Cell Tissue Res (1984) 236:27-33

a n d T't te R e s e a l T h �9 Springer-Verlag 1984

Intermediate flaments, microtubules and microfilaments in epidermis of sea urchin tube foot Patricia Harris 1' 2 and Gerry Shaw z

Department of Biology, University of Oregon, Eugene, USA; 2 Max-Planck-Institut fiir Biophysikalische Chemic, Grttingen, Bundesrepublik Deutschland

Summary. Tube foot epidermal cells of the sea urchin Stron- gylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to iden- tify the chemical nature of prominent bundles of cytoplas- mic filaments. Cross sections revealed filaments of roughly 7-8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reac- tion with the fluorescent F-actin binding drug NBD-phalla- cidin indicated that they were not actin. Indirect immuno- fluorescence microscopy of whole tissues and frozen sec- tions revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot pro- tein, a band of apparent molecular weight around 50000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate fila- ments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.

Key words: Intermediate filaments - Microtubules - Micro- filaments - Sea urchin - Cytoskeleton

The suckered tube feet of sea urchins are multi-purpose structures, serving locomotory, adhesive, and possibly also sensory and respiratory functions. They have been the sub- ject of several light microscope studies (Smith 1937, 1947; Nichols 1961) and more recently have been examined at the ultrastructural level (Kawaguti 1964; Coleman 1969a, b; Florey and Cahill 1977). The tube foot wall consists of several well-defined layers: an outer pigmented epidermis including a nerve plexus, a basal lamina separating the epi- dermis from a connective tissue layer comprised of both longitudinal and circular bands, another basal lamina sepa- rating the connective tissue from the longitudinal smooth muscle, and finally, a thin ciliated epithelium.

The epidermal cells are of particular interest, in that these cells would be expected to play a role in the presumed sensory functions of the tube feet. Contrary to expectations, however, neither Coleman (1969b) nor Florey and Cahill (1977) found any morphological evidence that these cells had any nervous function. They do, however, have long

Send offprint requests to : Dr. Patricia Harris, Department of Biolo- gy, University of Oregon, Eugene, Oregon 97403, USA

processes, each containing a bundle of electron-dense fila- ments. These processes extend from the apical region to a foot-like terminus on the basal lamina. The absence of any synaptic vesicles in these endings led Florey and Cahill (1977) to reject the idea that these cells were sensory and to propose that the cable-like extensions served as structural devices for holding the epidermis to the connective tissue layer. This, of course, would be an important function in tube feet, which undergo extreme length changes.

For a better understanding of the function of these epi- dermal structures, it seemed important to try to identify the chemical nature of the fiber bundles. In the present study we have combined light and electron microscopy with the use of specific fluorescent probes for the localization of actin, tubulin, and various intermediate filament proteins in whole tissue preparations. The results indicate that the bundles of filaments do not contain detectable amounts of actin, and that they are structurally and immunologically distinct from microtubules. However, they show a strong reaction with a monoclonal antibody raised against porcine stomach desmin. SDS-polyacrylamide gels of whole tube foot proteins revealed a band of approximately 50 kd, which also reacted with the desmin monoclonal antibody.

Materials and methods

Specimens of the sea urchin Strongylocentrotus purpuratus were obtained from Pacific Biomarine Labs, Inc. (Venice, California) and kept in aquaria with artificial sea water (Tropic Marine Neu, D-6072 Dreieich 3) at 12 ~ C. For bright field and phase contrast light microscopy, and elec- tron microscopy, tube feet were dissected and fixed in either the contracted condition or extended, by clamping the ex- tended tube foot with forceps to retain internal hydrostatic pressure. The fixative was a mixture of 1% OsO4, 2% glu- taraldehyde and 0.45 M Na acetate at pH 6.5. Following 1 2 h fixation at room temperature, tissues were dehydrated in a graded series of ethanol and embedded in Epon epoxy resin. Sections were cut with glass knives. For light micros- copy, 1-2 t~m sections were stained with methylene blue and Azure II (Richardson et al. 1960). Thin sections for electron microscopy were stained with uranyl acetate and lead citrate and examined in a Philips 301 electron micro- scope.

