Journal of Cell Science 103, 131-143 (1992) 131 Printed in Great Britain © The Company of Biologists Limited 1992
A gene family consisting of ezrin, radixin and moesin
Its specific localization at act in filameni/plasma membrane association sites
NARUKI SATO1'2, NORIKO FUNAYAMA1, AKIRA NAGAFUCHI1, SHIGENOBU YONEMURA1, SACHIKO TSUKITA1 and SHOICHIRO TSUKITA12
' Laboratory of Cell Biology, Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki, Aichi 444, Japan 2Department of Physiological Sciences, School of Life Sciences, The Graduate University of Advanced Studies, Myodaiji-cho, Okazaki, Aichi 444, Japan
Summary
Radixin is a barbed end-capping actin-modulating croscopy, we closely analyzed their distribution using protein which was previously reported to be concen- polyclonal and monoclonal antibodies, which could trated at cell-to-cell adherens junctions (AJ) and recognize all three members. In addition to cell-to-cell cleavage furrows. Recently, cDNA encoding mouse AJ and cleavage furrows, it was shown that they were radixin was isolated, showing that radixin is highly concentrated at microvilli and ruffling membranes in homologous to but distinct from ezrin. From mouse various types of cells. Furthermore, the cell-to-substrate teratocarcinoma cells we isolated and analyzed cDNA AJ (focal contacts) were clearly stained by anti-radixin encoding another radixin-related protein. Sequence pAb only after the apical/lateral membranes and analysis has demonstrated that this protein is a mouse cytoplasm were removed by the zinc method. We homologue of human moesin (98.3% identity) and that it conclude that at least one of the members of the ezrin- shares 71.7% and 80.1% identity with ezrin and radixin-moesin family is concentrated at specific regions radixin, respectively. Translation experiments in vitro where actin filaments are densely associated with plasma combined with immunoblot analyses led us to conclude membranes. that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence mi- Key words: ezrin, radixin, moesin, actin filament.
Introduction ation is still fragmentary. The interesting feature of this type of association is that actin filaments are always It is well documented that actin and myosin are unidirectionally polarized in respect of their attach- involved in many types of cell motility including ment, with the arrowheads of myosin heads pointing muscular contraction (Pollard and Weihing, 1974; away from the plasma membrane, suggesting that a Ishikawa, 1979; Craig and Pollard, 1982; Pollard and kind of barbed-end capping protein may be responsible Cooper, 1986; Vandekerckhove, 1990). In most of these for the end-to-membrane association of actin filaments actin-based cell motility systems, there is a close (Ishikawa, 1979). association between actin filaments and plasma mem- Adherens junctions (AJ) are defined as one of the branes, and this association is thought to be essential for typical cell contacts in which actin filaments are cell motility and the maintenance of cell shape. In associated with the plasma membrane through its well- respect of actin filaments, this association is classified developed undercoat (Geiger, 1983). These junctions into two types; side-to-membrane and end-to-mem- are divided into two types: cell-to-cell and cell-to- brane (Ishikawa, 1979). In the side-to-membrane substrate (Burridge and Connell, 1983; Geiger et al., association, the actin filaments are bound to the plasma 1985). The manner of association (side-to-membrane or membrane along their lengths through thin cross- end-to-membrane) of actin filaments with the plasma linkers. Some of these crosslinkers have been ident- membrane in both types of junctions remains unknown. ified; for example, a 110 kDa protein in intestinal However, it is clear that at least in the cell-to-cell AJ of epithelial cell microvilli (Matsudaira and Burgess, 1979; cardiac muscle cells (intercalated discs) and in the cell- Louvard, 1989). In contrast, our knowledge of the to-substrate AJ of skeletal muscle cells (myotendenous proteins responsible for the end-to-membrane associ- junctions), all actin filaments are bound to the under- 132 N. Sato and others coat in an end-to-membrane fashion by their barbed where actin filaments are densely associated with ends (Ishikawa, 1979). Therefore, many investigators plasma membranes. We believe that this study can give have attempted to identify the barbed-end capping us a clue to understanding the molecular basis for the protein located at the undercoat of AJ. Vinculin, one of end-to-membrane association of actin filaments with the major undercoat-constitutive proteins of AJ plasma membranes in general. (Geiger, 1979, 1983), was originally reported to have a barbed-end capping activity (Wilkins and Lin, 1982). However, it is known that this activity is attributed to contaminants and that highly purified vinculin does not Materials and methods cap the barbed ends of actin filaments (Evans et al., 1984; Wilkins and Lin, 1986). Lin and his colleagues Cells and antibodies have studied this contaminated protein mainly using Cell lines of mouse mammary tumour MTD-1A, which is a immunological methods and reported that the contami- subclone isolated from the original MTD-1 line (Enami et al., nated proteins may arise from proteolysis of 200 kDa 1984; Hirano et al., 1987), mouse fibroblast tk~L (Murayama- molecules called tensin (Wilkins et al., 1986; Davis et Okabayashi et al., 1971), mouse teratocarcinoma F9 (Bern- al., 1991). However, Miron et al. (1988, 1991) have stein et al., 1973), human epidermoid carcinoma A431 and KB were used. Cells were maintained in DMEM sup- purified a 25 kDa barbed-end capping protein (25 kDa plemented with 10% FBS. IAP) as a contaminated protein and found that this is Anti-radixin polyclonal antibodies (pAbs), pAb-ll and identical to a low molecular mass heat shock protein. pAb-p800, were prepared against purified radixin from rat Radixin is a barbed end-capping actin-modulating liver AJ, and against the fusion protein generated from a protein first identified as one of the major undercoat- fragment of mouse radixin cDNA in Escherichia coli, constitutive proteins isolated from cell-to-cell AJ in rat respectively, as described previously (Tsukita et al., 1989a; liver (Tsukita and Tsukita, 1989; Tsukita et al., 1989a). Funayama et al., 1991). Anti-radixin monoclonal antibody Recently, radixin was shown to be highly concentrated (mAb), CR-22, was generated against the purified radixin at the cleavage furrow during cytokinesis (Sato et al., from chicken gizzard as described previously (Sato et al., 1991). Considering that the most prominent feature 1991). Anti-vinculin mAb (V115) was purchased from Sigma shared by AJ and the cleavage furrow is the tight Co. association of actin filaments with plasma membranes, we were led to speculate that radixin plays a crucial role cDNA library screening and DNA sequencing in binding the barbed end of actin filaments to the Two different Agtll expression libraries made from mouse F9 plasma membrane, at least at these sites. More poly(A)+ RNAs were used in the following procedures recently, we have succeeded in isolating and sequencing (Nagafuchi et al., 1987). In preparing these libraries, either a cDNA encoding mouse radixin and found that radixin is random mixture of hexanucleotides or oligo(dT) was used as similar to ezrin (~75% identity) (Funayama et al., primer for the first-strand synthesis. The initial cDNA clone, 1991), which has been reported to be a member of the 22C (see Fig. 2, below), was isolated from a randomly primed band 4.1 "superfamily" (Gould et al., 1989; Turunen et library using a monoclonal CR-22 (Sato et al., 1991), al., 1989). As pointed out in a previous study, taking according to the method previously described (Nagafuchi et al., 1991). Then, the 22C fragment was radiolabeled with [a- into consideration that the amino acid sequence of 32P]dCTP. Using this fragment as a probe, the 22T4, 22T5 and radixin is highly homologous to that of ezrin and that 22T6 clones were isolated from the oligo(dT)-primed library the molecular mass of radixin is expected to be almost (see Fig. 2). equal to that of ezrin, previous western blotting All clones to be analyzed were subcloned into pBluescript analyses and immunolocalization studies on ezrin and SK(—) and sequenced with the 7-deaza Sequenase Version radixin should be re-evaluated because anti-ezrin 2.0 kit (U.S.Biochemical Corp., Cleveland, Ohio). Long antibodies may cross-react with radixin and vice versa inserts were sequenced from nested deletion subclones (Funayama et al., 1991). In this study, by western created using the Deletion Kit for Kilo-sequence (Takara blotting analyses and cDNA cloning, we first show that Shuzo Co., LTD., Kyoto, Japan). Both strands of all clones in addition to radixin and ezrin a third highly related were sequenced. protein is widely expressed in various type of cells, suggesting the existence of a gene family consisting of at Isolation of RNA and northern blot hybridization least three members. During the course of this study, Total RNAs from cultured F9 cells were isolated according to Lankes and Furthmayr (1991) isolated a cDNA en- the method for rapid isolation of total RNA from mammalian coding human "moesin". Sequence analysis of the third cells described by Sambrook et al. (1989). Approximately 8 ng radixin/ezrin-like protein has demonstrated that it is a per well of RNA were subjected to electrophoresis and mouse homologue of moesin (98.3% identity). There- blotted onto a nitrocellulose membrane. An RNA ladder fore, in this study, we have tentatively designated this (Bethesda Research Laboratories, Bethesda, MD) was used as size marker. The 22C fragment was labeled with [a- gene family as the ezrin-radixin-moesin (ERM) family. 32 Second, using anti-radixin pAb (polyclonal antibody) P]dCTP, using the Random Primer DNA Labeling Kit and mAb (monoclonal antibody), which can recognize (Takara Shuzo Co., LTD., Kyoto, Japan), and used as a probe. Hybridization was carried out at high stringency (50% three members of the ERM family, we examined their formamide/lOxDenhart's solution without BSA/5xSSC/50 distribution. The results show that at least one of the mM phosphate buffer (pH 6.5)/100 ng/fji boiled salmon sperm members of the ERM family is concentrated at regions DNA). The ezirin-radixin-moesin family 133
One- and two-dimensional gel electrophoresis and detection of actin filaments, rhodamine-phalloidin was mixed immunoblotting with the second antibody solution. After being washed with Proteins were separated by the one-dimensional SDS-PAGE PBS three times, the samples were examined in a Zeiss (Laemmli, 1970) or two-dimensional NEPHGE (O'Farrell, Axiophot photomicroscope. 1975) method. Immunoblotting was performed after one- or The basal membrane preparations were obtained from two-dimensional gel electrophoresis and electrophoretic cultured MTD-1A cells essentially according to the zinc transfer of the polypeptides to nitrocellulose sheets. Nitrocel- method developed by Avnur and Geiger (1981). The cells lulose transfers were incubated with pAb-Il, pAb-p800, CR- were rinsed briefly with buffer A (50 mM MES (pH 6.0), 5 22 (mAb), or a mixture of pAb-Il and CR-22. For antibody mM MgCl2, 3 mM EGTA), then incubated at room detection, the blotting detection kit with alkaline phospha- temperature for 3 minutes with buffer A containing 3 mM tase-conjugated immunoglobulin (Amersham Ltd., Amer- ZnCI2. They were rinsed with phosphate-buffered saline, pH sham, UK) was used. Cell-to-cell adherens junctions and the low-salt extract were prepared as previously described (Tsukita and Tsukita, 1989; Tsukita et al., 1989b, 1991; Itoh et al., 1991).
In vitro translation Translations in vitro were performed as reported previously (Pelham and Jackson, 1975). RNA was synthesized from each cDNA using an mCAP™mRNA Capping Kit (STRATA- GENE). Capped RNA was translated in a mRNA-dependent rabbit reticulocyte lysate in the presence of 35S-methionine using an In Vitro Express™ Translation Kit (STRATA- GENE). The 35S-methionine (1300 Ci/mmol, Amersham, Arlington Heights, IL) was present at ~2.6 mCi/ml in a final reaction volume of 25 (A. Each reaction product in the in vitro translation reaction was then analyzed using one- or two- dimensional electrophoresis followed by autoradiography.
Immunofluorescence microscopy The cells on coverglasses were fixedwit h 1% formaldehyde in PBS for 15 min. The fixed cells were treated with 0.2% Triton X-100 in PBS for 15 min and washed three times with PBS. After being soaked in PBS containing 1% BSA for 10 min, the sample was treated with the first antibodies in 1% BSA/PBS i f at room temperature for 1 h. As the first antibody, pAb-Il or CR-22 (mAb) was used for the single staining. For the double Fig. 1. Immunoblots of mouse F9 cell lysate with three staining, pAb-11 was mixed with anti-vinculin mAb. The distinct anti-radixin antibodies. Lane 1, pAb-Il; lane 2, samples were then washed with 1% BSA/PBS three times, pAb-p800; lane 3, CR-22 (mAb). Three bands (85 kDa, 82 followed by incubation with the second antibodies (FITC- kDa and 75 kDa) are recognized by pAbl, while pAb-p800 conjugated goat anti-rabbit IgG or anti-mouse IgG; FITC- is preferentially specific to the 82 kDa band. CR-22 can conjugated goat anti-rabbit TgG/rhodamine-conjugated goat recognize both 85 kDa and 82 kDa band and strongly anti-mouse IgG) in 1% BSA/PBS for 30 min. For the stains the 75 kDa band.
B AP Bg I
22C 22T4- 22T5' 22T6' I I I 1000 2000 3000 3835 bp B Fig. 2. (A) Restriction map and cDNA fragments of newly identified radixin/ezrin-like protein. The shadowed box indicates the coding region. P, Pst I; B, BamYR; A, Apal; X, Xbal; Bg, Sg/II. (B) Northern blot analysis of total RNA isolated from mouse F9 cells. Total RNA was probed at high stringency with 22C cDNA. Arrowheads on the right indicate the position of RNA markers of 9.5, 7.5, 4.4, 2.4 and 1.4 kb from the top. 134 N. Sato and others
7.2, and subjected to jets of 5-10 ml of the same buffer using a Karnovsky, 1966). The samples were washed in the same 5 ml syringe with a 23G needle. The isolated basal membranes buffer, postfixed in cold 1.8% OSO4 in 0.1 M cacodylate on glass were fixed and processed for indirect immunofluor- buffer for 1 h, and dehydrated and embedded in Epon 812. escence microscopy.
