Journal of Cell Science 109, 1229-1239 (1996) 1229 Printed in Great Britain © The Company of Biologists Limited 1996 JCS7092

Identification and characterization of espin, an -binding localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations

James R. Bartles*, Allison Wierda and Lili Zheng Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

Ectoplasmic specializations are membrane-cytoskeletal and two peptides which contain clusters of multiple gluta- assemblages found in Sertoli cells at sites of attachment to mates bracketed by arginines, lysines and glutamines in a elongate spermatids or neighboring Sertoli cells. They are pattern reminiscent of the repetitive motif found in the characterized in part by the presence of a unique junctional protein trichohyalin. The -like repeats and a 66 plaque which contains a narrow layer of parallel actin amino acid peptide in the C terminus show significant bundles sandwiched between the Sertoli cell plasma sequence similarity to encoded by the forked membrane and an affiliated cistern of endoplasmic of Drosophila. A fusion protein containing the C-terminal reticulum. Using a monoclonal antibody, we have identified 378 amino acids of espin was found to bind with high ‘espin,’ a novel actin-binding protein localized to ectoplas- affinity (Kd=~10 nM) to F-actin in vitro with a stoichiome- mic specializations. By immunogold electron microscopy, try of ~1 espin per 6 actin monomers. When expressed by espin was localized to the parallel actin bundles of ecto- transfected NRK fibroblasts, the same C-terminal plasmic specializations at sites where Sertoli cells contacted fragment of espin was observed to decorate actin fibers or the heads of elongate spermatids. The protein was also cables. On the basis of its structure, localization and prop- detected at the sites of ectoplasmic specializations between erties, we hypothesize that espin is involved in linking actin neighboring Sertoli cells. Espin exhibits an apparent filaments to each other or to membranes, thereby poten- molecular mass of ~110 kDa in SDS gels. It is encoded by tially playing a key role in the organization and function of an ~2.9 kb mRNA, which was found to be specific to testis the ectoplasmic specialization. among the 11 rat organs and tissues examined. On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein which contains 8 ankyrin-like repeats in its N- Key words: Espin, Junction, Seminiferous epithelium, , terminal third, a potential P-loop, two proline-rich peptides Ankyrin repeat, Forked, Spermiogenesis

INTRODUCTION It consists of two cell populations, spermatogenic cells and Sertoli cells, and undergoes cyclical changes in organization to The adhesion of cells to form functional tissues and organs is facilitate the continuous production of spermatozoa (Leblond mediated by junctions. Familiar examples include intercellular and Clermont, 1952a,b; Russell and Peterson, 1985; Vogl, junctions, such as the tight junction, the zonula adherens and the 1989). The structure of the seminiferous epithelium appears to desmosome, and cell-extracellular matrix junctions, such as the be determined largely by the Sertoli cells. These large, focal contact and the hemidesmosome. Although these junctions irregular epithelial cells span the seminiferous epithelium from differ from each other in many important respects, each is a site base to lumen (Fig. 1). Near the base of the epithelium, the where adhesion molecules in specialized domains of the plasma Sertoli cells are joined by junctional complexes, which contain membrane are connected to submembranous plaques which extensive tight junctions and divide the epithelium into basal contain peripheral membrane proteins and elements of the and adluminal compartments. The spermatogenic cells exhibit cortical cytoskeleton (reviewed by Gumbiner, 1996). This clus- a net displacement from the base of the epithelium toward the tering or tethering of adhesion molecules, peripheral membrane lumen as they undergo proliferation, meiosis and differen- proteins and cytoskeletal elements is believed to stabilize tiation (or spermiogenesis). They are tranlocated from the basal adhesion and afford a path along which forces and signals can compartment through the Sertoli cell-Sertoli cell junctional be relayed between the surface and the interior of the cell. complex into the adluminal compartment during meiosis, and Few tissues are as complex as the seminiferous epithelium. it is in the adluminal compartment where the resulting haploid 1230 J. R. Bartles, A. Wierda and L. Zheng spermatogenic cells (spermatids) undergo the stepwise trans- cialization. We have named the protein ‘espin’ (ectoplasmic formation into highly asymmetrical spermatozoa (Leblond and specialization + -in) on the basis of its localization. Clermont, 1952a,b). Although the spermatogenic cells remain intimately associ- MATERIALS AND METHODS ated with Sertoli cells throughout spermatogenesis, the inter- cellular junctions formed between these two cell populations Molecular biological procedures were carried out as described change dramatically (reviewed by Russell and Peterson, 1985; (Sambrook et al., 1989; Ausubel et al., 1989) with minor modifica- Vogl, 1989; Vogl et al., 1991; Pelletier and Byers, 1992). Sper- tions. Monoclonal antibodies were prepared in mice (Hubbard et al., matogenic cells up to and including early (‘round’) spermatids 1985) using Percoll gradient fractions enriched in rat late spermatids are linked to Sertoli cells via an ocassional desmosome or and testicular spermatozoa (Petruszak et al., 1991) as the immunogen. short, discontinous strip of tight junction. However, as the Most of the late spermatids in this preparation retain an ectoplasmic spermatids elongate and become positioned with their heads in specialization stuck to their head as a tightly adherent fragment of crypt plasma membrane derived from a Sertoli cell (see below). The deep invaginations of the apical surface of Sertoli cells known resulting monoclonal antibodies were screened by western blotting. as crypts, another type of intercellular junction becomes pre- One monoclonal antibody, an IgM, was selected on the basis of its dominant. This intercellular junction is called the ectoplasmic reactivity against a ~110 kDa protein (espin) present in the specialization. It is formed adjacent to the head of the elongate immunogen. An ~1.4 kb cDNA corresponding to the 3′ end of espin spermatid (Fig. 1) and is distinguished by the presence of a mRNA was identified using the espin monoclonal antibody in con- unique junctional plaque which contains a narrow layer of junction with 125I-labeled goat anti-mouse Ig to screen a λgt11 rat hexagonally packed, parallel actin bundles sandwiched testis cDNA library (Clontech, Palo Alto, CA). The ~1.4 kb cDNA between the Sertoli cell crypt plasma membrane