Antibodies and staining procedures. The monoclonal rat an- tibody (code: YL 1/2) against yeast ~-tubulin (Kilmartin

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Fig. 1. Cross section of tube foot epidermis, extending from branched microvilli at the outer surface (upper left corner) to the basal lamina (BL) which separates the epidermis and nerve (N) from the loose connective tissue (far right). A Golgi membrane complex (G) and numerous clear vesicles are found in the apical region of filament-containing cells. Mucous cells (MC) are scattered throughout the epidermis. Arrows indicate filament bundles, x 7000

Fig. 2. Higher magnification of the apical region of an epithelial cell. Typical cell junctions consist of an outer intermediate junction (/) immediately followed by a septate junct ion (S). Dense deposits (D) possibly resulting from the fixation, are sometimes found in the intercellular spaces. In this section a band of filaments extends from the intermediate junction parallel to the cell surface (upper arrows). Arrows in the lower part of the picture indicate an oblique section through a filament bundle. (FB). x 35000

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et al. 1982), known to recognize only the carboxyterminally tyrosinated molecule (Wehland et al. 1983), was available through J. Wehland and originally supplied by Dr. J. Kil- martin. A monoclonal mouse antibody (code: DE-U10) eli- cited with porcine stomach desmin was made available by Dr. E. Debus (Debus et al. 1983). Fluorescein-labelled anti- mouse and anti-rat-IgG antibodies were purchased from Cappel Laboratories (Cochranville, Pa., USA) and were used at a final concentration of 0.5 mg/ml. For actin detec- tion, 7-nitrobenz-2-oxa-l,3-diazole-phallacidin (NBD-phal- lacidin), a fluorescent derivative of the F-actin-specific drug phalloidin (Barak et al. 1980) was obtained from Molecular Probes, Inc. (Junction City, Oregon, USA) and diluted I : 5 with 1% bovine serum albumin to a final concentration of 20 units/ml. Purified porcine stomach desmin was made available by Dr. N. Geisler.

During the dissection procedures it was observed that the epidermis, including the basal lamina, is easily separated from the collagen and muscle at the level of the loose colla- gen layer immediately below the basal lamina. Therefore, to insure good penetration of antibodies, the epidermal layer was stripped from the entire tube foot, split open and spread out as a flat sheet on a polylysine-coated cover- slip. It was fixed in this position for 6-10 min in - 2 0 ~ methanol and then rehydrated in phosphate buffered saline solution (PBS: 140 mM NaC1, 3 mM KCI, 10raM Na/K phosphate, at pH 7.3). The tissue usually floated free of the coverslip during rehydration, and from this time on

Fig. 3. Epithelial cell processes in cross section show a loose sheath of microtubules (MT) surrounding each of the filament bundles (FB). x 74000

Fig. 4. A longitudinal section grazing the surface of a filament bundle (FB) with associated microtubules (MT). Additional fibrous material is also present around the microtubules. x 74000

was transferred from one solution to another either with a pipette or on a dissecting needle.

The epidermis was suspended in a 5-10 gl drop of anti- body on a coverslip and incubated for 1.5 h at 37~ in a moist chamber. Following each antibody incubation, the tissue was washed in 3 changes of PBS for a total of 5 min and finally mounted in Mowiol 4-88 (Hoechst, Frankfurt) under a coverslip. Pressure was applied to insure that the tissue layer was flat. Similar procedures were used for stain- ing of the stripped collagen and muscle layers. For NBD- phallacidin staining, tissues were used either unfixed, or treated with cold methanol for times not exceeding 1 min. Following I0-15 min incubation in the stain, the tissues were washed three times in PBS and mounted as above in Mowiol 4-88 under coverslips.

