The NF2 Tumor Suppressor Merlin and the ERM Proteins Interact with N-WASP and Regulate Its Actin Polymerization Function

Abstract

The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation. The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation. Ezrin, radixin, and moesin, known as ERM 1The abbreviations used are: ERM, ezrin, radixin, and moesin; FERM, band 4.1/ezrin/radixin/moesin-like; N-term, NH2-terminal half; C-term, COOH-terminal half; FL, full-length; WH1, WASP homology 1; NHE-RF, Na+/H+ exchanger-regulatory factor; WIP, WASP interacting protein; HA, hemagglutinin; GST, glutathione S-transferase; PIP2, polyphosphatidylinositol 4,5-bisphosphate; IP, immunoprecipitation; GTPγS, guanosine 5′-3-O-(thio)triphosphate; DAPI, 4′,6-diamidino-2-phenylindole; IVTT, in vitro transcribed and translated; VCA, verprolin homology, cofilin homology, acidic; aa, amino acids; GBD, G protein binding domain. proteins, act as membrane-cytoskeletal linkers that play important roles in cell morphology, polarity, and signal transduction (1.Bretscher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 586-599Crossref PubMed Scopus (1137) Google Scholar). ERMs bind membrane proteins through their N-terminal FERM (band 4.1, ERM) domain and actin filaments through their C terminus, thus linking the cell membrane to the underlying cytoskeleton. Merlin, the protein product of the Neurofibromatosis type 2 (NF2) tumor suppressor gene, is closely related to the ERMs (2.Trofatter 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. 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 (1106) Google Scholar, 3.Ramesh V. Nat. Rev. Neurosci. 2004; 5: 462-470Crossref PubMed Scopus (60) Google Scholar). The FERM domain of merlin and ERMs is the region of highest homology and can interact with various membrane proteins. The C terminus of ERMs contains a high affinity actin-binding site that is not conserved in merlin. However, merlin also directly binds F-actin through its FERM domain and stabilizes actin filaments in vitro (4.Brault E. Gautreau A. Lamarine M. Callebaut I. Thomas G. Goutebroze L. J. Cell Sci. 2001; 114: 1901-1912PubMed Google Scholar, 5.James M.F. Manchanda N. Gonzalez-Agosti C. Hartwig J.H. Ramesh V. Biochem. J. 2001; 356: 377-386Crossref PubMed Scopus (68) Google Scholar). ERMs and merlin are regulated by an intramolecular head-to-tail association between the FERM domain and the C terminus that prevents binding of membrane partners as well as F-actin (6.Hirao 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 (512) Google Scholar). The autoinhibited conformation of the ERMs can be disrupted by polyphosphatidylinositol 4,5-bisphosphate (PIP2) binding or phosphorylation at a conserved C-terminal threonine. The role of ERMs and merlin in actin cytoskeleton reorganization has been well documented. In cultured cells, they localize to actin-rich regions such as microvilli, lamellipodia, filopodia, and neuronal growth cones. Antisense suppression of ERMs can disrupt microvilli and slow growth cone advancement (7.Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (319) Google Scholar, 8.Paglini G. Kunda P. Quiroga S. Kosik K. Caceres A. J. Cell Biol. 1998; 143: 443-455Crossref PubMed Scopus (141) Google Scholar). The absence of merlin, either in schwannoma cells from NF2 patients, or mouse embryo fibroblasts from NF2-deficient mice, results in increased membrane ruffling and cell motility (9.Shaw R.J. Paez J.G. Curto M. Morse Pruit W. Saotome I. O'Bryan J.P. Gupta V. Ratner N. Der C.J. Jacks T. McClatchey A.I. Dev. Cell. 2001; 1: 63-72Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 10.Bashour A.M. Meng J.J. Ip W. MacCollin M. Ratner N. Mol. Cell. Biol. 2002; 22: 1150-1157Crossref PubMed Scopus (63) Google Scholar). Thus merlin may negatively regulate actin remodeling in vivo. These cellular phenotypes caused by merlin or ERM loss can be partially explained by their structural role as membrane-cytoskeleton linkers. In addition, merlin and ERMs are regulated by Rho family GTPases via phosphorylation and also alter GTPase function through a negative feedback mechanism, thereby regulating the actin cytoskeleton indirectly (9.Shaw R.J. Paez J.G. Curto M. Morse Pruit W. Saotome I. O'Bryan J.P. Gupta V. Ratner N. Der C.J. Jacks T. McClatchey A.I. Dev. Cell. 2001; 1: 63-72Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 11.Speck O. Hughes S.C. Noren N.K. Kulikauskas R.M. Fehon R.G. Nature. 