and an affili- was used to identify an overlapping ~1.6 kb cDNA upon rescreening ated cistern of endoplasmic reticulum (reviewed by Russell and of the library by Southern blotting. These cDNAs were introduced into the pBluescriptSK+ phagemid (Stratagene, La Jolla, CA) and Peterson, 1985; Vogl, 1989; Vogl et al., 1991). There is com- α ′ pelling evidence to suggest that each elongate spermatid propagated in E. coli DH5 . cDNAs corresponding to the 5 end of espin mRNA were obtained using the 5′ RACE System for Rapid remains tightly attached to the Sertoli cell via an ectoplasmic Amplification of cDNA Ends (Life Technologies, Gaithersburg, MD), specialization until near the end of spermiogenesis. Just prior total RNA isolated from rat testis and nested primers positioned ~200 to release of the very late spermatids into the lumen as sper- bp downstream from the 5′ end of the ~1.6 kb cDNA obtained by matozoa, these ectoplasmic specializations disappear, possibly library screening and engineered to contain a SalI site. cDNAs were through the transient formation of a related holdfast device introduced into M13mp cloning vectors (Life Technologies) and known as the tubulobulbar complex. propagated in Escherichia coli JM109. Single-stranded templates cor- Ectoplasmic specializations have also been observed as part responding to both the coding and noncoding strands were sequenced of the junctional complexes between neighboring Sertoli cells in their entirety using [35S]dATPαS and the Sequenase Version 2.0 (reviewed by Russell and Peterson, 1985; Vogl, 1989; Vogl et kit (Amersham, Cleveland, OH). Sequence comparisons were carried al., 1991). The contributions of these ectoplasmic specializa- out using the TFASTA program available through the University of tions to the Sertoli cell-Sertoli cell junctional complex remain Wisconsin Genetics Computer Group (GCG) Sequence Analysis Software Package (Devereux et al., 1984). Total RNA was isolated to be determined. Like other key elements of this junctional from rat organs and tissues by guanidine thiocyanate extraction and complex, however, these ectoplasmic specializations appear to centrifugation in CsCl gradients. Aliquots containing ~10 µg of RNA undergo periodic disassembly and reassembly as the meiotic were resolved in formaldehyde-denaturing agarose gels, transferred to spermatogenic cells are translocated from the basal to the nitrocellulose and probed with randomly primed 32P-labeled espin adluminal compartment of the seminiferous epithelium during cDNAs (Nehme et al., 1993) prepared using the ~1.4 kb cDNA that each successive round of spermatogenesis. encoded the C-terminal 378 amino acids of espin and the 3′ untrans- Even though there is a wealth of information currently lated sequence. available regarding the ultrastructure of the ectoplasmic spe- An espin fusion protein was prepared using the Protein Fusion and cialization, relatively little is known about the organization of Purification System (New England Biolabs, Beverly, MA). The ~1.4 this intercellular junction at the molecular level. Unlike other kb cDNA that encoded the C-terminal 378 amino acids of the espin actin-associated intercellular junctions (Gumbiner, 1996), protein was introduced into the pMAL-c2 vector and used to express α the corresponding maltose-binding protein fusion protein in E. coli typical cadherins, - and appear to be TB1. The fusion protein was isolated from Tris-buffered saline absent (Byers et al., 1994). However, the adhesion molecule extracts of frozen-thawed, sonicated bacteria by affinity chromatog- α6β1-integrin (Salanova et al., 1995) and the actin-binding raphy on amylose resin as described by the manufacturer. The maltose proteins α- (Franke et al., 1978) and (Grove et eluate was concentrated by ultrafiltration over a PM10 membrane al., 1990) have been localized to ectoplasmic specializations (Amicon, Beverly, MA) and either used to immunize three rabbits by immunofluorescence. In addition, the actin-bundling protein (Bartles et al., 1985) or dialyzed against 0.1 M KCl, 2 mM MgCl2, fimbrin has been detected on western blots of subcellular 0.005% (w/v) NaN3, 10 mM imidazole-HCl, pH 7.4, and clarified by fractions containing ectoplasmic specializations (Grove and centrifugation at 150,000 g for 90 minutes at 4¡C for use in binding Vogl, 1989). assays (see below). IgG was isolated from pooled preimmune and To identify novel proteins of the ectoplasmic specialization immune rabbit sera by chromatography on columns of diethy- that might account for the organization of its unique junctional laminoethyl-cellulose (Bartles et al., 1985). IgG, maltose-binding protein and espin fusion protein were coupled to cyanogen bromide- plaque and make important contributions to its function, we activated Sepharose 4B (Bartles et al., 1985, 1987). To deplete the have prepared monoclonal antibodies to fractions containing serum of antibodies directed against the maltose-binding protein, ectoplasmic specializations. In this article, we describe the iden- pooled rabbit immune serum was passed over a maltose-binding tification and characterization of a novel actin-binding protein protein-Sepharose 4B column. The rabbit polyclonal espin antibody localized to the parallel F-actin bundles of the ectoplasmic spe- was then affinity purified from the depleted serum by chromatogra- Espin and the ectoplasmic specialization 1231 phy on columns of espin fusion protein-Sepharose 4B using low pH glycine buffer as eluent (Bartles et al., 1987). The resulting affinity purified polyclonal espin antibody was neutralized immediately and dialyzed against phosphate-buffered saline containing 0.02% (w/v) NaN3 for use in western blotting and immunolocalization experi- ments. Western blotting was carried out using either monoclonal espin IgM followed by 125I-goat anti-mouse Ig or affinity purified rabbit polyclonal espin antibody followed by 125I-Protein A (Bartles et al., 1985, 1987). Espin was isolated from Triton X-100/n-octyl-β-D-glu- copyranoside extracts of rat testis homogenate by large-scale immunoprecipitation using rabbit polyclonal espin IgG-Sepharose 4B (Bartles et al., 1985, 1987), resolved in reducing SDS gels and trans- ferred to Problott membrane (Applied Biosystems, Inc., Foster City, CA). The espin band was identified by staining with Coomassie blue, cut out and sent to the Macromolecular Structure Facility in the Department of Biochemistry at Michigan State University (East Lansing, MI) for the preparation, isolation and gas-phase microse- quencing of tryptic peptides. In preparation for immunofluorescence, cells obtained by the Fig. 1. Schematic depiction of a cross-section of seminiferous mechanical dissociation of rat testis were fixed to poly-L-lysine- epithelium illustrating the architecture of the Sertoli cell and the coated slides or coverslips with 2% paraformaldehyde