THE ANATOMICAL RECORD 245157-64 (1996) Cytoskeletal Differences Between Stereocilia of the Human Sperm Passageway and Microvilli/Stereocilia in Other Locations DIRK HOFER AND DETLEV DRENCKHAHN Znstitute of Anatomy, Julius-Maximilians-University Wurzburg, 0-97070 Wiinburg, Germany ABSTRACT Background: Stereocilia of the human ductus epididymidis and ductus deferens display unique features in that they arise from an apical cell protrusion (hillock)and contain thick stem portions which are interconnected by cytoplasmic bridges. The molecular basis for this unique fusion and branching pattern is hitherto unknown. These morphologic specialities led us to study the cytoskeleton of male spermway stereocilia with respect to the major proteins that constitute the supportive cytoskeleton of intestinal microvilli and inner ear stereocilia. Methods: Samples of the human epididymidis and ductus deferens were studied by immunoblotting and immunocytochemistry at the light and electron microscope levels. Results: Spermway stereocilia are supported by an internal actin filament bundle crosslinked by fimbrin and associated with the membrane linker molecule ezrin. The stem portions and hillock area are supplied with the crossbridge forming molecule a-actinin. Spermway stereocilia differ from brush border microvilli of the intestine, kidney, and ductuli efferentes by the lack of the second bundling protein villin and the unusual expression of a-actinin in the stem region. They resemble inner ear stereocilia by the presence of fimbrin and absence of villin, but differ from them by expression of ezrin and a-actinin. Thus, the main molecular difference between spermway stereocilia and stereocilidmicrovilli of other locations is the presence of a-actinin in their stem portion and the hillock area. Conclusions: Since a-actinin can form crossbridges between adjacent actin filaments (bundles)at longer distances than the other crosslinker of the stereocilium core bundle, fimbrin, we assume that a-actinin is essential for both the formation of the stem portions of spermway stereocilia and for the generation of their striking branching pattern. A developmentally regulated temporal sequence of expression of fimbrin and a-actinin might control the unique architecture of spermway stereocilia. 0 1996 Wiley-Liss, Inc. Key words: Stereocilia, Male Reproductive System, Cytoskeleton The terminology of rod-shaped cell surface projections goes back to the end of the 19th century when histologists grouped these extensions into two main categories: i) motile hairs (kinocilia) and ii) non-motile hairs (stereocilia). The term stereocilium (Gr. stereos = stiff, Lat. cilium = hair, eyelash) comprised the rodshaped units of the intestinal and kidney tubular brush border as well as the stereocilia of inner ear hair cells (hair border) and the epithelium of the male sperm passageway (Ductus epididymidis, Ductus deferens) (Stohr, 1906). After introduction of the electron microscope the term “microvillus” was proposed to distinguish the short projections seen on many cell types from the long stereocilia of the inner ear and male sperm passageway (Fawcett and Porter, 1954). Stereocilia and microvilli in several locations contain an internal supportive cytoskeleton composed of 0 1996 WILEY-LISS, INC. parallel-aligned actin filaments with uniform polarity (Mooseker and Tilney, 1975; Flock and Cheung, 1977; Matsudaira, 1991). In microvilli of the kidney and intestinal brush border actin filaments are bundled by villin and fimbrin (Bretscher and Weber, 1980a,b). This extensively crosslinked core bundle is associated by an unknown mechanism with ezrin, a peripheral membrane protein which is interposed between actin filaments and proteins of the plasma membrane (Gould et al., 1986; Bretscher, 1989; Luna, 1991). Myosin I is another peripheral membrane protein involved in the Received October 18, 1995; accepted December 15, 1995. Address reprint requests to Prof. Dr. Drenckhahn, Institute of Anatomy, Julius-Maximilians-University, Koellikerstrasse 6, D-97070 Wiirzburg, Germany. 