Immunoelectron microscopy Results The cultured L cells were fixed with 4% formaldehyde in 0.1 . . ... , , , , , , ., ,. M HEPES (pH 7.5) for 15 min at room temperature followed Anti-radixin polydonal and monoclonal antibodies by treatment with 0.2% Triton X-100 in PBS for 15 min. The Originally, radixin was identified and purified from rat samples were then rinsed in PBS containing 1% BSA for 20 liver (Tsukita et al., 1989a), which was reported mostly min and incubated with the first antibody (CR-22) for 1 h. to lack ezrin (Bretscher, 1983); the contamination of They were then washed three times with PBS containing 1% ezrin in our purified radixin fraction, if any, can BSA, followed by incubation with the second antibody probably be ignored. However, considering that radixin (horseradish perox.dase-conjugated goat anti-mouse IgG) for and ezrjn ar£ sjmilar jn thejr deduced amino acid g SamP Wer H ^^ lf Sr'if't n, H sequences and in their calculated molecular masses fixed with 0.5% glutaraldehyde in PBS for 10 min, followed ,J? . ,~~.,x . ... ., , ., by the first incubation for 50 min at room temperature in a (Funayama et al., I99l), our ant.-rad.x.n pAb and mAb freshly prepared solution of 0.2 mg/ml 3,3'-diaminobenzidine should ^ re-evaluated with a special reference to the (DAB) of 0.05 M Tris-HCl buffer, pH 7.6, and then for an cross-reactivity with ezrin. In this study, we used two additional 5 min in the same solution containing 0.005% distinct anti-radixin pAbs, pAb-Il and pAb-p800 (see hydrogen peroxide for a second incubation (Graham and Materials and methods). Resolution around 80 kDa in CTTTGTAAAGTTCTGGCCCOTrAOCAGOAAlXXXMOCACTI^ 120 GCCCGAC*GTITCCA CACCACnxX^AGCAGCMbl lllJACCAQOTOCnX^AAACTATTOCnTTGAGOOAAOTTTtHjriVI 1 IUOlLIU^OTACCAOGACACAAAA0Criil'UTCTACTT0OCT0AAACTCAATAA M0 TTOKQLFDQVVKTIGLREVVPFGLQYQDTKAFSTWLKLNK 63 (G) GAAOOTCACTGCACAGOATOTGCGOAACOAAAOrCCA 11ULIC1 IUUUTrTCCOOO TCTCCAA(nt3AAOOAOOOCATTCTt^ATOACGACATrrACnntX^XrrOAAACTtXX(nu;i'(Jl' TOCCTTCTTATCCCCTCCACTtTAAOT'ATCOTCACTTCAATAAGGAACTOCACAA BOO LQVKEGILHDDIYCPPETAVLLASYAVQSKYCDPNKBVHIt 143 (TrCTGGCTACCTGGCTGGAGAT/UlonwnUXJ^AAOAUIlTllJQAOCAOCACAAA^^ T20 SCYLAODKLLPQRVLEQHKLNKDQWEERIQVWBEEHROML 183 CMGOQA«MT0CTGT«rrGGAATATCTCMAGATTTXTCAAGACCT0GAAATOTAT00TintUACrATT^ 840 REDAVLEYLKIAQDLENYGVNYFSIKNKKGSELILGVDAL 223 CCOTCTCAACATCTATOATOACAATOACACACTXaACTCCTAAOATTOOCTT^^ 960 GLNIYEQNDRLTPKIGFPWSEIRNIGFNOKKFVIKPIDKK 283 GGCCCCCGACM'I^UIVriVrATXKTrCCCCOOCTTCTCATTAACAAiXXXMlVritJCCCCTCTGCATGGOAAATC^ 1080 APDFVFYAPRLRINKRILALCMGNHELYHRRRKPDTIEVQ 303 QMKAQAREEKRQKQMERALLEHEKKKRELAEKEKEKIERE 343 (N) (D) (M) KEELMEKLKQIEEQTIIAQQELEEQTRRALELBQERKRAQ 343 (MTCCATGCTGAGAAUTGCGACTTMXMC(MGACAAATACAAaACCCT(XXX&^ 1920 IHAENMRLGRDKYKTLRQIRQGNTKQRIDEFESM* 577 CACGOACCaji'lt.TlVri'U'ritCnT.TlXXCACACTCCATAm 11IUJ1 AACTAACACrGTGTG 1.rl.1.1.1.rlvl<^A1|1.1.wl.1.1.1.1.1,AAATAA.1.[^|l|.1.ltJ0A000CT1TATOCTC^ M3S Fig. 3. Nucleotide sequence and deduced amino acid sequence of newly identified radixin/ezrin-like protein. The complete nucleotide sequence determined is 3,835 bases long including a poly(A)+ tail. The coding region is preceded by 172 nucleotides of 5'-untranslated sequence and followed by a 1,932-nucleotide-long 3'-untranslated region. The protein encoded by this cDNA and human moesin are identical in 98.3% of their residues. Amino acid residues of human moesin that are not identical to this mouse protein are shown in parentheses. The typical polyadenylation consensus sequence found near the end of cDNA is underlined. The ezirin-radixin-moesin family 135 one-dimensional SDS-PAGE showed that in the lysate Northern blot analysis with total mRNA obtained from of mouse teratocarcinoma F9 cells pAb-Il recognized mouse F9 cells was performed. As shown in Fig. 2B, the strongly two bands of 85 kDa and 82 kDa, and weakly band at 3.8 kb was hybridized to 22C. Since the size of one band of 75 kDa. The 82 kDa band was preferen- the major mouse radixin and ezrin mRNAs were tially recognized by pAb-800 (Fig. 1, lanes 1 and 2). reported to be 4.2 kb and 2.7 kb, respectively As has been shown, anti-radixin mAb, CR-22, clearly (Funayama et al., 1991), we were led to conclude that recognized purified radixin from isolated AJ (Sato et 22C was not part of radixin or ezrin cDNA. al., 1991). In F9 cells, CR-22 reacted with both the 85 Then, to isolate the full-length cDNA, -lxlO5 kDa and 82 kDa bands (Fig. 1, lane 3). Furthermore, colonies of the oligo(dT)-primed Agtll cDNA library this mAb strongly stained the 75 kDa band. This band were screened by DNA hybridization with the radiola- was weakly recognized by pAb-Il and had been thought beled 22C at high stringency. Three positive clones to be a proteolytic degradation product (Fig. 1, lane 1). were obtained: 22T4 (2.2 kb), 22T5 (2.6 kb) and 22T6 However, the immunoblotting pattern of F9 cells with (3.7 kb). Judging from their restriction maps, these CR-22 led us to consider the existence of a third clones overlapped (Fig. 2A), so they were sequenced. radixin/ezrin-like protein. Therefore, the isolation of The complete nucleotide sequence and deduced amino cDNA encoding this 75 kDa radixin/ezrin-like protein acid sequences of the cloned molecule are shown in Fig. was attempted. 3. The composite cDNA is 3,835 nucleotides long: 172 bp of the 5'-untranslated region, an open reading frame (ORF) of 1,731 bp encoding 577 amino acids, and 1,932 Isolation and sequencing of cDNA encoding a bp of the 3'-untranslated region with a poly(A)+ tail. radixin/ezrin-like protein The calculated molecular mass of the protein encoded Using anti-radixin mAb, CR-22, we screened ~lxlO5 by this cDNA is 67.8 kDa. plaques from a random-primed Agtll cDNA library In Fig. 4, the deduced amino acid sequence of this made from F9 cells, and cloned one positive phage protein was compared with those of ezrin and radixin, recombinant: 22C (1.8 kb). Using this 22C as a probe, indicating that this newly identified protein was very a 1# nPKPINVRVTTnDAFXEFAIQPNTTGKQLFDQVVKTVGUJF^TrFFGLQYVDSKCTSTWUaNKKVTQQDVKKENPI^ b 1' MPKTISVRVTTNDAELEFAIQPHTTGKQLFDQWKTIGIJ«F^WFFt3LQYQDTKAFSTIfLKLNKKVTAQDVRKESPLLFKF C1" MPKPINVRVTTmAFXEFAIQPNTTGKQLFDQVVKTIGLREVWYFGLPYVDNKGFrrtLKLDKKVSAQEVRKENPVQFKF 81# RAKFFPEDVSEELIQEITQRLFFLQVKEAILNDEIYCPPETAVLLASYAVQAKYGDYNKEIHKPGYLANDRLLPQRVLEQ 81' RAKFYPEDVSEEIIQDITQRLFFLQVKEGILNDDIYCPPETAVLLASYAVQSKYGDFJiKEVHKSGYLAGDKLLPQRVLEQ 