in phosphate- locations of ectoplasmic specializations. The unique junctional buffered saline, quenched with NH4Cl and bovine serum albumin and plaque of the ectoplasmic specialization includes a narrow layer of labeled with affinity purified polyclonal espin antibody or hexagonally packed, parallel bundles of F-actin (dotted line) and an preimmune IgG and fluorescein-labeled phalloidin (Molecular affiliated cistern of endoplasmic reticulum (thick black line adjacent Probes, Eugene, OR) followed by rhodamine-labeled goat anti-rabbit to dotted line). Spermatogenic cells at stages of development other IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) than the late spermatid and junctions other than ectoplasmic in the presence of 0.025% (w/v) saponin (Petruszak et al., 1991). specializations have been omitted for clarity. (Modified after Vogl et Immunogold labeling was carried out similarly, except that the phal- al., 1991.) loidin was left out and the rhodamine-labeled secondary antibody was replaced by 12 nm diameter colloidal gold conjugate of goat anti- rabbit IgG (Jackson ImmunoResearch Laboratories) which had been et al., 1989). The NRK cells were cultured on coverslips in preabsorbed against an excess of fixed-quenched testicular cells Dulbecco’s modified Eagle’s medium containing 10% (v/v) calf embedded in low-melting-temperature agarose (Cesario and Bartles, serum and penicillin/streptomycin. They were fixed directly on the 1994). After the final rinse, the specimens were postfixed and coverslips, with or without a 5 minute pretreatment in ice-cold 0.1% processed for electron microscopy (Cesario and Bartles, 1994). For Triton X-100 in phosphate-buffered saline, and processed for immunoperoxidase labeling, rat testis was fixed by perfusion with immunofluorescence as described above. Bouin’s fluid and embedded in paraffin (Cesario et al., 1995). Sections (5 µm) were deparaffinized with xylenes, labeled with affinity purifed polyclonal espin antibody or preimmune IgG RESULTS followed by horseradish peroxidase-conjugated donkey anti-rabbit F(ab′)2, reacted with H2O2 and 3,3′-diaminobenzidine and counter- stained with hematoxylin (Cesario et al., 1995). Identification, sequence and tissue distribution F-actin was prepared by dilution of rabbit skeletal muscle actin Espin was first identified on western blots as a ~110 kDa (Cytoskeleton, Inc., Denver, CO) into 0.1 M KCl, 2 mM MgCl2, 1 protein that reacted with an IgM monoclonal antibody prepared mM ATP, 0.005% (w/v) NaN3, 10 mM imidazole-HCl, pH 7.4, and from mice that had been immunized with a mixture of rat late incubation for 60 minutes at 37¡C. To assay for binding to F-actin spermatids and testicular spermatozoa (data not shown). Most (Menkel et al., 1994), an equal volume of solution containing different of the late spermatids in this fraction retain an ectoplasmic spe- amounts of affinity purified espin fusion protein or maltose-binding cialization stuck to their head in the form of a tightly adherent protein in the same buffer minus ATP were incubated with the fragment or ‘cap’ of crypt plasma membrane derived from a preformed actin filaments (at a final concentration of either 50 or 500 µ Sertoli cell (see below). Overlapping cDNA clones of ~1.4 kb g/ml) for 60 minutes at 37¡C. Following centrifugation for 90 ′ minutes at 150,000 g, the levels of espin fusion protein, maltose- and ~1.6 kb corresponding to the 3 end of espin mRNA were binding protein, and actin present in the supernatant and pellet obtained by screening an oligo-dT-primed λgt11 rat testis fractions were determined by laser densitometric scanning of cDNA library. cDNAs of ~1.1 kb corresponding to the 5′ end Coomassie blue-stained SDS gels using the rabbit skeletal muscle of espin mRNA were obtained from rat testis RNA by RACE- actin as the protein standard. The maltose-binding protein used as a PCR using nested primers corresponding to the 5′ end of the control in the actin-binding experiments was actually the MBP2* ~1.6 kb cDNA. protein, a bacterially expressed version of the wild-type maltose- The combined nucleotide sequence, the predicted amino binding protein that includes a few additional amino acids encoded acid sequence and a stick-figure model of espin are shown in by the pMAL-c2 polylinker (New England Biolabs). Fig. 2. The 2,783 bp combined cDNA was similar in size to For transient transfection, the ~1.4 kb espin cDNA that encoded the C-terminal 378 amino acids of the protein was introduced into the the ~2.9 kb espin transcript detected by northern blotting (see pcDNA3 vector (Invitrogen, San Diego, CA) and used to transiently below). It included a single large open-reading frame of 2,508 transfect cells of the rat NRK fibroblastic line (ATCC, Rockville, nt that was predicted to encode a 836 amino acid protein with MD) using a modification of the calcium precipitation method that an isoelectric point of 6.89 and a Mr of 90,950, which is a little includes treatment chloroquine and a brief glycerol shock (Sambrook less than the ~110 kDa apparent molecular mass noted for 1232 J. R. Bartles, A. Wierda and L. Zheng espin on western blots of reducing SDS gels (see below). Of analysis. Unambiguous assignments were obtained for tryptic the two possible initiation codons located near the 5′ end of the peptides or segments of tryptic peptides corresponding to cDNA (nt 17-19 and nt 35-37), only the first occurred within amino acids 20-39, 556-568, 662-684, 775-779 and 820-822 a Kozak consensus (Kozak, 1987). The first in-frame stop (see underlined peptides in Fig. 2). codon appeared at nt 2,524-2,526, 245 nt from what appeared The espin protein was predicted to contain 8 ankyrin-like to be the beginning of a poly(A) tail, even though no (A)ATAA repeats (Michaely and Bennett, 1993; Hoover et al., 1993) in consensus site for poly(A) addition (Sheets et al., 1990) was its N-terminal third, centered roughly on amino acids 31-60, noted. Stop codons arose at approximately this same position 65-94, 99-129, 133-162, 167-196, 201-230, 234-263, and 266- in all three reading frames. Espin was purified from nonionic 295 (peptides in brackets in Fig. 2). The presence of this motif detergent extracts of rat testicular homogenate by large-scale for protein-protein interaction made the N-terminal third of immunoprecipitation using a polyclonal fusion protein espin resemble segments of other proteins that contain antibody prepared in rabbits (see below) and subjected to multiple ankyrin-like repeats. Foremost among these was the amino acid sequence analysis. Although the amino terminus of large version of the protein encoded by the forked gene of espin proved to be blocked, the amino acid sequences of a Drosophila (Hoover et al., 1993) and the different forms of number of internal peptides were found to be in complete ankyrin (Michaely and Bennett, 1993). This region of espin agreement with those predicted on the basis of cDNA sequence was found to be 31-35% identical in amino acid sequence to