58 D. HOFER AND D.DRENCKHAHN linkage of the actin filament core bundle to the microvillus membrane (Glenney et al., 1982; Matsudaira and Burgess, 1979; Drenckhahn and Dermietzel, 1988; Rodman et al., 1986). One difference between the actin filament core bundles of inner ear stereocilia and brush border microvilli is the absence of villin from stereocilia. The only crosslinker of actin filaments in stereocilia appears to be fimbrin (Flock et al., 1982; Drenckhahn et al., 1985, 1991). The cytoskeleton of spermway stereocilia has not yet been analyzed. The sperm passageway that connects the testes with the urethra consists of ductuli efferentes, the ductus epididymidis, and the ductus deferens. Ductuli efferentes (that are located in the head portion of the epididymis) are lined by ciliated and nonciliated cells. The latter predominate and are distinguished by an apically located microvillus border that resembles structurally in many respects the brush border of enterocytes (Morita, 1966). The apical surface of the remaining sperm passageway, i.e., the ductus epididymidis and ductus deferens is occupied with long, nonmotile microvillus-like projections, the height of which decreases from 80 pm in the head region of the epididymal duct to 40 p,m in the epididymal tail and ductus deferens (Horstmann, 1962; Holstein, 1969; Popovic et al., 1973; Dym, 1988). The long and flexible spermway stereocilia differ in their morphology from the stiff inner ear stereocilia and brush border microvilli in two main aspects. First, the tuft of stereocilia arises from an apical cell protrusion (further denoted as hillock) that is absent from inner ear hair cells and from the intestinal and kidney brush border region. Second, the basal portions of spermway stereocilia frequently fuse to form thicker stem portions several pm in length. These stems of stereocilia may locally divide and fuse again. The stems often contain two or more individual actin filament bundles that are continuous with the actin filament bundles of the two or more stereocilia arising from each stem portion. The molecular basis controlling this unique fusion and branching pattern is hitherto unknown. To address this question and to reveal possible differences of the cytoskeleton between brush border microvilli, inner ear stereocilia, and spermway stereocilia we attempted to analyze the cytoskeleton of human epididymal stereocilia with respect to composition and localization of the major cytoskeletal components of the intestinal brush border and inner ear hair border, i.e., actin, villin, fimbrin, ezrin, and a-actinin. Myosin I was not included in this study because of the low degree of crossreactivity of antibodies against intestinal brush border myosin I with the many isoforms of myosin I expressed in different tissues (Goodson and Spudich, 1993). buffered saline (PBS: 10 mM sodium phosphate, 140 mM NaC1; pH 7.4) warmed to 56°C or with 0.2 M glycine-HC1(pH 2.8) at 4°C (Drenckhahn and Franz, 1986; Drenckhahn et al., 1993). In addition, we used a monoclonal mouse IgM antibody directed against a-actinin (Sigma, Deisenhofen, Germany), a monoclonal mouse IgM antibody against actin (Amersham, Braunschweig, Germany), and a monoclonal mouse IgG antibody against human villin (Camon, Immunotech, Marseille, France). Tissue pieces of the human epididymis were homogenized in 80°C sample buffer. Thirty minutes later, insoluble material was pelleted (lO,OOOg, 10 min) and the supernatant was subjected to sodium dodecyl sulphate polyacrylamide (10%) gel electrophoresis (SDS-PAGE)in the presence of 0.1 M dithiothreitol. The separated proteins were electroblotted on nitrocellulose membranes (Burnette, 1981) (Schleicher and Schull, Darmstadt, Germany) which were then incubated with antibodies. Bound immunoglobulins were visualized by the peroxidase-antiperoxidase method (Drenckhahn et al., 1983) using a-chloronaphthol (BioRad, Richmond, VA) as chromogen. lmmunostaining Small tissue pieces of human epididymis, 1-5 mm3 in size, were excised. Tissue pieces