SDS-polyacrylamide gels and blots. Homogenized whole tube feet were solubilized by boiling in sample buffer (con- taining 2% SDS, 1% mercaptoethanol, 67.5 mM Tris-HC1, and 5% glycerol) and resolved on 6% polyacrylamide slab gels, with adjacent lanes containing purified porcine des- min. The proteins were transferred from the gel to nitrocel- lulose sheets by a method modified from Towbin et al. (1979), using longer term diffusion rather than electrophor- esis. The nitrocellulose sheets were reacted with the same monoclonal antibody (DE-UI0) against desmin that had been used for indirect immunofluorescence staining of tis- sue preparations. The antibody bound to the nitrocellulose

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Figs. 5, 6. Fluorescence and phase- contrast pictures of the same field, where the epidermis was only partly stripped from the underlying muscle and collagen. Fluorescent NBD- phallacidin stained only the straight longitudinal muscle (Fig. 5). The overlying wavy collagen fibers seen in phase contrast (Fig. 6) are not stained, nor is there any staining in the remaining sheet of epidermal cells which covers the lower half of the picture, x 200

Fig. 7. Tubulin immunofluorescence showing the wavy processes of the epithelial cells overlying a fine array of parallel bands, possibly part of the nerve plexus, x 430

Fig. 8. Another preparation showing tubulin fluorescence in the nerve net. The large microtubule bundles running diagonally from top to bottom are parallel to the main longitudinal nerve (not shown), x 600

Fig. 9. Flattened epidermis stained with anti-desmin DE-U10 antibody, focussed on the uppermost apical region of the cells. The fiber bundles appear to follow the cell periphery. x 800

Fig. 10. A preparation similar to Fig. 9 focussed at the level of the cell processes, showing the brightly staining filament bundles, x 500

was reacted with 125I-labelled goat ant i -mouse second anti- body (Cappel Laborator ies , Cochranville, PA). Specific binding was visualized by au to rad iography using K o d a k R X-ray film. Exposure time was typically ~ 3 days at -- 70 ~ C.

Results

Electron microscopy

The fine structure of the tube foot epidermis from Strongy- locentrotus purpuratus is essentially identical to that de-

scribed for Diadema antillarum by Coleman (1969b) and for Strongylocentrotus franciscanus, Arbacia lixula and Echinus esculentus by Florey and Cahill (1977). Figure 1 is a low magnification cross section view of the epidermis, extending from the branched microvilli at the outer surface (upper left hand corner) to the basal lamina (BL). The lat ter structure separates the nerve plexus and epithelium from the loose connective tissue (far right). A large Golgi mem- brane complex (G) and numerous clear vesicles (V) are characteristic of the apical region of filament containing cells. The extreme interdigitat ion of the cell membranes of-

ten makes it difficult to trace the individual filament-con- taining processes (arrows) to their termination on the basal lamina. Mucous cells (MC) are scattered throughout the epidermis. Bundles of nerve axons (N) lie adjacent to the basal lamina and form a nerve net.

The epithelial cell junctions are similar to those de- scribed in Diadema (Coleman 1969b) and are located at the most apical region of the cells (Fig. 2). An outer inter- mediate junction (I), or "belt desmosome" (Spiegel and Howard 1983), with densities along the membrane, lies just apical to a septate junction (S). In this section a band of filaments can be seen extending from the intermediate junc- tion parallel to the cell surface (arrows). Arrows in the lower part of the picture indicate an oblique section through a filament bundle (FB).

The filament bundles of the epidermal cell processes are seen in cross section at higher magnification in Fig. 3. A loose sheath of microtubules surrounds each bundle. In- dividual filaments are difficult to measure in these prepara- tions because of close packing, although their diameters appear to be roughly the same as the thickness of the micro- tubule walls, i.e., 7-8 nm. Besides the filament bundles and microtubules, membranous elements of the endoplasmic re- ticulum and mitochondria are also generally found along the whole length of the epidermal cell processes, and are often seen in the foot-like endings. Figure 4 shows a longitu- dinal section grazing the surface of a filament bundle with its associated microtubules. Additional fibrous material ap- pears to be associated with the microtubules and may also be present in the bundles of the filaments as well.