81" RAKFYPEDVAEELIQDITQKLFFLQVKDGILSDEIYCPPETAVLLGSYAVQAKFGDYNKHUHKSGYLSSERLIPQRVMDQ 1G1# HKLTKEQfEERIQNWHEEHRGMLREDSMMEYLKIAQDLEHYGVNYFEIKNKKGTELWLGVDALGLNIYEHDDKLTPKIGF 161' HKLNKDQWEERIQVWHEEHRGflLREDAVLEYLKIAQDLEMYGVHYFSIKNKKGSELWLGVDALGLNIYEQNDRLTPKIGF 161" HKLSRDQWEDRIQVWHAEHRGMLKDSAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDDKLTPKIGF 241# PWSEIRNISFKDKKFVIKPIDKKAPDFVFYAPRLRINKRIULCnGNHELYMRRRKPDTIEVQQMKAQAREVLHQKQLER 241' PWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRIULCMGNHELYHRRRKPDTIEVQQMKAQAREEKHQKQHER 241" PfSFJRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLCMGNHELYIWRRKPDTIEVQQNKAQAREEKHQKQLER 321# AQLENEKKKREIAEKEKERIEREKEEIJIERLRQIEEQTVKAQKEIJIQTRKAUIEQERQRAKEEAERLDRERRAAEEAK Fig. 4. Comparison of amino • ••••••••* *••*•* *«•»•**«** * ****** ••• ******* «•**•**• ** •** • •• ***** acid sequences between three 321' ALLENEKKKRElJkEKEKEKIEREKEEL«EKlJ(QIEEOTKKA mouse moesin Turunen et al., 1989; Rees et al., 1990; Funayama et al., 756 lsee 2256 3886 3756 1991; Lankes and Furthmayr, 1991; Gu et al., 1991; Yang and Tonks, 1991). Identification and expression of three members of the ERM family In F9 cells, pAb-Il and CR-22 mainly recognize three bands (85 kDa, 82 kDa and 75 kDa) in SDS-PAGE (see Fig. 1), and we obtained three distinct cDNAs encoding highly related proteins from the F9 cell cDNA library. Then, to clarify the relationship between these bands and cDNAs, translation experiments in vitro were performed: RNA was synthesized from each cDNA, modified and translated in a mRNA-dependent rabbit reticulocyte lysate in the presence of [ Sjmethionine. Each reaction product in the in vitro translation reaction was then analyzed on a SDS-polyacrylamide gel next to the lysate of F9 cells. After transfer to a nitrocellulose sheet, each product was detected by autoradiography and three bands in the lysate of F9 3758 cells were stained with a mixture of pAb-Il and CR-22. As shown in Fig. 6A, the electrophoretic mobilities of Fig. 5. Comparison of nucleotide sequence of human the products from cDNAs encoding ezrin, radixin and moesin cDNA with that of mouse moesin cDNA. Diagonal moesin were identical to those of the 85 kDa, 82 kDa plots were made using the HARPLT2 program. The dots correspond to midpoints of 15-nucleotide spans with at and 75 kDa bands, respectively, recognized by the pAb- least 12 identical nucleotides between two cDNAs. The Il/CR-22 mixture. This relationship was further con- axes are labelled with nucleotide numbers. Note that they firmed by two-dimensional gel electrophoresis (Fig. are highly homologous not only with the coding regions 6B). (between two arrowheads) but also in 3'-noncoding Next, by immunoblot analyses with the pAb-Il/CR- regions. 22 mixture, we have examined the expression of each ERM family member in various types of cultured cells similar to but distinct from ezrin and radixin, and that and tissues (Fig. 7). In all types of mouse cultured cells their mRNAs were not derived from a single gene by an we have analyzed, ezrin, radixin and moesin were all alternative splicing mechanism. This newly identified clearly coexpressed. This was also the case in some protein shares 80.1% and 71.7% identity with mouse human cultured cells such as A431 and KB cells. In radixin and ezrin, respectively. The most characteristic contrast, in tissues their expression pattern was distinct aspect of the amino acid sequence of this radixin/ezrin- from tissue to tissue. For example, in the liver ezrin was like protein is that it lacks a polyproline stretch, which is not detected as previously reported (Bretscher, 1983), found both in ezrin and radixin. Amino acid substi- while the intestinal epithelial cells appeared mostly to tutions in the amino-terminal half are highly conserva- lack radixin. The skeletal muscle was characterized by a tive compared to the carboxyl-terminal half of these small amount of moesin. three molecules. Therefore, the overall structure of this newly identified protein must be similar to that of ezrin Ezrin-radixin-moesin at cell-to-cell AJ, cleavage and radixin. furrow and microvilli During the course of this study, cDNA encoding In previous studies using pAb-Il and CR-22 we human "moesin" was isolated and sequenced (Lankes reported that "radixin" is highly concentrated in the and Furthmayr, 1991). Close comparison revealed that cell-to-cell AJ and cleavage furrow in the interphase this human moesin is almost identical to the radixin/ez- and mitotic phase, respectively (Tsukita et al., 1989a; rin-like protein identified in this study (98.3% identity) Sato et al., 1991). However, it is now clear that both (see Fig. 3). Furthermore, the 3'-untranslated se- pAb-Il and CR-22 can more or less recognize all quences of these cDNAs were also highly homologous members of the ERM family. Therefore, all we can say (Fig. 5). These data conclusively show that the newly at present is that at least one member of the ERM identified radixin/ezrin-like protein is a mouse homol- family is highly concentrated in the undercoat of the ogue of human moesin. Therefore, we are led to cell-to-cell AJ and the cleavage furrow. conclude that there is a gene family consisting of at least So far ezrin (cytovillin) has been reported to be three members, ezrin, radixin and moesin, and that localized in microvilli in various types of cells three highly related proteins are coexpressed in F9 (Bretscher, 1983; Pakkanen et al., 1987). However, as cells. We tentatively designate this gene family as the reported previously, pAb-Il and CR-22 did not stain ezrin-radixin-moesin (ERM) family. As shown pre- the microvilli in the intestinal epithelial cells in viously, this ERM family is included in the band 4.1 conventional frozen sections (Tsukita et al., 1989a), "superfamily" (see Fig. 12, below) (Gould et al., 1989; although these antibodies can crossreact with ezrin. The ezirin-radixin-moesin family 137 B NEPHGE NEPHGE H* OH" H* OH" 85 SDS 82 75 12 3 4 Fig. 6. In vitro expression of each ERM family member from its cDNA clone. (A) The total reaction products obtained from each cDNA (lane 1, 22T6 cDNA; lane 2, radixin cDNA; lane 3, ezrin cDNA) in vitro were separated on one- dimensional SDS-PAGE and detected by autoradiography. Lane 4, immunoblotting of F9 cell lysate with a mixture of pAb-Il and CR-22. (B) A mixture of the total reaction products obtained from three different types of cDNA were separated on two-dimensional SDS-PAGE