whatever the specific function of IFs in RPE cells may be, it is clear that the specific functions of these cells that are common to the diverse vertebrate species cannot depend on a particular pattern of expression of IF-desmosome cytoskeleton: What birds may see using vimentin RPEs, bulls see with cytokeratin RPEs.

Acknowledgements--The author wishes to thank Dr W. W. Franke for critically reading and correcting the manuscript, Ms I. Purkert for typing and Drs S. Tsukita and S. Tsukita for Fig. 14. This work was supported in part by grants from the Ministry of Education, Science and Culture in Japan, and the Deutsche Forschungsgemeinschaft. Figs. 1 - 9, 11 - 13, 15 - 17 and 19- 21 are from the Journal o f Cell Biology, copyright by Rockefeller University Press (New York), and Figs. 22 and 3 0 - 3 2 are from Cell and Tissue Research, copyright by Springer-Verlag Inc. (Heidelberg) with permission.

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DEROSIER, D., MANDELKOW,E., SILLIMAN,A., TILNEY, L. and KANE, R. (1977) Structure of actin-contalning filaments from two types of non-muscle cells. J. molec. Biol. 113: 679 - 695. DEROSIER, D . J . and TILNEY, L . G . (1982) HOW actin filaments pack into bundles. Cold Spring Harbor Syrup. Quant. Biol. 46: 525- 540. DOCHERTY, R. J., EDWARDS, J . G . , GARROD, D . R . and MA'I'TEY,D. L. (1984) Chick embryonic pigmented retina is one of the group of epitheloid tissues that lack cytokeratins and desmosomes and have intermediate filaments composed of vimentin. J. Cell Sci. 71:61 - 74. DRENCKHAHN, D. and GROSCHEL-STEWART, U. (1977) Localization of myosin and actin in ocular nonmuscle cells. Immunofluorescence microscopic studies. Cell Tiss. Res. 181: 493-503. DRENCKHAHN, D. and WAGNER, H.-J. (1985) Relation of retinomotor responses and contractile proteins in vertebrate retinas. Fur. J. Cell Biol. 37: 156- 168. EDDS, K. T. (1979) Isolation and characterization of two forms of a cytoskeleton. J. Cell Biol. 83: 109-115. FARQUHAR, i . G . and PALAOE, G . E . (1963) Junctional complexes in various epithelia. J. Cell Biol. 17: 375-412. FRANKE, W. W., eDWIN, P., SCHMELZ, M. and KAPPRELL, H.-P. (1987a) The desmosomal plaque and the cytoskeleton. In: Junctional Complexes o f Epithelial Cells (G. Buck and S. Clark, eds) pp. 26-44. Ciba Foundation Symp. 125, John Wiley, Chichester. FRANKE, W. W., DENK, H., KALT, R. and SCHMID,E. (1981a) Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue. Expl. Cell Res. 131: 299-318. FRANKE, W . W . , HERGT, M. and GRUND, C. (1987b) Rearrangement of the vimentin cytoskeleton during adipose conversion: Formation of an intermediate filament cage around lipid globules. Cell 49:131 - 141. FRANKE, W. W., MOLL, R., SCHILLER, D. L., SCHMID, E., KARTENBECK, J. and MUELLER, H. (1982a) Desmoplakins of epithelial and myocardial desmosomes are immunologically and biochemically related. Differentiation 23: 115- 127. FRANKE, W. W., SCHMID, E., OSBORN, M. and WEBER, K. (1979) Intermediate-sized filaments of human endothelial cells. J. Cell Biol. 81: 570- 580. FRANKE, W. W., SCHMID, E., SCHILLER, D. L., WINTER, S., JARASCH, E.-D., MOLL, R., DENK, H., JACKSON,B. W. and ILLMENSEE, K. (1982b) Differentiation-related patterns of expression of proteins of intermediate-size filaments in tissues and cultured cells. Cold Spring Harbor Syrup. Quant. Biol. 46:431-453. FRANKE, W. W., WEBER, K., OSBORN, M., SCHMID, E. and FREUNDENSTEIN, C. 0978) Antibody to prekeratin: Decoration of tonofilament-like arrays in various cells of epithelial character. Expl. Cell Res. 116:429 - 445. FRANKE, W . W . , WINTER, S., GRUND, C., SCHM|D, E., SCHILLER, D. L. and JARASCH, E. D. (1981b) Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border. J. Cell Biol. 90: 116- 127. GEIGER, B., DUTTON, A. H., TOKUYASU, K. T. and SINGER, S . J . (1981) Membrane-microfilament interactions: Distribution of a-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J. Cell Biol. 91: 614-628.

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