were rapidly frozen in melting isopentane cooled with liquid nitrogen. The frozen tissue samples were carefully freeze-dried for 12 h at -40°C and Torr and were then immersed for 48 h a t lop3Torr and 20°C with Epon containing 1.8% (v/v) Epon accelerator. Afterwards, samples were transferred to gelatin capsules and embedded in Epon at 60°C for 24-48 h. Semithin sections (0.5-1 pm) in thickness were mounted on glass coverslips and heated to 80°C for 2 h. Removal of the resin was performed by placing the coverslips for 5 min in a 1:l mixture of methanol and toluene containing 10%sodium methoxide, which was prepared from metallic sodium as described by Major et al. (1961). The sections were then rinsed with a 1:l mixture of methanol and toluene (5 rnin), acetone (2 x 5 rnin), distilled water (5 rnin), and phosphate-buffered saline (PBS: 10 mM sodium phosphate, 140 mM NaC1, pH 7.4) for 5 min as described elsewhere (Drenckhahn et al., 1983; Drenckhahn and Franz, 1986). Antibody staining was done by overlaying each section for 2 h with 20 p1 of affinity-purified antibodies (-1-10 pg/ml PBS). After several washes with PBS, sections were incubated for 30 min at RT with the secondary antibodies. As secondary antibodies we used tetramethylrhodamine (TRITCI-labelled goat anti-rabbit IgG and TRITC-labelled goat anti-mouse IgG (Dianova, Hamburg, Germany; dilution 1 5 0 in PBS). Afterwards the slides were washed again with PBS (3 x 5 min) and mounted in 60%glycerol in PBS MATERIALS AND METHODS containing 1.5% n-propyl-gallate as antifading comAntibodies and lmmunoblotting pound (Serva, Heidelberg, Germany). Negative conSpecificities of the polyclonal rabbit antibodies raised trols included omission of primary antibodies and, in against chicken villin, fimbrin, and a-actinin have been case of the antibodies against villin, fimbrin, and actin described elsewhere (Drenckhahn and Dermietzel, absorption with an excess of the respective antigens. 1988). The antibody against human placenta ezrin was For double immunofluorescence, sections were incukindly provided by Dr. Bretscher (Ithaca, NY). The an- bated for 60 min at RT with a mixture of the polyclonal tibodies against villin, fimbrin, and a-actinin were af- a-actinin antibody and the monoclonal actin IgM antifinity-purified using the respective antigens immobi- body. As secondary antibodies a mixture was used conlized by transfer to nitrocellulose. The bound taining fluorescein isothiocyanate (F1TC)-labelledgoat immunoglobulins were eluted either with phosphate- anti-mouse IgM and Texas Red-labelled goat anti-rab- 59 SPERMWAY STEREOCILIA Fig. 1 . Immunocytochemical localization of ezrin (a) and fimbrin (b,c ) in sections of the epididymal duct and ductus deferens (c) (indirect immunofluorescence).Both antibodies show intense labelling of apical stereocilia projecting into the ductal lumen. Staining is present along the entire length of the stereocilia. L. lumen. Bar: 10 km. bit IgG (the final dilution of both antibodies was 1 5 0 in PBS). The secondary antibody mixture was applied for 1h at RT. lmmunoelectron Microscopy For immunoelectron microscopy tissue samples were fixed overnight with a mixture of 0.1 glutaraldehyde and 2% paraformaldehyde in PBS. After fixation, tissue pieces were treated for 15 min with 0.5 mg/ml sodium borohydride in PBS and embedded in the hydrophilic methacrylate resin LR-White (London Resin Co., Woking, UK).Ultrathin tissue sections were collected on gold grids and processed for immunogold labelling with affinity-purified anti-a-actinin exactly as described (Drenckhahn and Merte, 1987; Drenckhahn and Dermietzel, 1988). Goat anti-rabbit IgG coupled to colloidal gold particles (10 nM in diameter) were purchased from Janssen Pharmaceutica (Beerse, Belgium). RESULTS lmmunofluorescence Sections of the tail and head portion of the epididyma1 duct incubated with polyclonal antibodies to fimbrin, ezrin, and actin resulted in a bright staining of the long wavy bundles of stereocilia that project from the apical surface of the columnar principal