1 t gcc gag ggt gac acc atg gcc ctg gaa cag gcg atg cag gcg gca cgg 1 M A L E Q A M Q A A R 50 cgg ggc gac ctg gac gtg ctg agg tcc ctg cac gcc gcc ggc ctg ctg ggg 12 R G D L D V L R S L H A A G L L G 101 cct tct ctg cgc gac ccg cta gac gcc ctg ccg gtg cac cat gcg gcc cgc 29 P S {L R D P L D A L P V H H A A R 152 tca ggc aag ctg cac tgt ttg cgc tac ttg gtg gag gag gtt gcc ctc cca 46 S G K L H C L R Y L V E E V A} L P 203 gct gtg tcc cgc gcg cgc aac ggc gcc aca cca gcc cat gat gcc gct gcc 63 A V {S R A R N G A T P A H D A A A 254 acg ggc tac ctc tct tgc ctg cag tgg ctg ctc aca cag ggt ggc tgc agg 80 T G Y L S C L Q W L L T Q G G} C R 305 gtg cag gaa aaa gat aac tct ggt gcc acg gtc ctg cac ctg gct gcc cgc 97 V Q {E K D N S G A T V L H L A A R 356 ttt ggc cac ccg gac gtg gtg aac tgg ctg ctg tac cag ggc ggt gcg aac 114 F G H P D V V N W L L Y Q G G A} N 407 tct gcc atc acc aca gac acg ggc gcc ctg cct atc cac tat gct gcc gcc 131 S A {I T T D T G A L P I H Y A A A Fig. 2. The cDNA and amino acid sequence of 458 aaa gga gat ctc ccc tcc atg aag ctt ctt gtc ggg cac tac cct gag gga 148 K G D L P S M K L L V G H Y P} E G rat espin. The peptides verified by amino acid 509 gtg aat gcc caa acc aac aac ggt gcc acg ccc ctg tac ctg gcg tgc cag sequencing are underlined. The eight ankyrin- 165 V N {A Q T N N G A T P L Y L A C Q like repeats in the N-terminal third of espin are 560 gag ggc cac ctg gaa gtg acg aag tac ctt gtg cag gag tgc agt gca gat centered roughly within the peptides shown in 182 E G H L E V T K Y L V Q E C S} A D brackets. The 66 amino acid C-terminal peptide 611 ccg cac ctg cgc gcc caa gac ggc atg acc ccc ctg cat gcc gcg gcg cag 199 P H {L R A Q D G M T P L H A A A Q corresponding to amino acids 723-788 shows 662 atg ggc cac aac cca gtc ctg gtg tgg ctg gtg agc ttt gcg gac gtg agc 39% sequence identity to a peptide present in 216 M G H N P V L V W L V S F A D} V S both the large and small forked proteins; the 713 ttc gag cag gac cac gac ggc gcc aca gcc atg cac ttt gca gcc agc cgc identical amino acids are marked with an 233 F {E Q D H D G A T A M H F A A S R asterisk. The potential P-loop corresponds to 764 ggc cac acc aaa gtg ctc agc tgg ctc ctg ctg cac ggc gca gag atc tcc 250 G H T K V L S W L L L H G A} E I {S amino acids 640-647. The proline-rich peptides 815 cag gac ctg tgg ggc ggg acc ccg ctg cat gat gct gct gag aac ggg gaa correspond to amino acids 421-455 and 582-604. 267 Q D L W G G T P L H D A A E N G E The two peptides that contain clusters of multiple 866 ctg gag tgc tgc cag atc ctc gcg gtg aat ggt gcg ggg ctg gac gtc cgc glutamates bracketed by positively charged 284 L E C C Q I L A V N G A} G L D V R amino acids and glutamine in a fashion 917 gac cac gat ggg tat acg gct gcg gac ctg gct gat ttc aat ggc cac acc 301 D H D G Y T A A D L A D F N G H T reminscent of the trichohyalin repeat correspond 968 cac tgc tcc cgc tac cta cgt acg gtg caa acc ctg agc ttg gaa cac cga roughly to amino acids 743-765 and 778-802. 318 H C S R Y L R T V Q T L S L E H R These sequence data are available from 1019 gtc ctg tcc cgg gat cca tcc atg gac ctg gag gca aag cag ccg gac tca GenBank/EMBL/DDBJ under accession number 335 V L S R D P S M D L E A K Q P D S U46007. Beneath the sequence is a stick-figure 1070 ggc atg tct tca ccc aac acc acc atg tcg gtc cag ccg ccg aac ttt gac diagram depicting the relative positions of the 352 G M S S P N T T M S V Q P P N F D 1121 ctt ggc tca ccc acc agc acc ctc tcc aac tat gac tcc tgc tcc tcc agc eight ankyrin-like repeats (8 ANKYRIN 369 L G S P T S T L S N Y D S C S S S REPEATS), the two proline-rich peptides (PR), 1172 cat tcc agc agc aag ggt cag cgc tct act cga ggt gcc aga tcc tca gac the potential P-loop (P), the 66 amino acid 386 H S S S K G Q R S T R G A R S S D peptide that shows 39% identity to the peptide in 1223 tta cag agc tac atg gac atg ctg aac cca gag cct cgg agc aag caa ggg the forked proteins (FORK), and the two 403 L Q S Y M D M L N P E P R S K Q G 1274 aag cct tca tct cta cca cca ccg cca cca cca agc ttc cct cca cca cca trichohyalin-like repeats (TH). The arrowhead 420 K P S S L P P P P P P S F P P P P indicates the position of the N terminus of the 1325 ccc cca ggc aca cag ctg ccc cca cct cca cca ggc tac cca gct ccc aat espin C-terminal fragment represented in the 437 P P G T Q L P P P P P G Y P A P N espin fusion protein and expressed by transiently 1376 ccc cct gtg ggg ctg cat ttg gat aac att tac atg cag acc aag aac aaa transfected NRK cells. 454 P P V G L H L D N I Y M Q T K N K Espin and the ectoplasmic specialization 1233 regions containing ankyrin-like repeats in the large forked peptides that were especially rich in proline (amino acids 421- protein (GenBank/EMBL/DDBJ accession number D21203), 455 and 582-604; Williamson, 1994), and two peptides human ankyrin B (Z26634), mouse epithelial ankyrin 3 (amino acids 743-765 and 778-802) which contained clusters (L40632) and human ankyrin G (U13616). Only three of the of multiple glutamates bracketed by arginines, lysines and eight ankyrin-like repeats (the third, seventh and eighth; Fig. glutamines in a pattern reminiscent of the repetitive motif 2) more closely resembled the true ankyrin-like consensus found in trichohyalin (Fietz et al., 1993). than the forked version of the ankyrin-like consensus (Hoover The ~1.4 kb espin cDNA that encoded the C-terminal 378 et al., 1993). Outside of the region that included these ankyrin- amino acids of the protein and included the 3′ untranslated like repeats at the N terminus, however, espin showed rela- sequence was used to probe a northern blot of total RNA tively little in the way of significant sequence similarity to prepared from 11 different rat organs and tissues under con- other DNAs and proteins cataloged in the database. One ditions of high stringency. Intense labeling was noted for a notable exception was a stretch of 66 amino acids (723-788) single ~2.9 kb RNA, which was detected only in rat testis (Fig. near the C terminus of the protein (amino acids marked with 3). Overexposure of the blots revealed a minor cross-hybridiz- an asterisk in Fig. 2), which was found to be 39% identical to ing band at ~1.7 kb present in total RNA isolated from small a peptide present in both the large and the small versions of intestine and, to a lesser extent, kidney (Fig. 3). The charac- the Drosophila forked protein (encoded by nt 17,220-17,399 terization of these smaller mRNAs and the low-molecular mass of the forked gene; Hoover et al., 1993). Espin was also espin-related protein they encode will be the subject of a predicted to contain a P-loop consensus site for binding ATP separate article (J. R. Bartles, A. Wierda and L. Zheng, unpub- or GTP (amino acids 640-647; Saraste et al., 1990), two lished data).