followed by autoradiography (left). F9 cell lysate was separated on two- dimensional SDS-PAGE and three members of the ERM family were identified by the pAb-Il/CR-22 mixture (right). This may be due to a kind of masking problem in electron microscopic analyses revealed that these rod- immunofluorescence microscopy. On a previous study like structures corresponded to the elongated micro- using immunofluorescent staining on dividing cultured villi. This staining pattern was observed in other types cells, we noticed (and described) that in many cultured of cultured cells. Taken together with a report on cells some fuzzy structures on the cell surface are cytovillin localization (Pakkanen et al., 1987), we occasionally stained, suggesting that our antibodies may conclude that at least one ERM family member is stain some microvilli in cultured cells (Sato et al., 1991). concentrated at the cytoplasmic surface of the microvilli To evaluate this speculation, mouse fibroblast L cells plasma membrane. were stained with pAb-Il or CR22, at both light microscopic and electron microscopic levels (Fig. 8). In Ezrin/radixin/moesin at cell-to-substrate AJ and L cells, a large number of rod-like structures were ruffling membranes immunofluorescently stained on their surface, and The concentration of the ERM family members at the B 1 2 3 4 5 6 1 23456789 Fig. 7. Widespread occurrence of three members of the ERM family in various types of cells and tissues. Each sample was loaded on a gel with the same amount of total protein. (A) Immunoblot analysis of cultured cells with the pAb-ll/CR-22 mixture. In all types of cultured cells, ezrin (E), radixin (R), and moesin (M) are all coexpressed. Lane 1, low salt extract of isolated AJ from mouse liver; lane 2, mouse F9 cells; lane 3, mouse L cells; lane 4, mouse MTD-1A cells; lane 5, human A431 cells; lane 6, human KB cells. (B) Immunoblot analysis of mouse tissues with the pAb-Il/CR-22 mixture. The expression pattern of the ERM family members is distinct from tissue to tissue. Lane 1, spleen; lane 2, intestine; lane 3, brain, lane 4, skeletal muscle; lane 5, cardiac muscle; lane 6, uterus; lane 7, kidney; lane 8, liver; lane 9, low salt extract of isolated AJ from mouse liver. 138 N. Sato and others r \ Fig. 8. Concentration of ERM family members at microvilli in mouse L cells as revealed by anti-radixin mAb, CR-22. (A and B) Immunofluorescence microscopy. A large number of elongated microvilli (arrows) are clearly stained. Bar, 15 jjm. (C and D) Immunoelectron microscopy. The activity of horseradish peroxidase is detected at the microvilli along their lengths (arrows). No signals are detected in the control sample (D), in which nonimmune mouse IgG was used instead of CR-22. Bar, 500 nm. cell-to-cell AJ, microvilli and the cleavage furrow cells (Tsukita et al., 1989a). However, close inspection strongly suggests that this family is directly involved in revealed that only in a small number of cells did the the association of actin filaments with the plasma vinculin-positive cell-to-substrate AJ appear to be membranes. One must question whether these proteins stained with pAb-Il (Fig. 10A,B). This was also are concentrated in the undercoat of the cell-to- observed in other types of cultured cells, such as L cells substrate AJ, where actin filaments are densely associ- and MDBK cells, leading us to speculate that the ated with the plasma membrane. To answer this, we antigenic sites for pAb-Il are mostly masked in focal used pAb-Il to analyze the distribution of the ERM contacts and that this masking could be removed, family in mouse epithelial MTD-1A cells, which bear depending on the phase of the cell cycle or on other both belt-like cell-to-cell AJ and cell-to-substrate AJ. factors. Then, we attempted to remove the possible When MTD-1A cells were doubly stained with pAb-Il masking at the focal contacts. For this purpose, the and anti-vinculin mAb according to the conventional apical/lateral plasma membranes together with cyto- immunofluorescence method, the cell-to-cell AJ was plasm and nucleus were removed, leaving the basal clearly stained, while no signal was detected from the membranes containing cell-to-substrate AJ, according cell-to-substrate AJ in most cells (Fig. 9). This was to the zinc method developed by Avnur and Geiger consistent with our previous observations using MDBK (1981). When these basal membranes isolated on The ezirin-radixin-moesin family 139 Discussion We have previously isolated and sequenced cDNAs encoding radixin and ezrin from mouse teratocarci- noma F9 cell cDNA libraries (Funayatna et al., 1991). In this study, cDNA encoding another radixin/ezrin- like protein was isolated from same libraries and analyzed. Sequence analysis has revealed that this protein is a mouse homologue of "moesin", which was originally identified in human as an extracellular protein and was later found to be an intracellular protein (Lankes et al., 1988; Lankes and Furthmayr, 1991). Comparison of the nucleotide sequences of these cDNAs indicates a gene family consisting of ezrin, radixin and moesin (ERM family). By translation experiments in vitro combined with immunoblot ana- lyses, we have identified each member of ERM family at the protein level. Ezrin, radixin and moesin have molecular masses of 69.3 kDa, 68.5 kDa and 67.8 kDa, calculated from the deduced amino acid sequence, and of 85 kDa, 82 kDa and 75 kDa in SDS-PAGE, respectively. The existence of some ezrin-like proteins has been so far suggested. Bretscher (1989) reported that in placenta some bands with smaller molecular masses than ezrin were recognized by anti-ezrin pAb. Birgbauer and Solomon (1989) obtained an mAb, whose antigen appeared to be similar to ezrin in many respects. They reported that in immunoblot analyses of some cultured cells this mAb recognized several bands around 80 kDa. These proteins may be identical to one of the ERM family members mentioned above, but have not been analyzed. Fig. 9. Concentration of ERM family members at cell-to- Since three highly homologous proteins are coex- cell adherens junctions in mouse MTD-1A cells. After pressed in various types of cells and tissues, previous being fixed and permeabilized, cells were doubly stained immunolocalization works on ezrin and radixin (and with anti-vinculin mAb (A) and anti-radixin pAb, pAb-Il also moesin) should be re-evaluated. There is a (B). The belt-like cell-to-cell