cells (Figs. la,b, 2a). Immunofluorescence was observed along the entire length of the stereocilia. Identical results were obtained with stereocilia of the ductus deferens (Fig. lc). No immunostaining specific for villin was observed in stereocilia using both poly- and mono-clonal antibodies directed against villin. In this respect, sperm- way stereocilia differ from brush border microvilli of enterocytes and kidney tubules but resemble inner ear stereocilia that do also not contain villin. However, in contrast to inner ear stereocilia and brush border microvilli stereocilia of the epididymis reacted with two different antibodies specific for a-actinin. In sections double-labelled with antibodies to a-actinin and actin it became evident that the a-actinin label was restricted to the basal portions (stem regions) of the stereocilia and absent from their remaining mid and distal portions (Fig. 2a,b). Anti-a-actinin and anti-actin reacted also strongly with the hillock area of the apical cell pole. A dotted staining pattern along the lateral cell borders is most likely due to labelling of the zonula adhaerens and type I1 desmosomes (adhaerens plaques) (Drenckhahn and Franz, 1986). In sections of ductuli efferentes antibodies to actin, fimbrin, ezrin, and villin labelled the rather short microvilli of the nonciliated epithelial cells. a-Actinin-like immunoreactivity was confined to the zonula adhaerens and the lateral cell border but was absent from the microvillus border of ductuli efferentes (Fig. 3a,b,c,d). Thus, the microvillus border of ductuli efferentes resembles by these criteria the intestinal and kidney brush borders. lmmunoelectron Microscopy Immunofluorescence microscopy did not allow to resolve the subcellular location of a-actinin in the spermway stereocilia. In particular, we wanted to know whether a-actinin extends into the stereocilia or whether it is restricted to the cone-shaped hillock area that contains the rootlets of stereocilia. By immu- 60 D. HOFER AND D. DRENCKHAHN Fig. 2. Luminal border of the epididymal duct double-immuno-labelled with a monoclonal antibody against actin (a),and a polyclonal antibody against a-actinin (b).Whereas the actin antibody shows intense labelling of the entire length of stereocilia (indicated by arrowhead), the a-actinin immunostaining is restricted to the hillock area and proximal portion of the stereocilia. Note also staining of the area of the zonulae adhaerentes (small arrows) and numerous small dots along the basolateral cell margins probably representing type I1 desmosomes (puncta adhaerentia). Bar: 10 pm. nogold labelling of ultrathin sections of epididymal tissue we confirmed our impression that the immunolabel specific for a-actinin is present in both the hillock and the stem portions of stereocilia (Fig. 4).The label was strongest in the hillock area and in the stem region with little label extending into stereocilia beyond their branching points. Within the remaining mid and apical portion of stereocilia the immunogold label was negligible. Significant immunogold labelling was also seen in association with the zonula adhaerens (Fig. 4) and type I1 desmosomes (not shown). crease the membrane surface area available for integral membrane proteins that participate in resorption (e.g., kidney and intestinal brush borders, spermway stereocilia), secretion [microvilli of gastric parietal cells (Ito, 198713, or chemosensation [microvilli of receptor cells in taste buds and the vomeronasal organ (Vaccarezza et al., 1981; Roper, 198911. Other types of microvilli/stereocilia provide lever-like processes involved in mechanosensation [e.g., stereocilia of inner ear hair cells (Hudspeth, 1989) and microvilli of skin Merkel cells (Gould et al., 198511. According to these different functions, microvilli/stereocilia display pronounced morphological differences ranging from the uniformly sized and regularly arranged microvilli of the intestinal and kidney brush border to the thick and long stereocilia of the inner ear hair cells with their organpipe-like