A Gene Family Consisting of Ezrin, Radixin and Moesin

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.

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