1427 ctc cgc cac gtg gag gtg gac tcc ctc aag aag gag ccg agc tcc ggc gac 471 L R H V E V D S L K K E P S S G D 1478 ggc tac tcg ggg cta cgc agg cag gat tcc ggg ctg ctc agg cag gat tcg 488 G Y S G L R R Q D S G L L R Q D S 1529 gag ctg ctg ctc agg cac aac acc gga ctg cgc agg cag gac tcc gac cgc 505 E L L L R H N T G L R R Q D S D R 1580 aaa cag cgt tca ttc agt aaa cag ccc agc acg ggg gac tac tac cgc cag 522 K Q R S F S K Q P S T G D Y Y R Q 1631 ctg ggc cgc agc ccg ggg gag ccg ctg gcc gca cgc ccg ggc atg gcc cac 539 L G R S P G E P L A A R P G M A H 1682 agc gag gag gcg gcg ctg ctc ccc ggg aac cac gtg cac aac ggc tgc tcc 556 S E E A A L L P G N H V H N G C S 1733 gcg gac tcc aaa gcg tcc agg gag ctg ccg ccg cca ccg ccg ccg ccg ccc 573 A D S K A S R E L P P P P P P P P 1784 ctg ccc gag gcc ctg agt tcg ccg cca ccc gcc cca cct ctg ccc atc gag 590 L P E A L S S P P P A P P L P I E 1835 ggc gcg ggc gag ctg cgg aca gcg gcg ttc ctc atc ttc tac tgg cag cac 607 G A G E L R T A A F L I F Y W Q H 1886 caa atc ttt caa cat gat gtc cca acg ggt gac aac tcg gag ttg ctg gct 624 Q I F Q H D V P T G D N S E L L A 1937 gaa ata aaa gca ggc aag agt ctg aaa cca aca ccg cag agc aag ggg ctg 641 E I K A G K S L K P T P Q S K G L 1988 aca aca gtg ttc tca ggc agc ggg cag cca gcc tcc cag cct gag tca cca 658 T T V F S G S G Q P A S Q P E S P 2039 cag cct gcg gtg tca cct ggg cca tct cgg gcc cgg agc ccc acc cca cca 675 Q P A V S P G P S R A R S P T P P 2090 gcc tct ggg cct cag cca ctg ctc aat ggc agc ata gta ccg gca cca cct 692 A S G P Q P L L N G S I V P A P P 2141 gcc acc cta gca cca gga gtg cat ctg gat gtg gag gcc ctc atc ccc aca 709 A T L A P G V H L D V E A L I* P* T 2192 ctt gat gag cag ggc cgg ccc atc ccg gag tgg aag cgc cag gtg atg gtc 726 L D* E Q G* R P I* P* E W* K* R* Q* V M* V 2243 cgg aag ctg cag cag aaa atg cag gag gaa gag gag cag cgg agg aag gaa 743 R K* L Q Q K M Q E E E E* Q R* R K E 2294 gaa gag gag gag gcc cgg ctg gcc agc ctg ccc gcc tgg aga cga gac att 760 E* E E* E A R* L* A S L P* A W* R R* D* I 2345 ctt agg aag aag ctg gag gag gag agg gag cag aag cga aaa gag gag gag 777 L* R K K L E* E E* R E Q K* R K E E E 2396 cgg cag aag ctg gag gaa atc cag agg gcg aaa gaa cag tcg gag aag ctg 794 R Q K L E E I Q R A K E Q S E K L 2447 cgg aca cta gga tac gac gag gcc aag ctc gcg ccc tgg cag cga cag gtc 811 R T L G Y D E A K L A P W Q R Q V 2498 atc ttg aag aag ggg gag atc cct aag taa tag gag tct cgg cct ctt gcg 828 I L K K G E I P K 2549 tgt agc ctc acg agc tct gag aaa tgg gga gcg gcc ccc agc ccc cgc ccc 2600 atc ctg tca ggt tgg tgc gga agg ggg gga gcc ttg agc cct tcc ctt cgg 2651 tgc ctg ccg gaa tct tcc cta ttc ccc tcc ctg gcc cca cat ccc caa ccc 2702 tgc tgc tgg agt gct ctc gcg aac ccc tgc tgt cgc ctg gaa aaa aaa agt 2753 gcc cag gct gct gac gca aaa aaa aaa aaa a TH TH 8 ANKYRIN REPEATS PR PR P FORK N ------C ٙ 1234 J. R. Bartles, A. Wierda and L. Zheng Fusion protein and antibody Because the espin monoclonal antibody allowed neither immunoisolation of the protein nor its localization in aldehyde- fixed specimens, we expressed espin as a fusion protein in bacteria and then used the fusion protein to prepare polyclonal antibodies in rabbits. To avoid the region of the protein that contained the relatively common ankyrin-like repeats, the ~1.4 kb cDNA encoding the C-terminal 378 amino acids of espin was inserted into the pMAL-c2 protein fusion vector and used to express a maltose-binding protein fusion protein in E. coli. Upon induction, the transformant was able to express relatively large quantities of a fusion protein of ~90 kDa, which was labeled specifically with the espin monoclonal antibody on western blots (Fig. 4). The maltose-binding protein contributed ~43 kDa to this fusion protein (Kellerman and Szmelcman, 1974). The fusion protein was purified from extracts of lysed bacterial cells by affinity chromatography on amylose resin (Fig. 4). In addition to the 90 kDa fusion protein, the maltose eluate sometimes contained variable amounts of what appeared to be proteolytic breakdown products that migrated between 90 Fig. 3. Northern blot analysis of the tissue distribution of espin kDa and 43 kDa. Because we were unable to cleave the espin mRNA. Duplicate ~10 µg aliquots of total RNA isolated from the fragment from the fusion protein using Factor Xa, the purified designated rat organs and tissues were electrophoresed in a fusion protein was used in its entirety to immunize rabbits. The formaldehyde-denaturing agarose gel and transferred to nitrocellulose. The resulting blot was labeled under conditions of antibodies in the resulting pooled antisera were then affinity high stringency with randomly primed 32P-labeled espin cDNAs purified on columns of fusion protein-Sepharose 4B after first prepared using the ~1.4 kb espin cDNA as a template. BR, brain. LI, depleting the sera of any antibodies directed against the liver. HE, heart. KI, kidney. TE, testis. BA, brown adipose. SP, maltose-binding protein by passing the sera over a column of spleen. TH, thymus. SM, skeletal muscle. WA, white adipose. SI, maltose-binding protein-Sepharose 4B. On western blots, the small intestine. resulting affinity purified rabbit polyclonal antibody was found to react specifically with the fusion protein, but not the maltose-binding protein (Fig. 4). The affinity purified poly- cles’ on these large Sertoli cell fragments (Russell and clonal espin antibody was also found to react with a ~110 kDa Peterson, 1985; Vogl, 1989; Vogl et al., 1991); some of these espin protein present in the mixture of late spermatids and tes- crypts contained the heads of elongate spermatids, whereas ticular spermatozoa that served as the immunogen for others had apparently lost their spermatids during sample preparing monoclonal antibodies, but not in samples of mature preparation or, alternatively, represented fragments