arrangement and, finally, to the long flexible stereocilia of the male sperm passageway that display an unusual branching pattern in their basal portions. These morphologic differences indicate differences in the organization of the scaffolding cytoskeleton that is generally believed to control stiffness and size of microvilli/stereocilia. In the present study we investigated the microvillil stereocilia lining the male sperm passageway with respect to the major proteins that constitute the support- lrnrnunoblotting In immunoblots fimbrin (68 kD band) and a-actinin (100 kD band) were detected in both the tail and head portions of the epididymis (Fig. 5 ) . In contrast poly- and mono-clonal antibodies to villin did not detect any polypeptide band in the tail of the epididymis. However, both antibodies against villin detected a distinct villin band (95 kD band, comigrating with intestinal villin) in the head of the epididymis that is a mixed tissue containing both ductuli efferentes and the ductus epididymidis. DISCUSSION Microvilli and stereocilia are rod-shaped cellular projections serving various functions such as to in- SPERMWAY STEREOCILIA 61 Fig. 3. Localization of ezrin (a), fimbrin (b), a-actinin (c), and villin (d) in cross sections of ductuli efferentes from the head of the epididymis. Immunoreactivityis confined to the short apical microvilli in a, b and d. Note absence of a-actinin staining in apical microvilli but immunolabelling of zonulae adhaerentes (arrows) and type I1 desmosomes. L, lumen. Bar: 10 pm. ive cytoskeleton of microvilli/stereocilia a t other fimbrin but not villin (Flock et al., 1982; Drenckhahn locations. The main outcome can be summarized as fol- et al., 1985, 1991). Thus, spermway stereocilia provide lowed (see also Table 1): First of all we observed that another example of a microvillus type lacking one of the cytoskeleton of the microvilli associated with the the actin bundling proteins of the kidney and intestinal luminal surface of nonciliated epithelial cells of ductuli brush border. Beside this similarity to inner ear stereocilia, spermefferentes resembles strongly the cytoskeleton of brush border microvilli in other locations in that these mi- way stereocilia differ from their counterparts in the crovilli contain an actin core supplemented with the inner ear in two aspects. First, spermway stereocilia two actin-bundling proteins villin and fimbrin and contain ezrin, which we did not observe in inner ear with the plasmalemmal linker molecule, ezrin. In this stereocilia (unpublished observations). In this respect respect the microvillus border of nonciliated epithelial spermway stereocilia are related to brush border micells of ductuli efferentes can be considered as a further crovilli. Although ezrin has been recently shown in fiexample of a typical brush border. broblasts (Tsukita et al., 1994) to link actin filaments In contrast, stereocilia of the ductus deferens and to the cell surface glycoprotein CD 44, the binding site ductus epididymidis displayed some conspicuous differ- of ezrin at the microvillus membrane is still unknown. ences with respect to the composition of the cytoskele- Since ezrin is typically found in cell surface projections ton. Similarities shared by all three types of microvilli/ involved in resorptive processes as for example in mistereocilia exist in the actin filament core bundle crovilli of the intestinal and kidney brush border crosslinked by one actin filament bundling protein, (Bretscher, 1983; Gould et al., 1986) and in the amninamely, fibrin. Unlike brush border microvilli, sperm- otic epithelium (Wolf et al., 19911, it is likely that ezrin way stereocilia do not contain the second bundling pro- links membrane-bound enzymes or transporters to the tein, villin. In this respect spermway stereocilia resem- actin core bundle. ble inner ear stereocilia that do also contain only Spermway stereocilia differ from all other microvilli/ 62 D. HOFER AND D. DRENCKHAHN M,x103 1 2 3 4 5 6 10068 - 5 Fig. 5. Immunoblot analysis of the tail (lanes 2,4,6) and