Concentration of an Integral Membrane Protein, CD43 (Leukosialin, Sialophofin), in the Cleavage Furrow through the Interaction of Its Cytoplasmic Domain with Actin-based Cytoskeletons Shigenobu Yonemura,* Akira Nagafuchi,* Naruki Sato,** and Shoichiro Tsukita*r * Laboratory of Cell Biology, Department of Information Physiology,National Institute for Physiological Sciences, Okazaki, Aichi 444, Japan; and *Department of Physiological Sciences, School of Life Science, The Graduate University of Advanced Studies, Myodaiji-cho, Okazaki, Aichi 444, Japan Abstract. In leukocytes such as thymocytes and consisting of the extracellular domain of mouse Downloaded from http://rupress.org/jcb/article-pdf/120/2/437/1256004/437.pdf by guest on 24 September 2021 basophilic leukemia cells, a glycosilated integral mem- E-cadherin and the transmembrane/cytoplasmic do- brane protein called CIM3 (leukosialin or sialopho- main of rat CD43, and introduced it into mouse L rin), which is defective in patients with Wiskott- fibroblasts lacking both endogenous CD43 and Aldrich syndrome, was highly concentrated in the E-cadherin. In dividing transfectants, the chimeric cleavage furrow during cytokinesis. Not only at the molecules were concentrated in the cleavage furrow mitotic phase but also at interphase, CIM3 was pre- together with ERM, and both proteins were precisely cisely colocalized with ezrin-radixin-moesin family colocalized throughout the cell cycle. Furthermore, members (ERM), which were previously reported to using this transfection system, we narrowed down the play an important role in the plasma membrane-actin domain responsible for the CD43-concentration in the filament association in general. At the electron micro- cleavage furrow. Based on these findings, we conclude scopic level, throughout the cell cycle, both CIM3 and that CD43 is concentrated in the cleavage furrow ERM were tightly associated with microvilli, provid- through the direct or indirect interaction of its cyto- ing membrane attachment sites for actin filaments. We plasmic domain with ERM and actin filaments. constructed a cDNA encoding a chimeric molecule HE mechanism of cytokinesis has attracted increasing furrow is limited at the molecular level. This information is interest among cell biologists (for review see Mabuchi, indispensable in understanding the molecular basis for the T 1986; Rappaport, 1986; Satterwhite and Pollard, formation of cleavage furrows. 1992). Before cytokinesis, an unknown signal is thought to To clarify the molecular linkage from the actin filaments be transferred from the mitotic apparatus to the equatorial to the plasma membrane in the cleavage furrow, the first cortex, inducing the formation of a bundle of aligned actin protein that should be identified is an actin-modulating pro- filaments called the contractile ring just beneath the plasma tein. Many actin-modulating proteins reportedly show high membrane (Perry et al., 1971; Schroeder, 1973). At cytoki- affinity for the plasma membranes; for example, gelsolin nesis, the constriction of this ring results in the formation of (Janmey et al., 1987; Hartwig et al., 1989), lipocortin-85 the cleavage furrow and finally in the cell division. The me- (lkebuchi and Waisman, 1990), myosin-I (Adams and Pol- chanical force for the constriction is generated through the lard, 1989; Miyata et al., 1989; Mooseker et al., 1989), interaction between actin filaments and myosin-II inside the actin-binding protein (Fox, 1985; Ohta et al., 1991), spectrin ring (Fujiwara and Pollard, 1976; Mabuchi and Okuno, (for review see Bennet, 1989), the myristoylated, alanine- 1977; Yumura and Fukui, 1985; DeLozanne and Spudich, rich C kinase substrate (Hartwig et al., 1992), and ponticu- 1987; Knecht and Loomis, 1987; Karess et al., 1991). So far, lin (Shariff and Luna, 1990). However, none of them are various types of proteins, including actin and myosin-H, are reportedly concentrated in the cleavage furrow during cyto- reportedly concentrated in the cleavage furrow (Sanger et kinesis. Recently, an 82-kD protein called radixin, a barbed al., 1987, 1989; Ishidate and Mabuchi, 1988; Schweisguth end-capping, actin-modulating protein, has been reported to et al., 1990; Earnshaw and Cooke, 1991; Yonemura et al., be highly concentrated in the cleavage furrow in various 1991). However, despite these intensive studies, knowledge types of cells (Tsukita et al., 1989; Sato et al., 1991). Se- of the molecular architecture of the contractile ring remains quence analysis of radixin eDNA has revealed that radixin fragmentary. Especially, information on the way actin fila- is highly homologous to ezrin and moesin (Gould et al., ments associate with the plasma membrane in the cleavage 1989; Turunen et al., 1989; Funayama et al., 1991; Lankes The Rockefeller University Press, 0021-9525/93/01/437/13 $2.00 The Journal of Cell Biology, Volume 120, Number 2, January 1993 437-449 437 and Furthmayr, 1991), and that there is a gene family consist- ERM. The results