AJ is clearly stained by both likelihood that anti-ezrin and anti-radixin antibodies anti-vinculin mAb and anti-radixin pAb. Note that no cross-react with other members of the ERM family. As radixin-staining can be detected at the vinculin-positive shown in this study, pAb-Il and CR-22 recognized all cell-to-substrate adherens junctions (focal contacts) the members. The other technical difficulty encoun- (arrowheads in A), though at this focal plane they are out of focus. Bar, 30 ^m. tered in the immunolocalization of the ERM family members was a masking problem. For example, pAb-Il could recognize ezrin in immunoblot analyses, but in substrates were doubly stained with pAb-Il and anti- immunofluorescence microscopy of the conventional vinculin mAb, strong signals with pAb-Il were detected frozen sections it never stained the microvilli of in the vinculin-positive cell-to-substrate AJ in most cells intestinal epithelial cells (Tsukita et al., 1989a), where (Fig. 10C,D). ezrin was reported to be highly concentrated Next, we examined whether the ERM family mem- (Bretscher, 1983). In a previous study, CR-22 did not bers are concentrated in the ruffling membranes, where stain the cell-to-cell AJ, whereas the cleavage furrow actin filaments are thought to be densely associated was clearly stained. When the cultured epithelial cells with the plasma membrane. It is known that in KB cells were exposed to the medium with a low calcium level, the formation of ruffling membranes is remarkably the cell-to-cell AJ was stained with CR-22, indicating induced by insulin (Goshimaetal., 1984). KB cells were that the epitope for CR-22 was masked in intact cell-to- thus cultured in the presence of insulin and immuno- cell AJ (Sato et al., 1991). Furthermore, in this study, it fluorescently stained with pAb-Il or CR22. As shown in became clear that the epitopes for pAb-Il were masked Fig. 11, the ridges of typical ruffling membranes were mostly in the cell-to-substrate AJ and that this masking clearly stained with these antibodies. The ruffling was removed by the zinc method. One must then membrane-like structures were also stained in other consider this masking problem when working with the types of cultured cells. Thus we conclude that at least ERM family members in immunolocalization studies. one of the ERM family members is concentrated at the The next step in immunolocalization work is to obtain cell-to-substrate AJ and the ruffling membranes. mAbs specific to each member of the ERM family. This 140 N. Sato and others Fig. 10. Concentration of ERM family members at cell-to-substrate adherens junctions in mouse MTD-IA cells. (A and B) After being fixed and permeabilized, cells were doubly stained with anti-radixin pAb-11 (A) and anti-vinculin mAb (B). In most cells (*) the vinculin-positive cell-to-substrate AJ are not stained by pAb-Il, while on rare occasions clear staining with pAb-Il can be detected at the vinculin-positive cell-to-substrate AJ (arrows). Bar, 21 j«n. (C and D) After the apical/lateral plasma membranes were removed by the zinc method, together with cytoplasm and nucleus, leaving the basal membranes, cells were doubly stained with pAb-Il (C) and anti-vinculin mAb (D). In most cells, strong signals with pAb-Il are detected in the vinculin-positive cell-to-substrate AJ (arrows). Bar, 13 /zm. The ezirin-radixin-moesin family 141 pAb-p800, which preferentially recognized radixin in immunoblots, was not potent enough for immunolocal- ization work in various cell types. In this study, we examined the distribution of the members of the ERM family. Taking both cross- reaction and masking problems into consideration, we conclude that at least one ERM family member is concentrated in the specific regions where actin fila- ments are densely associated with plasma membranes, namely, cell-to-cell AJ, cell-to-substrate AJ, microvilli, ruffling membranes, and cleavage furrows. The signifi- cance of the coexpression of more than two ERM family members in single cells is not clear at present. It could be that the three members are sorted out in the above five actin filament-associated regions. Another possibility is that this coexpression is a redundancy for the sake of safety. Further experiments with cDNAs and mAbs for each member are needed. Among the ERM family members, only radixin is shown to be directly bound to the barbed ends of actin filaments (Tsukita et al., 1989a). The direct interaction of ezrin or moesin with actin has not been reported. Taking the distribution of the ERM family members shown in this study into consideration, it is likely that not only radixin but also ezrin and moesin directly interact with actin filaments in vitro. In this connection, it is of note that the ERM family is included in the band 4.1 superfamily (Fig. 12) (Gould et al., 1989; Rees et al., 1990; Funayama et al., 1991; Lankes and Furth- mayr, 1991; Gu et al., 1991; Yang and Tonks, 1991). In erythrocyte membranes, the band 4.1 protein forms a complex with actin filaments and spectrin molecules, binding to glycophorin A or glycophorin C (glycocon- nectin)(Anderson and Lovrien, 1984; Bennett, 1989). The binding of band 4.1 protein with glycophorin was reported to be regulated by the phosphorylation of the band 4.1 protein (Danilov et al., 1990) and its association with polyphosphoinositide (Anderson and Marchesi, 1985). Therefore, further studies on the interaction between actin filaments and ERM family members in vitro should consider the effects of spectrin molecules, the possible glycophorin-like protein, poly- phosphoinositide, and their phosphorylation. It appears that the ERM family members may play a crucial role in the association of actin filaments with the plasma membrane, especially in the end-to-membrane association. Therefore, the distribution of these mem- bers in microvilli is discussed. As shown in Fig. 8, in L cells, CR-22 clearly stained elongated microvilli along Fig. 11. Concentration of ERM family members at ruffling their lengths. Also in microvilli of intestinal epithelial membranes in human KB cells. Ruffling membranes cells, ezrin was reported to be distributed at the (arrows) were induced by insulin. A. Phase-contrast image; (B and C) immunofluorescence microscopic images, cytoplasmic surface of the plasma membrane along double-stained with rhodamine-phalloidin (B) and anti- their lengths (Bretscher, 1983). Therefore, it could