head portion (lanes 1,3,5) of the human epididymis incubated with antibodies to fimbrin (lanes 1,2),a-actinin (3,4), and villin (5,6).Note that fimbrin and a-actinin are expressed in both the tail and head portion whereas villin is only expressed in the head but absent from the tail. tuft-like arrangement of the stereocilia including the formation of the apical hillock from which the stereocilia arise. It has been recently shown that the actin binding sites for a-actinin and fimbrin overlap (Holtzman et al., 1994; Honts et al., 1994; McGough et al., 1994), suggesting that a-actinin and fimbrin might compete for the same binding site in the stem portions and rootlets of spermway stereocilia. Taking into account that fimbrin forms crosslinks of about 15 nm in Fig. 4. Electron micrograph of the apical portion of the epithelium of width, whereas a-actinin can form crossbridges of up to the epididymal duct (head portion) labelled with an affinity-purified 100 nm in size, it is possible that a-actinin occupies antibody against a-actinin and 10 nm gold particles coated with antiare separabbit IgG. Beside the zonula adhaerens (ZA) the a-actinin label is binding sites where adjacent also present in both the coneshaped hillock area (H) and the stem rated by a distance greater than the length of the fimregions (*) of stereocilia. Note negligible immunolabel in remaining brin molecule. At such places the long flexible a-actinin portions of stereocilia (arrows). Bar: 1 km. molecule could serve to hold adjacent core bundles together, thereby creating the thicker stem portions (Fig. 6). ~I stereocilia so far investigated in that they contain in their basal portions a-actinin. a-Actinin is a widespread actin filament crosslinking protein, found in the muscular Z-lines, densities of smooth muscle, densities of stress fibres, adhaerens type intercellular junctions, and focal cell-to-substrate contacts (Critchley, 1993). The function of a-actinin is to crosslink actin filaments of both opposite and uniform polarity (Meyer and Aebi, 1990). In addition, a-actinin can bind actin filaments to integral membrane proteins of the plasma membrane, such as the p,-subunit of integrins (Otey et al., 1990). Since in spermway stereocilia a-actinin was confined to the branching stem portions of stereocilia in the apical hillock, it appears reasonable to assume that a-actinin is involved in this unique anastomosing morphology in that it forms crosslinks between the core bundles of adjacent stereocilia. These crosslinks might be important for generation of the thicker stem portions and for stabilization of the cytoplasmic adhesive bridges that exist between neighbouring stereocilia. Such crosslinks could play an important role in the formation of the Once separated into individual stereocilia enveloped by the plasma membrane, a-actinin would lose its bivalent (cross-linking) binding sites and detach from the tightly packed core bundles crosslinked by fimbrin. Such a mechanism could explain why a-actinin is more or less restricted to the basal portion (stem portion) of stereocilia and is absent from the distal two thirds of their length. However, such a mechanism could not explain why the stem portions branch. One way of how this branching pattern might be controlled would be by a temporally separated initiation of the expression of fimbrin and a-actinin during development. If fimbrin is expressed at high levels early during stereociliogenesis, individual stereocilia might grow out lacking stem portions and cytoplasmic bridges. The expression of a-actinin might be low at this early stage. As soon as a-actinin becomes expressed a t higher levels, the rootlet portions of stereocilia would become crosslinked and, during further growth of stereocilia, the rootlets might become incorporated into the hillock and stem region. 