Actin

The actin-binding protein NBD-phallacidin stained strongly the bands of longitudinal smooth muscle and in favorable preparations the microvilli were also visibly stained. Figures 5 and 6 show the same preparation in fluo- rescence and in phase contrast, respectively. In this case the epithelium was not completely stripped from the colla- gen and muscle. In Fig. 5 only the straight longitudinal muscle fibers are brightly fluorescent. The overlying wavy collagen fibers seen in phase contrast (Fig. 6) are not stained, nor is there any staining of the filament bundles in the epithelium covering the lower half of the picture (compare with Figs. 9 and 10).

Tubulin

The YL 1/2 antibody against tyrosylated yeast ~-tubulin stained strongly the microtubules associated with the fila- ment bundles in the epidermal cells, as well as the many neurotubules of the nerve net. Although it was possible to stain the intact flattened tube foot, the resolution, as well as penetration of antibodies into the tissue was greatly facilitated by stripping the epidermis from the underlying collagen and muscle, and treating each of these layers sepa- rately. The degree of pressure used in the stripping proce- dure determined to what degree the nerve net was retained with the epidermis or with the collagen layer. Figure 7 shows the wavy processes of the epithelial cells, as well as a pattern of fine parallel microtubule bands, possibly part of the nerve net, aligned with the collagen layer. In another preparation (Fig. 8), more of the overlying nerve net is visible. The larger microtubule bundles running diag- onally from upper left to lower right are parallel to the

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Fig. 11. A SDS-polyacrylamide gel stained with Coomassie blue showing 53 Kd purified desmin (lane 1) and whole tube foot pro- teins (lane 2). B Nitrocellulose blot of gel in A, reacted with desmin antibody DE-U10 and visualized with autoradiography. Both the purified desmin (lane 3) and a band at approximately 50 Kd (markers, lanes 2 and 4) show a positive reaction

main longitudinal nerve (not shown here). No attempt was made here to investigate the structure of the nerve net or the possible differences between the innervation of the oral tube feet and the aboral sensory tube feet. The results sug- gested, however, that tubulin staining could be a useful tool in the investigation of tube foot neural anatomy.

Intermediate filaments

Several antibodies raised against vertebrate intermediate fil- ament proteins from each of the main classes were tested on frozen sections of tube foot or on stripped epidermis. Although most of these did not give a positive reaction, one mouse monoclonal antibody (DE-U10) prepared against porcine stomach desmin reacted very strongly with the filament bundles in the epidermal cells, both in frozen sections and in whole epidermis mounts. Examination of the stained whole epidermis at different levels of focus re- vealed a circumferential arrangement of the stained fila- ments in the uppermost apical region of the cells (Fig. 9). In a slightly lower optical section at the level of the cell processes the brightly stained bundles are clearly seen (Fig. 10). It is possible that the apical fibers are identical with those revealed by electron microscopy as associated with the intermediate junctions, and represent the anchor- ing of the filament bundles of the cell processes.

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Identification of the reacting protein

To identify the protein which gave the positive reaction to the desmin monoclonal ant ibody, whole tube foot pro- teins were resolved on SDS-polyacrylamide gels, transferred from the gel to nitrocellulose and subsequently labelled with the ant ibody. A single band was labelled, running slightly ahead o f purified desmin in an adjacent lane, giving an est imated molecular weight of about 50 kd (Fig. 11).

Discussion

Vertebrate intermediate filaments, whose size (about 10 nm) lies between that of act in-based microfi laments (6 nm) and microtubules (25 nm), can be segregated biochemically, im- munological ly, and to a certain extent histologically, into five distinct classes; these are epithelial keratins, neurofila- ments, muscle desmin, glial f ibril lary acidic protein and mesenchymal vimentin (for reviews see Ander ton 1981; Lazarides 1980; Osborn et al. 1982).

