Normal Development of Mice and Unimpaired Cell Adhesion/Cell Motility/Actin-based Cytoskeleton without Compensatory Up-regulation of Ezrin or Radixin in Moesin Gene Knockout

Ezrin/radixin/moesin (ERM) proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand, experiments at the cellular level and in vitro have suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level. Ezrin/radixin/moesin (ERM) proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand, experiments at the cellular level and in vitro have suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level. ezrin/radixin/moesin embryonic stem monoclonal antibody polyclonal antibody kilobase(s) splicing acceptor polyadenylation signal base pair(s). Three closely related proteins, ezrin, radixin, and moesin constitute a gene family called the ERM1 family. Ezrin, radixin, and moesin were identified in different directions (1Bretscher A. J. Cell Biol. 1983; 97: 425-432Crossref PubMed Scopus (217) Google Scholar, 2Pakkanen R. Hedman K. Turunen O. Wahlström T. Vaheri A. J. Histochem. Cytochem. 1987; 35: 809-816Crossref PubMed Scopus (53) Google Scholar, 3Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar, 4Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 5Hunter T. Cooper J.A. Cell. 1981; 24: 741-752Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 6Tsukita S. Hieda V. Tsukita S. J. Cell Biol. 1989; 108: 2369-2382Crossref PubMed Scopus (155) Google Scholar, 7Lankes W.T. Griesmacher A. Grünwald J. Schwartz-Albiez R. Keller R. Biochem. J. 1988; 251: 831-842Crossref PubMed Scopus (100) Google Scholar), but isolation and sequencing of their cDNAs revealed that they were closely related (amino acid sequence identity of 70–80% in the mouse) (8Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (207) Google Scholar, 9Turunen O. Winqvist R. Pakkanen R. Grzeschik K.H. Wahlström T. Vaheri A. J. Biol. Chem. 1989; 264: 16727-16732Abstract Full Text PDF PubMed Google Scholar, 10Funayama N. Nagafuchi A. Sato N. Tsukita S. Tsukita S. J. Cell Biol. 1991; 115: 1039-1048Crossref PubMed Scopus (137) Google Scholar, 11Sato N. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Sci. 1992; 103: 131-143PubMed Google Scholar, 12Lankes W.T. Furthmayr H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8297-8301Crossref PubMed Scopus (211) Google Scholar). Independently of the lines of study on ERM proteins, another ERM-like protein was identified as a tumor suppressor or hereditary neurofibromatosis type 2 and named merlin (moesin/ezrin/radixin-like protein) or schwannomin (13Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Pulst S.M. Lenoir G. Bijlsma E. Fashold R. Dumanski J. de Jong P. Parry D. Eldrige R. Aurias A. Delattre O. Thomas G. Nature. 1993; 363: 515-521Crossref PubMed Scopus (1181) Google Scholar, 14Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Haase V.H. Ambrose C.M. Munroe D.M. Bove C. Haines J.L. Martuza R.L. MacDonald M.E. Seizinger B.R. Short M.P. Buckler A.J. Gusella J.F. Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1088) Google Scholar).It is now widely accepted that ERM proteins function as general cross-linkers between plasma membranes and actin filaments (for reviews see Refs. 15Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 16Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 17Tsukita S. Yonemura S. Tsukita S. Trends. Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 18Tsukita S. Yonemura S. Tsukita S. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (308) Google Scholar, 19Vaheri A. Carpen O. Heiska L. Helander T.S. Jaaskelainen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (161) Google Scholar). The highly conserved NH2-terminal half of ERM proteins directly binds to the cytoplasmic domains of integral membrane proteins such as CD44, CD43, ICAM-1, and ICAM-2 (20Yonemura S. Nagafuchi A. Sato N. Tsukita S. J. Cell Biol. 1993; 120: 437-449Crossref PubMed Scopus (131) Google Scholar, 21Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. Tsukita S. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (677) Google Scholar, 22Helander T.S. Carpén O. Turunen O. Kovanen P.E. Vaheri A. Timonen T. Nature. 1996; 382: 265-268Crossref PubMed Scopus (200) Google Scholar, 23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 24Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (503) Google Scholar). On the other hand, ERM proteins directly interact with actin filaments (25Turunen O. Wahlström T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (346) Google Scholar, 26Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 27Roy C. Martin M. Mangeat P. J. Biol. Chem. 1997; 272: 20088-20095Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 28Martin M. Roy C. Montcourrier P. Sahuquet A. Mangeat P. Mol. Biol. Cell. 1997; 8: 1543-1557Crossref PubMed Scopus (55) Google Scholar). The co-existence of plasma membrane- and actin filament-binding domains in individual molecules may allow ERM proteins to function as plasma membrane/actin filament cross-linkers. Furthermore, ERM proteins are also thought to be involved in plasma membrane/actin filament cross-linkage through hetero- and/or homo-dimerization (29Gary R. Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10846-10850Crossref PubMed Scopus (93) Google Scholar, 30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (373) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar, 32Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and through binding to EBP (ERM-binding phosphoprotein) 50/NHE-RF (regulatory cofactor of Na+-H+ exchanger) (33Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar, 34Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (310) Google Scholar). There is accumulating evidence that the cross-linking activity of ERM proteins is regulated by the Rho-dependent signaling pathway through binding to Rho-GDP dissociation inhibitor and/or Rho-dependent phosphorylation (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar, 37Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 38Shaw R.J. Henry M. Solomon F. Jacks T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar).One of the important questions regarding ERM proteins that have not yet been addressed is the extent to which ezrin, radixin, and moesin are functionally redundant. Targeted disruption of ERM protein genes would be one of the most direct ways to approach this issue. Among the ERM proteins, we expected moesin to be rather functionally unique, partly because this molecule lacks the polyproline stretch found in both ezrin and radixin and partly because only moesin is not tyrosine phosphorylated by the epidermal growth factor receptor (39Franck Z. Gary R. Bretscher A. J. Cell Sci. 1993; 105: 219-231Crossref PubMed Google Scholar). Therefore, to address the redundancy problem in ERM proteins we generated mice with a targeted null mutation of the moesin gene located on the X chromosome.DISCUSSIONERM (ezrin/radixin/moesin) proteins have been implicated as general cross-linkers between the plasma membrane and actin filaments (for reviews see Refs. 15Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 16Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 17Tsukita S. Yonemura S. Tsukita S. Trends. Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 18Tsukita S. Yonemura S. Tsukita S. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (308) Google Scholar, 19Vaheri A. Carpen O. Heiska L. Helander T.S. Jaaskelainen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (161) Google Scholar). In this study, we generated male and female mice hemi- and homozygous, respectively, for a null mutation in the moesin gene located on the X chromosome. Surprisingly, the mutant mice exhibited no obvious abnormalities in appearance or fertility, and a systemic histological scan of mutant tissues revealed no abnormalities. Our results clearly demonstrated that moesin is not required for normal mouse development or for survival in the laboratory environment. This is surprising in view of the degree of conservation of the moesin gene, for example, the occurrence of a moesin gene inDrosophila (55Edwards K.A. Montague R.A. Shepard S. Edgar B.A. Erikson R.L. Kiehart D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4589-4593Crossref PubMed Scopus (59) Google Scholar, 56McCartney B.M. Fehon R.G. J. Cell Biol. 1996; 133: 843-852Crossref PubMed Scopus (129) Google Scholar) and the tissue-specific regulated expression of this gene (57Amieva M.R. Wilgenbun K.K. Furthmayr H. Exp. Cell Res. 1994; 210: 140-144Crossref PubMed Scopus (61) Google Scholar, 58Berryman M. Franck Z. Bretscher A. J. Cell Sci. 1993; 105: 1025-1043Crossref PubMed Google Scholar, 59Schwartz-Albiez R. Merling A. Spring H. Moller P. Koretz K. Eur. J. Cell Biol. 1995; 67: 189-198PubMed Google Scholar).To date immunofluorescence microscopy and immunoblotting analyses have revealed that the ratio of the levels of ezrin, radixin, and moesin expression in individual cells varies between different tissues in wild-type mice. Furthermore, ezrin, radixin, and moesin are not always colocalized. From these in situ observations in wild-type mice, ezrin, radixin, and moesin have been suggested to have specific functions. However, experiments in vitro or at the cellular level have not clearly identified differences in function between these molecules. When the expression of any one or two ERM proteins was selectively suppressed by antisense oligonucleotides, no phenotypic changes were detected, and cell-cell/cell-matrix adhesion and microvillar formation were affected only when expression of all family members was suppressed (40Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar). Furthermore, the NH2-terminal halves of all ERM proteins directly bound to the cytoplasmic domains of CD44, Rho-GDP dissociation inhibitor, and PIP2 (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and formed both homo- and heterodimers (29Gary R. Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10846-10850Crossref PubMed Scopus (93) Google Scholar, 30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (373) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar).The present results favor the notion that ERM proteins are functionally redundant also at the whole body level. Interestingly, in moesin-deficient mice as well as isolated moesin-deficient cells, targeted disruption of the moesin gene did not induce compensatory up-regulation of the other members of the ERM family. In any of the tissues examined in mutant mice, no changes were detected in the expression levels or subcellular distributions of ezrin or radixin. More importantly, in isolated moesin-deficient cells such as platelets and mast cells, in which moesin was predominantly expressed in wild-type cells, moesin completely disappeared, leaving relatively small amounts of ezrin and radixin. The aggregation activity of moesin-deficient platelets and the microvillar formation of moesin-deficient mast cells were not affected. Considering that ERM proteins were thought to be directly involved in platelet aggregation (54Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1996; 270: 31377-31385Abstract Full Text Full Text PDF Scopus (184) Google Scholar) and microvillar formation (4Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar, 40Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar, 60Bretscher A. Curr. Opin. Cell Biol. 1993; 5: 653-660Crossref PubMed Scopus (48) Google Scholar, 61Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar), we concluded that only a small fraction of total ERM proteins in wild-type platelets and mast cells are sufficient for their physiological functions. Similar observations, i.e. no phenotypic changes in knockout mice without up-regulation of other family members, has been reported in various systems. For example, mice devoid of components of intermediate-sized filaments such as vimentin and glial fibrillary acidic protein developed normally without compensatory expression of other intermediate filament components (62Colucci-Guyon E. Porter M.M. Dunia I. Paulin D. Pournin S. Babinet C. Cell. 1994; 79: 679-694Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 63Gomi H. Yokoyama T. Fujimoto K. Ikeda T. Katoh A. Itoh T. Itohara S. Neuron. 1995; 14: 29-41Abstract Full Text PDF PubMed Scopus (229) Google Scholar).Another issue that we should discuss here is the function of moesin in intracellular signaling. We proposed that the cross-linking activities of ERM proteins were regulated by the Rho-dependent signaling pathway through direct binding to Rho-GDP dissociation inhibitor and/or through Rho-dependent posphorylation (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar, 37Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 38Shaw R.J. Henry M. Solomon F. Jacks T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar). Ezrin and radixin bound to Rho-GDP dissociation inhibitor with similar binding constants to moesin and were phosphorylated by Rho kinase with similar efficiency to moesin in vitro. Mackay et al. (36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar) found that moesin was required for the Rho-dependent formation of stress fibers and focal contacts in permeabilized Swiss 3T3 cells. Also in this case, moesin was able to be replaced by ezrin and radixin. From the present results, we concluded that the functions of ERM proteins in the Rho-dependent signaling pathway are redundant also at the whole body level.In conclusion, the present observations were consistent with the notion that ERM proteins are functionally redundant. The possibility cannot be excluded that a more distantly related protein to moesin in the band 4.1 superfamily functionally compensates for the lack of moesin. Further generation of mutant mice lacking ERM proteins, both singly and in combination, will lead to a better understanding of the physiological relevance of the occurrence of these three closely related proteins. Three closely related proteins, ezrin, radixin, and moesin constitute a gene family called the ERM1 family. Ezrin, radixin, and moesin were identified in different directions (1Bretscher A. J. Cell Biol. 1983; 97: 425-432Crossref PubMed Scopus (217) Google Scholar, 2Pakkanen R. Hedman K. Turunen O. Wahlström T. Vaheri A. J. Histochem. Cytochem. 1987; 35: 809-816Crossref PubMed Scopus (53) Google Scholar, 3Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar, 4Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 5Hunter T. Cooper J.A. Cell. 1981; 24: 741-752Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 6Tsukita S. Hieda V. Tsukita S. J. Cell Biol. 1989; 108: 2369-2382Crossref PubMed Scopus (155) Google Scholar, 7Lankes W.T. Griesmacher A. Grünwald J. Schwartz-Albiez R. Keller R. Biochem. J. 1988; 251: 831-842Crossref PubMed Scopus (100) Google Scholar), but isolation and sequencing of their cDNAs revealed that they were closely related (amino acid sequence identity of 70–80% in the mouse) (8Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (207) Google Scholar, 9Turunen O. Winqvist R. Pakkanen R. Grzeschik K.H. Wahlström T. Vaheri A. J. Biol. Chem. 1989; 264: 16727-16732Abstract Full Text PDF PubMed Google Scholar, 10Funayama N. Nagafuchi A. Sato N. Tsukita S. Tsukita S. J. Cell Biol. 1991; 115: 1039-1048Crossref PubMed Scopus (137) Google Scholar, 11Sato N. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Sci. 1992; 103: 131-143PubMed Google Scholar, 12Lankes W.T. Furthmayr H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8297-8301Crossref PubMed Scopus (211) Google Scholar). Independently of the lines of study on ERM proteins, another ERM-like protein was identified as a tumor suppressor or hereditary neurofibromatosis type 2 and named merlin (moesin/ezrin/radixin-like protein) or schwannomin (13Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Pulst S.M. Lenoir G. Bijlsma E. Fashold R. Dumanski J. de Jong P. Parry D. Eldrige R. Aurias A. Delattre O. Thomas G. Nature. 1993; 363: 515-521Crossref PubMed Scopus (1181) Google Scholar, 14Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Haase V.H. Ambrose C.M. Munroe D.M. Bove C. Haines J.L. Martuza R.L. MacDonald M.E. Seizinger B.R. Short M.P. Buckler A.J. Gusella J.F. Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1088) Google Scholar). It is now widely accepted that ERM proteins function as general cross-linkers between plasma membranes and actin filaments (for reviews see Refs. 15Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 16Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 17Tsukita S. Yonemura S. Tsukita S. Trends. Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 18Tsukita S. Yonemura S. Tsukita S. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (308) Google Scholar, 19Vaheri A. Carpen O. Heiska L. Helander T.S. Jaaskelainen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (161) Google Scholar). The highly conserved NH2-terminal half of ERM proteins directly binds to the cytoplasmic domains of integral membrane proteins such as CD44, CD43, ICAM-1, and ICAM-2 (20Yonemura S. Nagafuchi A. Sato N. Tsukita S. J. Cell Biol. 1993; 120: 437-449Crossref PubMed Scopus (131) Google Scholar, 21Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. Tsukita S. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (677) Google Scholar, 22Helander T.S. Carpén O. Turunen O. Kovanen P.E. Vaheri A. Timonen T. Nature. 1996; 382: 265-268Crossref PubMed Scopus (200) Google Scholar, 23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 24Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (503) Google Scholar). On the other hand, ERM proteins directly interact with actin filaments (25Turunen O. Wahlström T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (346) Google Scholar, 26Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 27Roy C. Martin M. Mangeat P. J. Biol. Chem. 1997; 272: 20088-20095Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 28Martin M. Roy C. Montcourrier P. Sahuquet A. Mangeat P. Mol. Biol. Cell. 1997; 8: 1543-1557Crossref PubMed Scopus (55) Google Scholar). The co-existence of plasma membrane- and actin filament-binding domains in individual molecules may allow ERM proteins to function as plasma membrane/actin filament cross-linkers. Furthermore, ERM proteins are also thought to be involved in plasma membrane/actin filament cross-linkage through hetero- and/or homo-dimerization (29Gary R. Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10846-10850Crossref PubMed Scopus (93) Google Scholar, 30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (373) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar, 32Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and through binding to EBP (ERM-binding phosphoprotein) 50/NHE-RF (regulatory cofactor of Na+-H+ exchanger) (33Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar, 34Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (310) Google Scholar). There is accumulating evidence that the cross-linking activity of ERM proteins is regulated by the Rho-dependent signaling pathway through binding to Rho-GDP dissociation inhibitor and/or Rho-dependent phosphorylation (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar, 37Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 38Shaw R.J. Henry M. Solomon F. Jacks T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar). One of the important questions regarding ERM proteins that have not yet been addressed is the extent to which ezrin, radixin, and moesin are functionally redundant. Targeted disruption of ERM protein genes would be one of the most direct ways to approach this issue. Among the ERM proteins, we expected moesin to be rather functionally unique, partly because this molecule lacks the polyproline stretch found in both ezrin and radixin and partly because only moesin is not tyrosine phosphorylated by the epidermal growth factor receptor (39Franck Z. Gary R. Bretscher A. J. Cell Sci. 1993; 105: 219-231Crossref PubMed Google Scholar). Therefore, to address the redundancy problem in ERM proteins we generated mice with a targeted null mutation of the moesin gene located on the X chromosome. DISCUSSIONERM (ezrin/radixin/moesin) proteins have been implicated as general cross-linkers between the plasma membrane and actin filaments (for reviews see Refs. 15Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 16Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 17Tsukita S. Yonemura S. Tsukita S. Trends. Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 18Tsukita S. Yonemura S. Tsukita S. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (308) Google Scholar, 19Vaheri A. Carpen O. Heiska L. Helander T.S. Jaaskelainen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (161) Google Scholar). In this study, we generated male and female mice hemi- and homozygous, respectively, for a null mutation in the moesin gene located on the X chromosome. Surprisingly, the mutant mice exhibited no obvious abnormalities in appearance or fertility, and a systemic histological scan of mutant tissues revealed no abnormalities. Our results clearly demonstrated that moesin is not required for normal mouse development or for survival in the laboratory environment. This is surprising in view of the degree of conservation of the moesin gene, for example, the occurrence of a moesin gene inDrosophila (55Edwards K.A. Montague R.A. Shepard S. Edgar B.A. Erikson R.L. Kiehart D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4589-4593Crossref PubMed Scopus (59) Google Scholar, 56McCartney B.M. Fehon R.G. J. Cell Biol. 1996; 133: 843-852Crossref PubMed Scopus (129) Google Scholar) and the tissue-specific regulated expression of this gene (57Amieva M.R. Wilgenbun K.K. Furthmayr H. Exp. Cell Res. 1994; 210: 140-144Crossref PubMed Scopus (61) Google Scholar, 58Berryman M. Franck Z. Bretscher A. J. Cell Sci. 1993; 105: 1025-1043Crossref PubMed Google Scholar, 59Schwartz-Albiez R. Merling A. Spring H. Moller P. Koretz K. Eur. J. Cell Biol. 1995; 67: 189-198PubMed Google Scholar).To date immunofluorescence microscopy and immunoblotting analyses have revealed that the ratio of the levels of ezrin, radixin, and moesin expression in individual cells varies between different tissues in wild-type mice. Furthermore, ezrin, radixin, and moesin are not always colocalized. From these in situ observations in wild-type mice, ezrin, radixin, and moesin have been suggested to have specific functions. However, experiments in vitro or at the cellular level have not clearly identified differences in function between these molecules. When the expression of any one or two ERM proteins was selectively suppressed by antisense oligonucleotides, no phenotypic changes were detected, and cell-cell/cell-matrix adhesion and microvillar formation were affected only when expression of all family members was suppressed (40Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar). Furthermore, the NH2-terminal halves of all ERM proteins directly bound to the cytoplasmic domains of CD44, Rho-GDP dissociation inhibitor, and PIP2 (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and formed both homo- and heterodimers (29Gary R. Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10846-10850Crossref PubMed Scopus (93) Google Scholar, 30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (373) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar).The present results favor the notion that ERM proteins are functionally redundant also at the whole body level. Interestingly, in moesin-deficient mice as well as isolated moesin-deficient cells, targeted disruption of the moesin gene did not induce compensatory up-regulation of the other members of the ERM family. In any of the tissues examined in mutant mice, no changes were detected in the expression levels or subcellular distributions of ezrin or radixin. More importantly, in isolated moesin-deficient cells such as platelets and mast cells, in which moesin was predominantly expressed in wild-type cells, moesin completely disappeared, leaving relatively small amounts of ezrin and radixin. The aggregation activity of moesin-deficient platelets and the microvillar formation of moesin-deficient mast cells were not affected. Considering that ERM proteins were thought to be directly involved in platelet aggregation (54Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1996; 270: 31377-31385Abstract Full Text Full Text PDF Scopus (184) Google Scholar) and microvillar formation (4Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar, 40Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar, 60Bretscher A. Curr. Opin. Cell Biol. 1993; 5: 653-660Crossref PubMed Scopus (48) Google Scholar, 61Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar), we concluded that only a small fraction of total ERM proteins in wild-type platelets and mast cells are sufficient for their physiological functions. Similar observations, i.e. no phenotypic changes in knockout mice without up-regulation of other family members, has been reported in various systems. For example, mice devoid of components of intermediate-sized filaments such as vimentin and glial fibrillary acidic protein developed normally without compensatory expression of other intermediate filament components (62Colucci-Guyon E. Porter M.M. Dunia I. Paulin D. Pournin S. Babinet C. Cell. 1994; 79: 679-694Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 63Gomi H. Yokoyama T. Fujimoto K. Ikeda T. Katoh A. Itoh T. Itohara S. Neuron. 