of the spermatozoa collected from the vas deferens (Fig. 4). A single Sertoli cell-Sertoli cell ectoplasmic specializations (Fig. 5D,E). band at ~110 kDa band was also recognized by the antibody In no case were more mature spermatozoa isolated from the in samples of testicular homogenate (Fig. 4). No bands were epididymis or vas deferens found to be labeled with the poly- labeled when preimmune IgG was substituted for affinity clonal espin antibody (data not shown). purified polyclonal espin antibody (data not shown). Pre-embedment immunogold electron microscopy was used to establish the localization of espin at a higher level of reso- Localization lution on paraformaldehyde-fixed, saponin-permeabilized late The affinity purified polyclonal espin antibody or preimmune spermatids like those shown in Fig. 5A-C. Specimens labeled IgG was used to label formaldehyde-fixed, saponin-permeabi- with preimmune IgG showed no bound gold particles (data not lized preparations of mechanically dissociated rat testicular shown), but those labeled with affinity purified polyclonal cells by immunofluorescence. No labeling was noted when espin antibody showed large numbers of bound gold particles using the preimmune IgG (data not shown), but the affinity on the cytoplasmic side of the Sertoli cell plasma membrane purified polyclonal espin antibody was found to label the mod- at sites where it surrounded the heads of the elongate sper- erately phase-dense fragments or ‘caps’ of Sertoli cell crypt matids (Fig. 6). Upon closer examination, the gold particles plasma membrane that remained adherent to the highly refrac- were found to decorate bundles of electron-dense filaments that tile, sickle-shaped heads of the late spermatids (Fig. 5A,B). were intimately associated with membranous cisternae or This is precisely where one would normally find ectoplasmic vesicles. On the basis of its well-documented ultrastructure in specializations in this type of preparation (Russell and situ and following mechanical dissociation of testicular tissue Peterson, 1985; Vogl, 1989; Vogl et al., 1991). In fact, when (Russell and Peterson, 1985; Vogl, 1989; Vogl et al., 1991), we used fluorescent phalloidin as a marker for the F-actin we concluded that these densely labeled structures were in fact bundles of the ectoplasmic specialization, we found the two the parallel bundles of actin filaments and associated cisternae patterns of fluorescence to be indistinguishable (cf. Fig. 5B,C). of endoplasmic reticulum which comprised the junctional Occasionally, we encountered a much larger fragment of a plaques of ectoplasmic specializations. However, in this case, Sertoli cell in these preparations. As would be expected for a the conditions employed for the pre-embedment immunogold protein localized to ectoplasmic specializations, the polyclonal labeling appeared to have caused some segments of the junc- espin antibody was found to label multiple crypts or ‘recepta- tional plaque to be pulled slightly away from the Sertoli cell Espin and the ectoplasmic specialization 1235

To establish the localization of espin in situ, the immunoper- oxidase technique was used to localize espin on paraffin sections of Bouin’s-fixed rat testis. Sections labeled with preimmune IgG showed no brown reaction product (data not shown), whereas those labeled with affinity purified polyclonal espin antibody showed an accumulation of brown reaction product at the two sites expected for ectoplasmic specializa- tions (Russell and Peterson, 1985; Vogl, 1989; Vogl et al., 1991). In the adluminal compartment, labeling was found in close association with the heads of elongate spermatids in the different stages of differentiation. Consistent with the time course of appearance of these ectoplasmic specializations (Russell and Peterson, 1985; Vogl, 1989; Vogl et al., 1991), this pattern of labeling was first clearly discerned around the oval-shaped heads of elongate spermatids in mid-spermiogen- Fig. 4. Characterization of espin, espin fusion proteins and espin esis (Fig. 7, tubule at right center, paired small arrowheads) antibodies on western blots. The following specimens were resolved in reducing SDS gels and either examined by staining with Coomassie and was maintained as the spermatids continued to differenti- blue (CB), or transferred to nitrocellulose and examined by western ate, with their heads assuming the more streamlined sickle blotting using monoclonal espin IgM and 125I-goat anti-mouse Ig shape (e.g. Fig. 7, tubule at left center, paired small arrow- (mAb) or affinity purified rabbit polyclonal espin antibody and 125I- heads). Then, very late in spermiogenesis, at the time when Protein A (pAb): E. coli TB-1 cells transformed with the pMAL-c2 these ectoplasmic specializations begin to disappear (Russell vector carrying the ~1.4 kb espin cDNA grown in the absence (−) or and Peterson, 1985; Vogl, 1989; Vogl et al., 1991), the labeling presence (+) of the inducer isopropyl-β-D-thiogalactopyranoside; appeared to become distributed more diffusely within the affinity purified maltose-binding protein (MBP) or maltose-binding apical cytoplasm of the Sertoli cells (Fig. 7, partial tubule at protein fusion protein that includes the C-terminal 378 amino acids of top). The other type of labeled structure was present in the rat espin (EFP); mixture of isolated rat testicular spermatozoa and late basal compartment of the seminiferous epithelium and took the spermatids (TS); isolated rat vas deferens spermatozoa (VS); and rat testicular homogenate (TH). Arrowhead at left, position of espin fusion form of intermittent stripes or a ‘scalloped’ pattern which protein. Arrowhead at right, position of espin (~110 kDa). Solid circle appeared to pass around or between adjacent cells (e.g. Fig. 7, at left, position of maltose-binding protein. tubule at right center, single large arrowhead). This pattern was similar to those observed for F-actin and vinculin at the sites of those ectoplasmic specializations which are part of the junc- tional complexes between adjacent Sertoli cells (Vogl, 1989; plasma membrane. After viewing many specimens, we Grove et al., 1990; Vogl et al., 1991). surmised that this separation was necessary to optimize access of the colloidal gold-labeled secondary antibody and achieve Binding to actin in vitro and in vivo a high density of labeling. On the basis of the localization results described above, we