2003; 421: 83-87Crossref PubMed Scopus (203) Google Scholar). However, it remains unknown whether these proteins can directly alter actin nucleation and assembly downstream of Rho, Rac, or Cdc42. We examined whether merlin and ERMs could directly regulate the actin polymerization pathway downstream of these GTPases. The WASP (Wiskott-Aldrich syndrome protein) family proteins, upon activation by Rac and Cdc42, are capable of activating actin assembly by the actin nucleating Arp2/3 complex (12.Millard T.H. Sharp S.J. Machesky L.M. Biochem. J. 2004; 380: 1-17Crossref PubMed Scopus (212) Google Scholar). Cdc42 binds and activates N-WASP, a ubiquitously expressed member of the WASP family, which in turn stimulates Arp2/3 complex activity. WIP (WASP interacting protein) interacts with the WH1 domain of N-WASP and inhibits its function in vitro and in vivo (13.Martinez-Quiles N. Rohatgi R. Anton I.M. Medina M. Saville S.P. Miki H. Yamaguchi H. Takenawa T. Hartwig J.H. Geha R.S. Ramesh N. Nat. Cell Biol. 2001; 3: 484-491Crossref PubMed Scopus (227) Google Scholar, 14.Ho H.Y. Rohatgi R. Lebensohn A.M. Le M. Li J. Gygi S.P. Kirschner M.W. Cell. 2004; 118: 203-216Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). We report here that merlin and ERMs are able to directly bind N-WASP. Furthermore, merlin and ERMs can regulate N-WASP function in vitro and in vivo, demonstrating a novel, direct mechanism for their effect on actin reorganization and a new mode of N-WASP regulation. Materials and Antibodies—HA-radixin full-length (FL) and N-term and C-term plasmids were a gift from F. Solomon (MIT, Cambridge, MA). Mutants FL HA-radixin (T564E and T564A) were provided by S. Tsukita (Kyoto University, Kyoto, Japan). Myc-tagged N-WASP constructs were described previously (15.Rohatgi R. Ho H.Y. Kirschner M.W. J. Cell Biol. 2000; 150: 1299-1310Crossref PubMed Scopus (499) Google Scholar). Mini-N-WASP was provided by W. Lim (University of California, San Francisco, CA). The anti-WAVE, anti-N-WASP, and anti-WIP antibodies have been described (13.Martinez-Quiles N. Rohatgi R. Anton I.M. Medina M. Saville S.P. Miki H. Yamaguchi H. Takenawa T. Hartwig J.H. Geha R.S. Ramesh N. Nat. Cell Biol. 2001; 3: 484-491Crossref PubMed Scopus (227) Google Scholar, 16.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar, 17.Eden S. Rohatgi R. Podtelejnikov A.V. Mann M. Kirschner M.W. Nature. 2002; 418: 790-793Crossref PubMed Scopus (652) Google Scholar). Anti-NHE-RF IC270 and anti-merlin antibodies 1C4 and C26H were described previously (18.Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 19.Wiederhold T. Lee M.F. James M. Neujahr R. Smith N. Murthy A. Hartwig J. Gusella J.F. Ramesh V. Oncogene. 2004; 23: 8815-8825Crossref PubMed Scopus (58) Google Scholar). Anti-Nck and anti-Myc 9E10 antibodies were purchased from BD Transduction Laboratories and Developmental Studies Hybridoma Bank, respectively. Phospho-ERM antibody 3141 and anti-HA antibody were purchased from Cell Signaling Technologies and Covance, respectively. Pull-down Assays—Merlin and moesin GST fusion proteins were expressed and purified as described (18.Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). 293T lysates were incubated with 200pmol of immobilized merlin or moesin GST fusion protein. Lysate lane shows 10% of input. For pull-down assays using in vitro transcribed and translated N-WASP truncations, the SP6 Quick Coupled kit (Promega) was used with 2 μg of each N-WASP plasmid. The lysate lane shows 5% of input. Direct binding in solution was carried out with recombinant N-WASP (0.32 μg) and GST-purified moesin constructs (10 μg) as described (15.Rohatgi R. Ho H.Y. Kirschner M.W. J. Cell Biol. 2000; 150: 1299-1310Crossref PubMed Scopus (499) Google Scholar). The input N-WASP lane contains 40% of total input. Immunoprecipitation—IP was performed using 293T lysate and 5 μg of merlin antibody 1C4 or mouse IgG followed by Western blotting with anti-N-WASP antibody. Merlin IP was detected by anti-merlin C26H antibody. The reverse IP used 5 μg affinity purified N-WASP rabbit antibody or normal rabbit serum as a control and was detected by merlin antibody. For phospho-ERM interaction, HeLa cells were treated for 5 min with calyculinA (50 nm) before harvesting in lysis buffer (as above with 100 nm calyculinA). The N-WASP IP was performed with 8 μg of affinity-purified N-WASP or rabbit IgG, followed by Western blotting with the phospho-ERM and N-WASP antibodies. Actin Polymerization Assays—N-WASP and Cdc42 were purified as described (16.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). Actin from rabbit skeletal muscle and purified Arp2/3 complex were a generous gift of F. Nakamura and J. H. Hartwig (Brigham and Women's Hospital, Boston, MA). G-actin was freshly thawed at 4 °C and centrifuged at 400,000 × g for 1 h to remove F-actin. Polymerization assays were carried out as described (16.