be radixin mAb, CR-22 (C). Note that the ridges of ruffling that the ERM family members are distributed evenly at membranes are clearly stained with CR-22. Bar, 15 /zm. the cytoplasmic surface of microvilli plasma membrane, and are not restricted to the tip of the microvilli. In microvilli of cultured cells, the arrangement of actin accomplished, however, the masking problem would filaments is not well known in contrast to that in make it difficult for immunofluorescence microscopy to intestinal microvilli. In intestinal microvilli, the barbed- determine conclusively the localization of each member ends of actin filaments are associated with the plasma of the family inside cells. In fact, an anti-radixin pAb, membrane in an end-to-membrane fashion only at the 142 N. Sato and others 20 280 Fig. 12. ERM family and band 4.1 superfamily. Mouse ezrin, Ezrin 1 mouse radixin, mouse moesin, 20 280 1 human erythrocyte band 4.1 Radix in 84% 67% ERM family protein (Conboy et al., 1986), 1 mouse talin (Rees et al., 1990) 20 280 and human protein-tyrosine Moesin 83% 62% | phosphatase HI (PTPH1) (Yang 1 J and Tonks, 1991) were listed Band 4.1 superfamily here as members of band 4.1 superfamily. Each protein has a 20 280 homologous domain (shadowed box) in its N-terminal region. Band 4.1 31% 18% The identity of the homologous 45 330 and nonhomologous domains of Talin f each protein with those of ezrin 20% was calculated and shown in 43 283 each box. This type of PTPH1 33% comparison clearly points to a gene family, the ERM family. tip of the microvilli (Ishikawa et al., 1969; Mooseker membranes and the associated cytoskeleton. J.Mol.Biol. 153, 361- and Tilney, 1975). In spite of intensive studies, 379. Bennett, V. (1989). The spectrin-actin junction of erythrocyte however, a barbed-end capping protein localized at the membrane skeletons. Biochim.Biophys.Acta 988, 107-121. tip of intestinal microvilli could not be identified, Bernstein, E. G., Hopper, M. L., Grandchamp, S. and Ephrussi, B. though purified microvilli were available. However, the (1973). Alkaline phosphatase activity in mouse teratoma. distribution of ezrin along the lengths of intestinal Proc.Nat.Acad.Sci.USA 70, 3899-3903. microvilli does not support our speculation that ERM Birgbauer, E. and Solomon, F. (1989). A marginal band-associated protein has properties of both microtubule- and microfilament- family members play a crucial role in the end-to- associated proteins. J.Cell Biol. 109, 1609-1620. membrane association of actin filaments with plasma Bretscher, A. (1983). Purification of an 80,000-dalton protein that is a membrane. The discrepancy between the "possible" component of the isolated microvillus cytoskeleton, and its capping activity of the ERM family members in vitro localization in nonmuscle cells. J.Cell Biol. 97, 425-432. and their distribution in microvilli remains to be Bretscber, A. (1989). Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A- clarified. Further studies on the properties in vitro and 431 cells by epidermal growth factor. J.Cell Biol. 108, 921-930. localization of each member of the ERM family in vivo Burridge, K. and Connell, L. (1983). A new protein of adherens are needed to understand better the molecular bases of plaques and ruffling membranes. J.Cell Biol. 97, 359-367. the actin filament/plasma membrane association in Conboy, J., Kan, Y. W., Shohet, S. B. and Mohandas, N. (1986). general. Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. Proc.Nat.Acad.Sci.USA 83, 9512-9516. We are particularly grateful to Dr.T.Obinata (Chiba Craig, S. W. and Pollard, T. D. (1982). Actin-binding proteins. University) for his helpful discussions and encouragement Trends Biochem.Sci. 7, 88-92. throughout this study. Our thanks are also due to the Danilov, Y. N., Fennell, R., Ling, E. and Cohen, C. M. (1990). members of our laboratory for their help and advice. We Selective modulation of band 4.1 binding to erythrocyte thank Miss K. Oishi and N. Sekiguchi (National Institute for membranes by protein kinase C. J.Biol.Chem. 265, 2556-2562. Physiological Sciences) for their technical assistance. A431 Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., and KB cells were supplied by JCRB (Japanese Cancer Roberts, T. M., An, Q. and Chen, L. B. (1991). Presence of an SH2 Research Resources Bank), and MTD-1, tk~L and F9 cells domain in the actin-binding protein tensin. Science 252, 712-715. Knami, J., Enami, S. and Koga, M. (1984). Isolation of an insulin- were kindly provided by Dr. M. Takeichi (Kyoto University). responsive preadipose cell line and a mammary tumor-producing, This study was supported in part by research grants from dome-forming epithelial cell line from a mouse mammary tumor. the Ministry of Education, Science and Culture of Japan; by Develop.Growth Differ. 26, 223-234. research grants for cancer research from the Ministry of Evans, R. R., Robson, R. M. and Stromer, M. H. (1984). Properties Health and Welfare of Japan; and by research grants from the of smooth muscle vinculin. J.Biol.Chem. 259, 3916-3924. Naitoh Research Foundation to Shoichiro Tsukita. Funayama, N., Nagafuchi, A., Sato, N. and Tsukita, Sh. (1991). Radixin is a novel member of the band 4.1 family. J.Cell Biol. 115, 1039-1048. Geiger, B. (1979). A 130K protein from chicken gizzard: its References localization at the termini of microfilament bundles in cultured chicken cells. CeU 18, 193-205. Anderson, R. A. and Lovrien, R. E. (1984). Glycophorin is linked by Geiger, B. (1983). Membrane-cytoskeletal interaction. Biochim. band 4.1 protein to the human erythrocyte membrane skeleton. Biophys. Acta 737, 305-341. Nature 307, 655-658. Geiger, B., Voik, T. and Volberg, T. (1985). Molecular heterogeneity Anderson, R. A. and Marchesi, V. T. (1985). Regulation of the of adherens junctions. J.Cell Biol. 101, 1523-1531. association of membrane skeletal protein 4.1 with glycophorin by a Goshima, K., Masuda, A. and Owaribe, K. (1984). Insulin-induced polyphosphoinositide. Nature 318, 295-298. formation of ruffling membranes of KB cells and its correlation Avnur, Z. and Geiger, B. (1981). Substrate-attached membranes of with enhancement of amino acid transport. J. Cell Biol. 98,801-809. cultured cells. Isolation and characterization of ventral cell Gould, K. L., Bretcher, A., Esch, F. S. and Hunter, T. (1989). cDNA The ezirin-radixin-moesin family 143 cloning and sequencing of the protein-tyrosine kinase substrate, Nagafuchi, A., TakeichI, M. and Tsukita, Sb. (1991). The 102 kd ezrin, reveals homology to band 4.1. EM BO J. 8, 4133-4142. cadherin-associated protein: similarity to vinculin and Gould, K. L., Cooper, J. A., Bretscher, A. and Hunter, T. (1986). The posttranscriptional regulation of expression. Cell 65, 1-20. protein-tyrosine kinase substrate, p81, is homologous to a chicken O'Farrell, P. H. (1975). High resolutional two-dimensional microvillar core protein. J.Cell Biol. 102, 660-669. electrophoresis of proteins. J.Biol.Chem. 250, 4007-4021. Graham, R. C. Jr and Karnovsky, M. J. (1966). The early stages of Pakkanen, R., Hedman, K., Turunen, O., Wahlstrom, T. and Vaheri, absorption of injected horseradish peroxidasc in the proximal A. (1987). Microvillus-specific Mr 75,000 plasma membrane protein tubule of mouse kidney: Ultrastructural cytochemistry by a new of human choriocarcinoma cells. J.Histochem. Cytochem. 