63 SPERMWAY STEREOCILIA TABLE 1. Components of the actin filament cytoskeleton of microvillVstereociliain different locations Intestine, enterocvtesl Actin Villin Fimbrin Myosin-I Ezrin a-Actinin Microvilli Retina, Kidney, proximal tubule' Miiller cells3 + + + + +- + + + + +- + + + - - Ductuli efferentes + + + + +- stereocilia Ductus epididymidis ductus deferens + + nd5 + - + Inner ear4 +- + 26 - 'Drenckhahn and Dermietzel, 1988. *Rodman et al., 1986. 3Hofer and Drenckhahn, 1993. 4Drenckhahn et al., 1982; 1991 and additional personal data. 'nd not detected. 'Not detected in chicken (Drenckhahn et al., 1991), but present in stereocilia of frog saccular macula (Gillespie et al., 1993). Stereocilia Actin filaments bundled by fimbrin Stems with actin bundles interconnected by a-actinin (-) Hillock --. Zonula occludens Zonula adhaerens ype II - Desmosome Fig. 6. Summarizing diagram illustrating the location and possible role of a-actinin in generation and/or maintenance of the unique architecture of spermway stereocilia. In order to test this hypothesis, we have begun to study the development of spermway stereocilia in laboratory animals. ACKNOWLEDGMENTS We thank Martina Zink for skillful technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft. LITERATURE CITED Bretscher, A. 1983 Purification of an 80000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J . Cell Biol., 97:425-432. Bretscher, A. 1989 Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J. Cell Biol., 108:921-930. Bretscher, A. and K. Weber 1980a Fimbrin, a new microfilamentassociated protein present in microvilli and other cell surface structures. J . Cell Biol., 86:335-340. Bretscher, A. and K. Weber 1980b Villin is a major protein of the microvillus cytoskeleton which binds both G and F actin in a calcium dependent manner. Cell, 20:839-847. Burnette, W.N. 1981 Western blotting: Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radionated protein A. Anal. Biochem., 112:195-203. Critchley, D. 1993 a-Actinins. In: Guidebook to the Cytoskeletal and Motor Proteins. T. Kreis and R. Vale, eds. Sambrook and Tooze, Oxford, pp. 22-23. Drenckhahn, D. and R. Dermietzel 1988 Organization of the actin filament cytoskeleton in the intestinal brush border: A quantitative and qualitative immunoelectron microscope study. J . Cell Biol., 107:1037-1048. Drenckhahn, D., K. Engel, D. Hofer, C. Merte, L. Tilney, and M. Tilney 1991 Three different actin filament assemblies occur in every hair cell: Each contains a specific actin crosslinking protein. J . Cell Biol., 112:641-651. Drenckhahn, D. and H. Franz 1986 Identification of actin-, a-actinin-, and vinculin-containing plaques a t the lateral membrane of epithelial cells. J. Cell Biol., 102:1843-1852. Drenckhahn, D., H.-D. Hofmann, and H.G. Mannherz 1983 Evidence for the association of villin with core filaments and rootlets of intestinal epithelial microvilli. Cell Tissue Res., 228:409414. Drenckhahn, D., Th. Jons, and F. Schmitz 1993 Production of polyclonal antibodies against proteins and peptides. Methods. Cell Biol., 37:7-56. Drenckhahn, D., J. Kellner, H.G. Mannherz, U. Groschel-Stewart, J . Kendrick-Jones, and J. Scholey 1982 Absence of myosin-like immunoreactivity in stereocilia of cochlear hair cells. Nature, 300: 531-532. Drenckhahn, D. and C. Merte 1987 Restriction of the human kidney band 3-like anion exchanger to specialized subdomains of the basolateral plasma membrane of intercalated cells. Eur. J . Cell Biol., 45:107-115. Drenckhahn, D., T. Schafer, and M. Prim 1985 Actin, myosin, and associated proteins in the vertebrate auditory and vestibular organs: Immunocytochemical and biochemical studies. In: Auditory Biochemistry. D. Drescher, ed. Charles C. Thomas Publisher, Springfield, IL, pp. 312-335. Dym, M. 1988 The male reproductive system. In: Cell and Tissue Biology. L. Weiss, ed. Urban & Schwarzenberg, Baltimore, Munich, pp. 931-971. Fawcett, D.W. and K.R. Porter 1954 A study of the fine structure of ciliated epithelia. J. Morphol., 94:221-264. Flock, A., A. Bretscher, and K. Weber 1982 Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells. Hear. Res., 6:75-89. Flock, A. and H. Cheung 1977 Actin filaments in sensory hairs of the inner ear receptor cells. J. Cell Biol., 