1995; 14: 29-41Abstract Full Text PDF PubMed Scopus (229) Google Scholar).Another issue that we should discuss here is the function of moesin in intracellular signaling. We proposed that the cross-linking activities of ERM proteins were regulated by the Rho-dependent signaling pathway through direct binding to Rho-GDP dissociation inhibitor and/or through Rho-dependent posphorylation (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar, 37Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 38Shaw R.J. Henry M. Solomon F. Jacks T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar). Ezrin and radixin bound to Rho-GDP dissociation inhibitor with similar binding constants to moesin and were phosphorylated by Rho kinase with similar efficiency to moesin in vitro. Mackay et al. (36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar) found that moesin was required for the Rho-dependent formation of stress fibers and focal contacts in permeabilized Swiss 3T3 cells. Also in this case, moesin was able to be replaced by ezrin and radixin. From the present results, we concluded that the functions of ERM proteins in the Rho-dependent signaling pathway are redundant also at the whole body level.In conclusion, the present observations were consistent with the notion that ERM proteins are functionally redundant. The possibility cannot be excluded that a more distantly related protein to moesin in the band 4.1 superfamily functionally compensates for the lack of moesin. Further generation of mutant mice lacking ERM proteins, both singly and in combination, will lead to a better understanding of the physiological relevance of the occurrence of these three closely related proteins. ERM (ezrin/radixin/moesin) proteins have been implicated as general cross-linkers between the plasma membrane and actin filaments (for reviews see Refs. 15Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 16Bretscher A. Reczek D. Berryman M. J. Cell Sci. 1997; 110: 3011-3018Crossref PubMed Google Scholar, 17Tsukita S. Yonemura S. Tsukita S. Trends. Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 18Tsukita S. Yonemura S. Tsukita S. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (308) Google Scholar, 19Vaheri A. Carpen O. Heiska L. Helander T.S. Jaaskelainen J. Majander-Nordenswan P. Sainio M. Timonen T. Turunen O. Curr. Opin. Cell Biol. 1997; 9: 659-666Crossref PubMed Scopus (161) Google Scholar). In this study, we generated male and female mice hemi- and homozygous, respectively, for a null mutation in the moesin gene located on the X chromosome. Surprisingly, the mutant mice exhibited no obvious abnormalities in appearance or fertility, and a systemic histological scan of mutant tissues revealed no abnormalities. Our results clearly demonstrated that moesin is not required for normal mouse development or for survival in the laboratory environment. This is surprising in view of the degree of conservation of the moesin gene, for example, the occurrence of a moesin gene inDrosophila (55Edwards K.A. Montague R.A. Shepard S. Edgar B.A. Erikson R.L. Kiehart D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4589-4593Crossref PubMed Scopus (59) Google Scholar, 56McCartney B.M. Fehon R.G. J. Cell Biol. 1996; 133: 843-852Crossref PubMed Scopus (129) Google Scholar) and the tissue-specific regulated expression of this gene (57Amieva M.R. Wilgenbun K.K. Furthmayr H. Exp. Cell Res. 1994; 210: 140-144Crossref PubMed Scopus (61) Google Scholar, 58Berryman M. Franck Z. Bretscher A. J. Cell Sci. 1993; 105: 1025-1043Crossref PubMed Google Scholar, 59Schwartz-Albiez R. Merling A. Spring H. Moller P. Koretz K. Eur. J. Cell Biol. 1995; 67: 189-198PubMed Google Scholar). To date immunofluorescence microscopy and immunoblotting analyses have revealed that the ratio of the levels of ezrin, radixin, and moesin expression in individual cells varies between different tissues in wild-type mice. Furthermore, ezrin, radixin, and moesin are not always colocalized. From these in situ observations in wild-type mice, ezrin, radixin, and moesin have been suggested to have specific functions. However, experiments in vitro or at the cellular level have not clearly identified differences in function between these molecules. When the expression of any one or two ERM proteins was selectively suppressed by antisense oligonucleotides, no phenotypic changes were detected, and cell-cell/cell-matrix adhesion and microvillar formation were affected only when expression of all family members was suppressed (40Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar). Furthermore, the NH2-terminal halves of all ERM proteins directly bound to the cytoplasmic domains of CD44, Rho-GDP dissociation inhibitor, and PIP2 (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and formed both homo- and heterodimers (29Gary R. Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10846-10850Crossref PubMed Scopus (93) Google Scholar, 30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (373) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar). The present results favor the notion that ERM proteins are functionally redundant also at the whole body level. Interestingly, in moesin-deficient mice as well as isolated moesin-deficient cells, targeted disruption of the moesin gene did not induce compensatory up-regulation of the other members of the ERM family. In any of the tissues examined in mutant mice, no changes were detected in the expression levels or subcellular distributions of ezrin or radixin. More importantly, in isolated moesin-deficient cells such as platelets and mast cells, in which moesin was predominantly expressed in wild-type cells, moesin completely disappeared, leaving relatively small amounts of ezrin and radixin. The aggregation activity of moesin-deficient platelets and the microvillar formation of moesin-deficient mast cells were not affected. Considering that ERM proteins were thought to be directly involved in platelet aggregation (54Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1996; 270: 31377-31385Abstract Full Text Full Text PDF Scopus (184) Google Scholar) and microvillar formation (4Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar, 40Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar, 60Bretscher A. Curr. Opin. Cell Biol. 1993; 5: 653-660Crossref PubMed Scopus (48) Google Scholar, 61Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar), we concluded that only a small fraction of total ERM proteins in wild-type platelets and mast cells are sufficient for their physiological functions. Similar observations, i.e. no phenotypic changes in knockout mice without up-regulation of other family members, has been reported in various systems. For example, mice devoid of components of intermediate-sized filaments such as vimentin and glial fibrillary acidic protein developed normally without compensatory expression of other intermediate filament components (62Colucci-Guyon E. Porter M.M. Dunia I. Paulin D. Pournin S. Babinet C. Cell. 1994; 79: 679-694Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 63Gomi H. Yokoyama T. Fujimoto K. Ikeda T. Katoh A. Itoh T. Itohara S. Neuron. 1995; 14: 29-41Abstract Full Text PDF PubMed Scopus (229) Google Scholar). Another issue that we should discuss here is the function of moesin in intracellular signaling. We proposed that the cross-linking activities of ERM proteins were regulated by the Rho-dependent signaling pathway through direct binding to Rho-GDP dissociation inhibitor and/or through Rho-dependent posphorylation (23Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 35Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar, 37Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 38Shaw R.J. Henry M. Solomon F. Jacks T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar). Ezrin and radixin bound to Rho-GDP dissociation inhibitor with similar binding constants to moesin and were phosphorylated by Rho kinase with similar efficiency to moesin in vitro. Mackay et al. (36Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (266) Google Scholar) found that moesin was required for the Rho-dependent formation of stress fibers and focal contacts in permeabilized Swiss 3T3 cells. Also in this case, moesin was able to be replaced by ezrin and radixin. From the present results, we concluded that the functions of ERM proteins in the Rho-dependent signaling pathway are redundant also at the whole body level. In conclusion, the present observations were consistent with the notion that ERM proteins are functionally redundant. The possibility cannot be excluded that a more distantly related protein to moesin in the band 4.1 superfamily functionally compensates for the lack of moesin. Further generation of mutant mice lacking ERM proteins, both singly and in combination, will lead to a better understanding of the physiological relevance of the occurrence of these three closely related proteins. We thank Dr. S. Nishikawa (Department of Molecular Genetics, Kyoto University) for helpful discussions. We thank Dr. T. Murata and Dr. F. Ushikubi (Department of Cell Pharmacology, Kyoto University) for technical advice regarding platelet aggregation assay. Y. Doi thanks Dr. Y. Matsuzawa (Second Department of Internal Medicine, Osaka University) for providing him with the opportunity for this study.