In termediate fi lament character izat ion and subclassifi- cation, however, has been carried out for the most par t on vertebrate, and par t icular ly on warm-b looded vertebrate tissues. However, many studies clearly indicate the existence of intermediate filaments in invertebrates. The giant axon of the mar ine fan worm Myxicola infundibulum contains fi laments morphologica l ly identical to mammal ian neurofil- aments (Gilber t et al. 1975), which can be stained with anti- IFA, an an t ibody known to recognize all classes of interme- diate fi laments (Pruss et al. 1981). Squid neurofi laments are also structural ly similar, but biochemically distinct from vertebrate intermediate filaments (see for example Lasek et al. 1979). More recent studies on tissue culture cells de- rived from Drosophila indicate the presence of 46 kd and 40 kd components which show immunological cross reac- t ion with desmin and vimentin (Falkner et al. 1981). There is evidence for echinoderm proteins related to vertebrate intermediate filaments from histological studies. These show that echinoderm neurones can be visualized with neu- rofibri l lary stains, such staining apparent ly being an evolu- t ionari ly conserved proper ty of the neurofi lament proteins (Bodian 1939; Gambet t i et al. 1981; Pot ter 1971).

The subdivision of intermediate filaments made routine- ly for higher vertebrates, however, has not yet been explored in other phyla, nor has there been an a t tempt to see how sequence differences and similarities drift among different species. This is a par t icular problem raised by the sequence similarities between distinct intermediate fi lament proteins. The positive react ion of the fi lament bundles with a mono- clonal an t ibody to desmin does not necessarily mean that sea urchin epithel ium contains desmin rather than the cy- tokerat ins known to occur in ver tebrate epithelia. In fact, no desmin an t ibody react ion was associated with the tube foot longi tudinal smooth muscle, even though desmin is known to be abundan t in vertebrate smooh and striated muscle (Lazarides 1980; Small and Sobieszek 1977). Neither was there a react ion with 4-day pluteus larva esophagus and sphincter muscle, whose structure as shown in the sand dol lar Dendraster excentricus is a simple striated form (Burke 1981). I t seems much more likely that a par t icular intermediate f i lament epi tope characterist ic of myogenic desmin in warm-b looded vertebrates is present in a related intermediate f i lament protein found in sea urchin tube foot epithelium.

Thus, the assignment of the prominent filament bundle of the sea urchin epithelial cell to the intermediate filament class relies more on its morphology in the electron micro- scope and on its lack of phallacidin reaction than on its reaction with a part icular ant ibody against mammal ian in- termediate f l a me n t protein. Nevertheless, the positive reac- t ion with a desmin ant ibody tends to suppor t the electron microscopical identification.

An unexpected morphological result concerns the super- posit ion of microtubules and intermediate filaments seen in cross sections of the filament bundles. Indirect evidence of microtubule- intermediate fi lament interaction has long been known from electron microscopical studies, where in- termediate filaments collapse into large aggregates in cells treated with MT-disrupt ing drugs (Robbins and Gona tas 1964; Wisniewski et al. 1968; Go ldman 1971). The associa- t ion of microtubules with several intermediate fi lament types and their coincidence of localization has been ex- plored with double immunofluorescence labelling (e.g., Geiger and Singer 1980). These observations suggest that specific structural interactions may be physiologically regu- lated and uncoupled, for example, during mitosis. Intracel- lular motil i ty also appears to be related to the distr ibution and organizat ion of MTs and 10 nm filaments in BHK-21 cells (Wang and Go ldman 1978). Whether the microtubule- intermediate filament association in the sea urchin epider- mal cell processes serves a structural function or is involved in some intracellular t ransport mechanism remains to be investigated.

Acknowledgements. P. Harris wishes to thank Klaus Weber and Mary Osborn for the opportunity to spend a year in their laborato- ry in G6ttingen. For the many people who have helped during this period, thanks go to Dr. Elke Debus for supplying the desmin antibody, Dr. J. Kilmartin for the yeast tubulin antibody, Dr. Nor- bert Geisler for purified desmin, Sabine Schiller for cutting frozen sections and Regina Hasse for her help with the manuscript. This work was supported in part by grant PCM 81-02884 from the U.S. National Science Foundation to P. Harris.

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Accepted November 29, 1983