75:339-343. Gillespie, P.G., M.C. Wagner, and A.J. Hudspeth 1993 Identification of a 120 kD hair-bundle myosin located near stereociliary tips. Neuron 11.581-594. Glenney, I.R., M. Osborn, and K. Weber 1982 The intracellular localization of the microvillus llOk protein, a component considered to be involved in side-on membrane attachment of F-actin. Exp. Cell Res., 138:199-205. Goodson, H.V. and J.A. Spudich 1993 Molecular evolution of the my- 64 D. HOFER AND D. DRENCKHAHN osin family: Relationships derived from comparisons of amino acid sequences. Roc. Natl. Acad. Sci. U.S.A., 90:659-663. Gould, K.L., J.A. Cooper, A. Bretscher, and T. Hunter 1986 The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein. J . Cell. Biol., 102:660-669. Gould, V.E., R. Moll, J. Moll, J . Lee, and W.W. Franke 1985 Neuroendocrine (Merkel) cells of the skin: Hyperplasias, dysplasias, and neoplasms. Lab. Invest., 52r334-353. Hofer, D. and D. Drenckhahn 1993 Molecular heterogeneity of the actin filament cytoskeleton associated with microvilli of photoreceptors, Miiller’s glial cells and pigment epithelial cells of the retina. Histochemistry, 99:29-35. Holstein, A.F. 1969 Morphologische Studien am Nebenhoden des Menschen: Zwanglose Abhandlung aus dem Gebiet der normalen und pathologischen Anatomie. Hrsg. W. Bargmann und W. Doerr, Heft 20. Thieme, Stuttgart. Holtzman, D.A., K.J. Wertman, and D.G. Drubin 1994 Mapping actin surface required for functional interaction in vivo. J . Cell Biol., 126:423-432. Honts, J.E., T.S. Sandrock, S.M. Browner, J.L. O’Nell, and A.E.M. Adams 1994 Actin mutations that show sumression with fimbrin mutations identify a likely fimbrin-binding site on actin. J . Cell Biol., 126:413-422. Horstmann, E. 1962 Elektronenmikroskopie des menschlichen Nebenhodenepithels. Z. Zellforsch. 57t692-718. Hudspeth, A.J. 1989 How the ear’s works work. Nature, 341 :397-404. Ito, S. 1987 Functional gastric morphology. In: Physiology of the Gastrointestinal Tract, 2nd ED. Johnson L.R., Raven Press, New York, pp. 817-851. Luna, E.J. 1991 Molecular links between the cytoskeleton and membranes. Curr. Opin. Cell Biol., 3.120-126. Major, H.D., J.C. Hampton, and B. Rosario 1961 A simple method for removing the resin from epoxy-embedded tissue. J. Biophys. Biochem. Cytol., 9:909-910. Matsudaira, P.T. and D.R. Burgess 1979 Identification and organiza- tion of the components in the isolated microvillus cytoskeleton. J . Cell Biol., 83r667-673. Matsudaira, P.T. 1991 Molecular organization of actin crosslinking proteins. TIBS, 16:87-92. Meyer, R.K. and U. Aebi 1990 Bundling of actin filaments by a-actinin depends on its molecular length. J. Cell Biol., 110:2013-2024. McGough, A,, M. Way, and D. DeRosier 1994 Determination of the a-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis. J. Cell Biol., 126:433-443. Mooseker, M.S., and L.G. Tilney 1975 Organization of an actin filament-membrane complex. J . Cell Biol., 67:725-743. hlorita, J . 1966 Some observations on the fine structure of the human ductuli efferentes testis. Arch. Histol. Jpn., 26t341-365. Otey, C.A., F.M. Paralko, and K. Burridge 1990 An interaction between a-actinin and the @,-integrinsubunit in vitro. J . Cell Biol., 111:721-729. Popovic, N.A., D.G. McLeod, and A.A. Borski 1973 Ultrastructure of human vas deferens. Invest. Urol., 10:266-277. Rodman, J.S., M.S. Mooseker, and M.G. Farquhar 1986 Cytoskeletal proteins of the rat kidney proximal tubule brush border. Eur. J . Cell Biol., 42:319-327. Roper, S.D. 1989 The cell biology of vertebrate taste receptors. Annu. Rev. Neurosci., 12:329-353. Stohr, Ph. 1906 Lehrbuch der Histologie. Gustav Fischer Verlag, Jena. Tsukita, S., K. Oishi, N. Sato, J. Sagara, A. Kawai, and S. Tsukita 1994 ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J . Cell Biol., 126:391-401. Vaccarezza, O.L., L.N. Sepich, and J.H. Tramezzani 1981 The vomeronasal organ of the rat. J. Anat., 1321167-185. Wolf, H.J., W. Schmidt, and D. Drenckhahn 1991 Immunocytochemical analysis of the cytoskeleton of the human amniotic epithelium. Cell Tissue Res., 266:385-389.