Fig. 5. Localization of espin by immunofluorescence microscopy. Cells prepared by mechanical dissociation of rat testis were fixed onto poly-L-lysine-coated slides and labeled with affinity purified polyclonal espin antibody and fluorescein-labled phalloidin followed by rhodamine-labeled secondary antibody in the presence of 0.025 % saponin. (A-C) Late spermatids with moderately phase- dense fragments of Sertoli cell plasma membrane attached to their highly refractile, sickle-shaped heads. (D and E) Sertoli cell fragment with very late spermatids attached. (A and D) Phase contrast. (B and E) Rhodamine channel (espin). (C) Fluorescein channel (F- actin). Bar, 33 µm. 1236 J. R. Bartles, A. Wierda and L. Zheng

Fig. 6. Localization of espin by immunogold electron microscopy. Cells prepared by mechanical dissociation of rat testis were fixed onto poly-L-lysine- coated coverslips and labeled with affinity purified polyclonal espin antibody followed by 12 nm diameter colloidal gold-labeled secondary antibody in the presence of 0.025% saponin. This micrograph depicts a section cut through the head of a late spermatid oriented like the righthand spermatid in Fig. 5A. The head of the spermatid enters the figure from the lower righthand corner of the figure, where one can observe the electron- dense nucleus (n). The plasma membranes of the spermatid and Sertoli cell are closely apposed and together form a line of moderate electron density, which has been marked by arrowheads. The arrowheads point to the cytoplasmic face of Sertoli cell plasma membrane. The space between the spermatid nucleus and plasma membrane largely represents the extracted acrosome. In some places (e.g. at the right arrowhead), all three membranes, the Sertoli cell plasma membrane, the spermatid plasma membrane and the outer acrosomal membrane, can be clearly delineated. Bar, 0.3 µm. decided to test whether espin would bind to F-actin directly using bind to the F-actin, because only very small amounts of fusion a cosedimentation assay. Since it is not yet possible to obtain the protein were recovered in the pellet fraction in the absence of F- intact espin protein in a native state in the relatively large quan- actin (Fig. 8A). The binding of the fusion protein was due to the tities desired for such binding studies, we used the espin fusion espin fragment and not the maltose-binding protein, because the protein in these experiments. The affinity purified maltose- vast majority of the maltose-binding protein control remained binding protein fusion protein containing the C-terminal 378 soluble in the supernatant fraction even in the presence of F-actin amino acids of espin was incubated with preformed filaments of (Fig. 8A). The same was true of the maltose-binding protein- rabbit skeletal muscle actin for 1 hour at 37¡C in 0.1 M KCl, 2 sized proteolytic fragments which were present as contaminants mM MgCl2, 0.5 mM ATP, 0.005% (w/v) NaN3, 10 mM in the affinity purified fusion protein (Fig. 8A). When examined imidazole-HCl, pH 7.4. Unless using relatively high ratios of over a range of concentrations, the binding analysis yielded a fusion protein to actin, the vast majority of the fusion protein was roughly linear Scatchard plot, suggestive of a dissociation recovered with F-actin in the pellet fraction following high-speed constant of ~10 nM and a capacity for binding ~1 espin fusion centrifugation (Fig. 8A). The espin fusion protein was judged to protein per 6 actin monomers at saturation (Fig. 8B).

Fig. 7. Immunoperoxidase localization of espin on sections of testis. Rat testis was fixed by perfusion with Bouin’s fluid and embedded in paraffin. Sections (5 µm) were deparaffinized, labeled with affinity purified polyclonal espin antibody and horseradish peroxidase-labeled secondary antibody, processed for peroxidase histochemistry and counterstained with hematoxylin. Note that brown reaction product accumulates around the heads of elongate spermatids in the adluminal compartment (paired small arrowheads) and more basally in areas between adjacent Sertoli cells (single large arrowhead). Bar, 65 µm. Espin and the ectoplasmic specialization 1237

imposed upon the more diffuse component of cytoplasmic labeling. This filamentous pattern was especially evident in flattened regions of cells that expressed lower levels of espin (Fig. 9C) and appeared to correspond to the localization F-actin as determined by double labeling with fluorescent phalloidin (cf. Fig. 9C,D). Irrespective of the level of expression, strict colocalization of espin fragment and F-actin was always evident when the cells were subjected to a brief treatment with ice-cold Triton X-100 prior to fixation. This can be seen to special advantage in the well spread cell shown in Fig. 9E,F. These findings suggested that the espin C-terminal fragment could also associate with F-actin in vivo. Furthermore, compared to nonexpressing control cells (the cells shown in the bottom portions of Fig. 9A-D), those which contained the espin C-terminal fragment consistently showed higher levels of labeling with fluorescein-labeled phalloidin (Fig. 9B,D), suggesting that this peptide may bring about the accumulation, stabilization and/or bundling of F-actin in the NRK cells.