135,809- technique. J.Hislochem.Cylochem. 14, 291-302. 816. Gu, Y., York, J. D., Warshawsky, I. and Majerus, P. W. (1991). Pelham, H. R. and Jackson, R. J. (1976). An efficient mRNA- Identification, cloning, and expression of a cytosolic dependent translation system from reticulocyte lysates. Eur.J. megakaryocyte protein-tyrosine-phosphatase with sequence Biochem. 67, 247-256. homology to cyotoskeletal protein 4.1. Proc.Nat.Acad.Sci. USA 88, Pollard, T. D. and Cooper, J. A. (1986). Actin and actin-binding 5867-5871. proteins. A critical evaluation of mechanisms and functions. Hirano, S., Nose, A., Hatta, K., Kawakami, A. and Takelchi, M. Annu.Rev.Biochem. 55, 987-1035. (1987). Calcium-dependent cell-cell adhesion molecules Pollard, T. D. and Weihing, R. R. (1974). Actin and myosin in cell (cadherins): Subclass specificities and possible involvement of actin movement. CRC Crit.Rev.Biochem. 2, 1-65. bundles. J.Cell Biol. 105, 2501-2510. Rees, D. J. G., Ades, S. E., Singer, S. J. and Hynes, R. O. (1990). Ishikawa, H. (1979). Identification and distribution of intracellular Sequence and domain structure of talin. Nature 347, 685-689. filaments. In Cell Motility: Molecules and Organization (ed. Sambrook, J., Maniatis, T. and Fritsch, E. F. (1989). Molecular Hatano, S. Ishikawa, H. and Sato, H.), pp. 417-444. University of Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Tokyo Press, Tokyo. Laboratory Press, Cold Spring Harbor, New York. Ishikawa, H., BischofT, R. and Holtzer, H. (1969). Formation of Sato, N., Yonemura, S., Obinata, T., Tsukita, Sa. and Tsukita, Sh. arrowhead complexes with heavy meromyosin in a variety of cell (1991). Radixin, a barbed end-capping actin-modulating protein, is types. J.Cell Biol. 43, 312-328. concentrated at the cleavage furrow during cytokinesis. J.Cell Biol. ltoh, M., Yonemura, S., Nagafuchl, A., Tsukita, Sa. and Tsukita, Sh. 113, 321-330. (1991). A 220kD undercoat-constitutive protein: Its specific Tsukita, Sa., Hleda, Y. and Tsukita, Sh. (1989a). A new 82-kD localization at the cadherin-based cell-cell adhesion sites. J. Cell barbed end-capping protein (radixin) localized in the cell-to-cell Biol. 115, 1449-1462. adherens junction: Purification and characterization. J. Cell Biol. Laemmli, U. K. (1970). Cleavage of structural proteins during the 108, 2369-2382. assembly of the head of bacteriophage T4. Nature 227, 680-685. Lankes, W. and Furthmavr, H. (1991). Mocsin: A member of the Tsukita, Sa., Oishi, K., Akiyama, T., Yamanashi, Y., Yamamoto, T. protein 4.1-talin-ezrin family of proteins. Proc.Nat.Acad.Sci.USA and Tsukita, Sh. (1991). Specific proto-oncogenic tyrosine kinases 88, 8297-8301. of sre family are enriched in cell-to-cell adherens junctions where Lankes, W., Griesmacher, A., Grunwald, J., Schwartz-Albiez, R., the level of tyrosine phosphorylation is elevated. J.Cell Biol. 113, and Keller, R. (1988). A heparin-binding protein involved in 867-879. inhibition of smooth-muscle cell proliferation. Biochem.J. 251, 831- Tsukita, Sh., ltoh, M. and Tsukita, Sa. (1989b). A new 400-kD 842. protein from isolated adherens junctions: Its localization at the Louvard, D. (1989). The function of the major cytoskeletal undercoat of adherens junctions and at microfilament bundles such components of the brush border. Curr.Opin. Cell Biol. 1, 51-57. as stress fibers and circumferential bundles. J.Cell Biol. 109, 2905- Matsudaira. P. T. and Burgess, D. R. (1979). Identification and 2915. organization of the components in the isolated microvillus Tsukita, Sh. and Tsukita, Sa. (1989). Isolation of cell-to-cell adherens cytoskeleton. J.Cell Biol. 83, 667-673. junctions from rat liver. J.Cell Biol. 108, 31-41. Miron, T., Vancompernolle, K., Vandekerckhove, J., Wilchek, M. Turunen, O., Wlnqvist, R., Pakkanen, R., Grzeschik, K. H., and Geiger, B. (1991). A 25-kD inhibitor of actin polymerization is Wahlstrom, T. and Vaheri, A. (1989). Cytovillin, a microvillar Mr a low molecular mass heat shock protein. J.Cell Biol. 114,255-261. 75,000 protein. cDNA sequence, prokaryotic expression, and Miron, T., Wilchek, M. and Geiger, B. (1988). Characterization of an chromosomal localization. J.Biol.Chem. 264, 16,727-16,732. inhibitor of actin polymerization in vinculin-rich fraction of turkey Vandekerckhove, J. (1990). Actin-binding proteins. Curr.Opin. Cell gizzard smooth muscle. Eur.J.Biochem. 178, 543-553. Biol. 2, 41-51. Mooseker, M. S. and Tilney, L. G. (1975). The organization of an Wilkins, J. A. and Lin, S. (1982). High-affinity interaction of vinculin actin filament-membrane complex: filament polarity and with actin in vitro. Cell 28, 83-90. membrane attachment in the microvilli of intestinal epithelial cells. Wilkins, J. A. and Lin, S. (1986). A re-examination of the interaction J.Cell Biol. 67, 725-743. of vinculin with actin. J.Cell Biol. 102, 1085-1092. Murayama-Okabayashi, F., Okada, Y. and Tachibana, T. (1971). A Yang, Q. and Tonks, N. K. (1991). Isolation of a cDNA clone scries of hybrid cells containing different ratios of parental encoding a human protein-tryrosine phosphatase with homology to chromosomes formed by two steps of artificial fusion. Proc. the cyotskeletal-associated proteins band 4.1, ezrin, and talin. Nat.Acad.Sci.USA 68, 38-42. Proc.Nat.Acad.Sci.USA 88, 5949-5953. Nagafuchl, A., Shirayoshi, V., Okazaki, K., Yasuda, K. and Takcichi, M. (1987). Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature 329, 341-343. {Received 31 March 1992 - Accepted 28 May 1992)