of this study will lead us to a better under- ing of ezrin, radixin, and moesin, which we call the ERM ~ standing of the molecular mechanism of the cleavage furrow family (Sato et al., 1992; Tsukita et al., 1992). Recent close formation. immunofluorescence microscopic analyses have shown that the ERM family plays a crucial role in the association of ac- tin filaments with the plasma membrane in general. Con- Materials and Methods sidering that our anti-radixin pAb and mAb more or less crossreacted with ezrin and moesin, we should strictly say Cells and Antibodies that ERM family members are concentrated at the cleavage Thymocytes from 2-wk-old Wistar rats were collected by centrifugation, furrow (see details in Sato et al., 1992). Interestingly, the resuspended in RPMI-1640 supplemented with 10% FCS, and incubated for 30-60 rain at 37~ RBL-2H3 cells were obtained from the Japanese Can- NH2-terminal half of ERM significantly resembles that of cer Research Resources Bank (Tokyo, Japan) and cultured in RPMI-1640 band 4.1 protein, a major undercoat constituent of the eryth- with 10% FCS. Mouse fibroblastic L cells (Earle, 1943) and their E-cad- rocyte membrane (Sato et al., 1992). In erythrocyte mem- herin transfectants, EL8 cells (Nagafuchi et al., 1987), were cultured in branes, band 4.1 protein binds to an integral membrane pro- DME with 10% FCS. tein called glycophorin C through its NHe-terminal domain The following antibodies were used: anti-rat CD43 mAb, W3/13 (Cedar- lane Laboratories Limited, Hornby, Ontario, Canada); anti-radixin pAb, (Leto et al., 1986). Therefore, it is tempting to speculate that pAb-I1 (Tsukita et al., 1989); anti-radixin mAb, CR22 (Sato et al., 1991); in the cleavage furrow, ERM directly binds actin filaments anti-E-cadherin pAb (Nagafuchi et al., 1987); and anti-E-cadherin mAb, to a membrane protein (glycophorin-like?) at its NH2- ECCD-2 (Shirayoshi et al., 1986). Since pAb-I1 and mAb-CR22 were re- terminal domain. cently found to more or less crossreact with ezrin and moesin (Sato et al., 1992), we tentatively call these antibodies anti-ERM pAb and mAb, respec- Downloaded from http://rupress.org/jcb/article-pdf/120/2/437/1256004/437.pdf by guest on 24 September 2021 Since actin filaments are recruited preferentially into the tively. Rhodamine-phaUoidin (Molecular Probes, Inc., Eugene, OR) was cleavage furrow by migration of preexisting filaments in the used to stain aetin filaments. Chromosomes were visualized with 4'6- cell cortex and are not all assembled de novo in the furrow diamidino-2-phe~'Iindole dihydrochloride. (Cao and Wang, 1990a), the membrane protein such as the above putative ERM-anchoring protein, which is responsi- Immunofluorescence Microscopy ble for linking actin filaments to the plasma membrane, is All procedures were carried out at room temperature. Cells were fixed with likely to accumulate in the cleavage furrow. However, the oc- 4% fresh formaldehyde in 0.1 M Hepes buffer (pH 7.5) for 15 min. After currence of such a cleavage furrow-accumulated membrane washing three times with PBS containing 30 mM glycine (glycine-PBS), protein has not often been discussed in the field of cytokine- cells were soaked in blocking solution ~lycine-PBS containing 2 % normal goat serum) for 5 rain and incubated with W3/13 or anti-E-cadherin pAb sis. We recently noticed .that de Petris (1984), during the diluted with the blocking solution for 30 min. They were then washed with course of his studies on capping of the cell surface glycopro- glycine-PBS three times, treated with 0.2 % Triton X-100 in glycine-PBS for teins in lymphoid cells, described the concentration of 15 rain, and washed with glycine-PBS three times. After being soaked in W3/13 antigen in the cleavage furrow in dividing rat thymo- the blocking solution for 5 min, cells were incubated with anti-ERM pAh cytes. The W3/13 antigen is a rat homologue of human CIM3 or mAb diluted with the blocking solution for 1 h, washed three times, and soaked in the blocking solution for 5 min. They were then incubated with (leukosialin or sialophorin) (Killeen et al., 1987), which is secondary antibodies diluted with the blocking solution for 30 min. FITC- an integral membrane protein expressed in most leukocytes conjugated sheep anti-mouse Ig antibody and rhodamine-conjugated don- and platelets, and is defective in patients with the X-chromo- key anti-rabbit Ig (Amersham International plc, Amersham, UK) were used some-linked immunodeficiency disorder, Wiskott-Aldrich as secondary antibodies. For some studies, rhodamine-phalloidin and/or 4'6-diamidino-2-phenylindole dihydrochloride were mixed with the second- syndrome (for review see Remold-O'Donnell and Rosen, ary antibody. Cells were washed three times and then mounted in 90% 1990). Considering that CD43 is heavily sialoglycosylated glycerol-PBS containing 0.1% para-phenylendiamine. Some samples were just like glycophorin C (Brown et al., 1981) and that one anti- not treated with detergent or with anti-ERM antibody.