DISCUSSION

On the basis of its localization to the actin filament bundles of the ectoplasmic specialization (Fig. 6) and the ability of its C- terminal fragment to bind to F-actin in vitro (Fig. 8) and in vivo (Fig. 9), we conclude that espin is an actin-binding protein of the ectoplasmic specialization. Thus, espin joins α-actinin, vinculin and fimbrin in the list of actin-binding proteins believed to be associated with ectoplasmic specializations α Fig. 8. Binding of espin fusion protein to F-actin in vitro. (A) The (Vogl et al., 1991). However, unlike -actinin, vinculin, affinity purified espin fusion protein that includes the C-terminal 378 fimbrin and many other actin-binding proteins (Geiger, 1993; amino acids of rat espin (EFP; 35 µg/ml) or the affinity purified Critchley, 1993; Bretscher, 1993; Pollard, 1993), espin appears maltose-binding protein (MBP; ~100 µg/ml) were incubated for 60 to be expressed in a cell type-specific fashion. Our northern minutes at 37¡C, either alone (−) or in the presence (+) of 0.5 mg/ml blotting (Fig. 3) and immunolocalization studies (Figs 4-7) preformed filaments of rabbit skeletal muscle F-actin in 0.1 M KCl, 2 suggest that espin is present exclusively within the Sertoli cells mM MgCl2, 0.005% (w/v) NaN3, 0.5 mM ATP, 10 mM imidazole- of the seminiferous epithelium, where it is concentrated at the HCl, pH 7.4. The samples were sedimented at 150,000 g for 90 sites of ectoplasmic specializations, both those between Sertoli minutes to form high-speed supernatant (S) and pellet (P) fractions, cells and elongate spermatids and those between neighboring which were then resolved in reducing SDS gels and examined after staining with Coomassie blue. Arrowhead, position of the espin Sertoli cells. However, our detection of smaller espin-related fusion protein. Solid circle, position of the maltose-binding protein. mRNAs (Fig. 3) and proteins (J. R. Bartles, A. Wierda and L. Note that the maltose-binding protein migrates just below actin in the Zheng, unpublished data) in the small intestine and kidney gel. (B) Scatchard plot analysis of the binding observed between hints that espin is a member of a newly discovered family of different concentrations of the affinity purified espin fusion protein proteins which shows a wider distribution. and preformed filaments of F-actin under the same conditions as The relatively uniform distribution of espin along the described above under A, except that the final concentration of actin parallel actin filament bundles of the ectoplasmic specializa- µ was 50 g/ml. From the slope, the Kd was estimated to be ~10 nM, tion (Fig. 6) and the binding stoichiometry observed for the and from the intercept on the abscissa the maximum amount of espin fusion protein (Fig. 8B) suggest that espin binds to the fusion protein bound at saturation was estimated to be ~1 espin lateral surfaces of the actin filaments. This mode of interaction fusion protein/6 actin monomers. is commonly observed for proteins which crosslink actin filaments or anchor them to membranes (Matsudaira, 1991; Pollard, 1993). Providing that substitution of the N-terminal To determine whether espin could also associate with F- half of espin for the maltose-binding protein does not change actin in vivo, transient transfection was used to express the C- the interaction significantly, the results of our cosedimentation terminal fragment of espin represented in the fusion protein in experiments with the fusion protein (Fig. 8B) suggest that cells of the NRK rat fibroblastic line, which do not ordinarily espin binds with an affinity that is 1-2 orders of magnitude express espin. When localized in tranfected NRK cells that had greater than that reported for many other proteins which been labeled without prior extraction with Triton X-100, the associate with the lateral surfaces of F-actin (Pollard, 1993). espin C-terminal fragment appeared to be distributed more or Through the use of the fusion protein (Fig. 8) and the less uniformly throughout the cytoplasm of the vast majority expression of the corresponding C-terminal fragment in NRK of transfected cells (Fig. 9A). Occasionally, however, one cells (Fig. 9), we have tentatively mapped this high affinity could also discern a filamentous, stress fiber-like pattern super- actin-binding site to the C-terminal 378 amino acids of the 1238 J. R. Bartles, A. Wierda and L. Zheng

Fig. 9. Transient transfection of NRK cells with the C- terminal fragment of espin. The ~1.4 kb cDNA that encodes the C-terminal 378 amino acids of espin was introduced into the pcDNA3 vector and used to transiently transfect cells of the NRK fibroblastic line using a modification of the calcium precipitation procedure. At 2 days after transfection, the cells were fixed, either without (A and B, C and D) or with (E and F) a brief pretreatment with ice-cold 0.1% Triton X- 100 in phosphate-buffered saline, and labeled with affinity purified polyclonal espin antibody and fluorescein- labeled phalloidin followed by rhodamine-labeled goat anti- rabbit IgG. (A,C,E) Rhodamine channel (espin). (B,D,F) Fluorescein channel (F-actin). Bar in F, 50 µm (A-D) or 25 µm (E and F). protein. The C-terminal peptide in question shows no signifi- ments will be required to determine if the full-length espin cant sequence similarity to other proteins known or suspected protein can elicit the bundling of F-actin and, if so, whether it to interact with actin, with the exception of the 66 amino acid does so through the expression of more than one actin-binding peptide (723-788), which was found to be 39% identical to a site or a propensity to form dimers or higher oligmers (Mat- peptide present in both the large and small proteins encoded sudaira, 1991). by the forked gene of Drosophila (Hoover et al., 1993). The The ankyrin-like repeat is a relatively common motif for forked proteins have been implicated in the formation of protein-protein interaction that is shared by proteins as diverse plasma membrane-associated parallel bundles of F-actin in the as cytoskeletal linkers, cell cycle regulators and transcription bristles of Drosophila pupae from the analysis of mutants and factors (Michaely and Bennett, 1993). Despite this generality, from immunolocalization studies (Hoover et al., 1993; the observation that the ankyrin-like repeats of espin are, on Petersen et al., 1994; Tilney et al., 1995). An explanation the whole, more similar to those of the large forked protein proffered by Tilney et al. (1995) is that the forked proteins are than those of the mammalian suggests that the N- actin-bundling proteins which, together with another actin- termini of espin and the large forked protein could interact with bundling protein (a homolog of fascin encoded by the singed similar non-actin proteins or structures in the cell. A function gene of Drosophila), are responsible for the formation of the for the large forked protein is yet to be identified. It arises parallel actin bundles found in bristles. In a similar way, espin through the use of an alternative site for transcription initiation could be an actin-bundling protein of the ectoplasmic special- and appears to be less abundant than smaller versions of the ization and help give rise to the hexagonally packed, parallel forked protein which lack the ankyrin-like repeats (Hoover et actin bundles present within the plaque of this intercellular al., 1993; Petersen et al., 1994). Furthermore, experiments with junction (Russell and Peterson, 1985; Vogl, 1989; Vogl et al., transgenic pupae have indicated that one of these small forked 1991). A role for espin as an actin-bundling protein would be proteins is sufficient to restore normal bristle morphology to compatible with its localization (Fig. 6) and actin-binding pupae carrying the forked mutation (Petersen et al., 1994). In properties (Figs 8, 9) and may even explain the increase in light of the precedent for ankyrin-like repeats linking the labeling by fluorescent phalloidin noted in NRK cells express- cytoskeleton to integral membrane proteins (Michaely and ing the espin C-terminal fragment (Fig. 9). Additional experi- Bennett, 1993), one intriguing possibility is that espin con- Espin and the ectoplasmic specialization 1239 tributes to the organization of the ectoplasmic specialization by Hoover, K. K., Chien, A. J. and Corces, V. G. (1993). Effect of transposable anchoring the parallel actin bundles to the underlying cistern elements on the expression of the forked gene of Drosophila melanogaster. of endoplasmic reticulum or the overlying plasma membrane. Genetics 135, 507-526. Hubbard, A. L., Bartles, J. R. and Braiterman, L. T. (1985). Identification of Further experiments will be required to determine whether the rat hepatocyte plasma membrane proteins using monoclonal antibodies. J. full-length espin protein or its N-terminal fragment can interact Cell Biol. 100, 1115-1125. with integral membrane proteins. Finally, the detection of a Kellerman, O. and Szmelcman, S. (1974). Active transport of maltose in potential P-loop and proline-rich peptides in the sequence of Escherichia coli K12. Involvement of a ‘periplasmic’ maltose binding protein. Eur. J. Biochem. 47, 139-149. espin suggests ways that nucleotides or protein ligands might Kozak, M. (1987). 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