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). Briefly, actin was mixed with pyrene-labeled actin at a ratio of 3:2 and was used at a final concentration of 1 μm in a total volume of 80 μl of Xenopus buffer. Merlin and moesin proteins were preincubated with other proteins such as N-WASP, Arp2/3 complex, and Cdc42-GTPγS before the reaction was initiated by addition of actin mixture. The increase in fluorescence was measured by a spectrofluorimeter. Shigella Infections and Immunofluorescence—SV40 transformed mouse embryo fibroblast-like cells from N-WASP-deficient animals (28.Snapper S.B. Takeshima F. Anton I. Liu C.H. Thomas S.M. Nguyen D. Dudley D. Fraser H. Purich D. Lopez-Ilasaca M. Klein C. Davidson L. Bronson R. Mulligan R.C. Southwick F. Geha R. Goldberg M.B. Rosen F.S. Hartwig J.H. Alt F.W. Nat. Cell Biol. 2001; 3: 897-904Crossref PubMed Scopus (270) Google Scholar) were transduced with pLNCX-GFP-N-WASP retrovirus and sorted to enrich rescued cells. 18 h after radixin transfection, cells were infected with Shigella as described by T. Takeshima F. F. Alt F.W. S.B. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). was performed after lysis with using anti-HA antibody to radixin, followed by with antibody and to actin. were in to cells at were examined and for and at actin The of cells with actin was to cells with on the were from independent in which a total of cells were examined for each for the constitutively active radixin cells were The were by using and using the between and proteins in to performed pull-down assays using GST various merlin and moesin merlin and moesin N-WASP from and 293T cell lysates and WASP family and the Arp2/3 complex were not detected in these The full-length (FL) and N-terminal FERM of merlin and moesin N-WASP, the C-terminal and GST The binding of FL moesin is a of its The intramolecular association of merlin, moesin, is which (1.Bretscher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 586-599Crossref PubMed Scopus (1137) Google Scholar). We binding to NHE-RF, an FERM in the merlin and moesin FERM NHE-RF, C-term and GST not and N-WASP bind the FERM domain in affinity for moesin FERM, as has been previously (18.Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). the N-WASP FERM suggesting that it binds NHE-RF, which are conserved between the FERM We also whether other proteins that with N-WASP in the an protein that N-WASP function through its domain R. P. Ho H.Y. Kirschner M.W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, F. V. S. S. I. G. M. Nature. 1999; PubMed Scopus Google not with N-WASP WIP in a complex with N-WASP is expressed in cells, and could not a in the cells used in N-WASP from various by interacting with partners through each of its which of these the FERM domain interaction, used in vitro transcribed and translated N-WASP in a GST pull-down Myc-tagged N-WASP were for binding to the merlin FERM domain GST fusion protein WH1 and G were capable of the homology, cofilin homology, was that the WH1 domain is This is a region conserved between N-WASP and not proteins, which that not interact with merlin or whether the is performed a solution binding using purified and N-WASP, followed by of GST fusion proteins. moesin FERM domain and FL moesin direct binding as with C-term and FERM moesin FL N-WASP in a the between merlin and N-WASP in vivo, performed using 293T cell antibody N-WASP with merlin and the N-WASP antibody merlin and N-WASP moesin and merlin were capable of interacting with N-WASP in pull-down were to ERMs in N-WASP suggesting that their conformation in vivo of a conserved C-terminal has been to the conformation of ERMs T. M. Y. Yonemura S. M. K. Tsukita S. Tsukita S. J. Cell Biol. 1998; PubMed Scopus Google Scholar, Gautreau A. C. L. P. D. M. J. Cell Biol. 2004; PubMed Scopus Google Scholar). enrich treated HeLa cells with a and used an ERM antibody that the conserved C-terminal the N-WASP antibody for detected a association between the active ERMs and N-WASP in vivo We also performed using FERM domain and WH1 which that these regions were to the in vivo. Manchanda and V. The ERMs are to each other the FERM domain to merlin high of and the that ERMs and merlin can with N-WASP, it is that ERMs interact with N-WASP. that of FERM binding to ezrin, radixin, and the of the between and N-WASP, examined the effect of these proteins on N-WASP Arp2/3 actin polymerization in The of actin polymerization can be using pyrene-labeled which an increase in fluorescence into N-WASP, in vitro, binding of such as Cdc42 or (16.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). We examined the effect of on full-length N-WASP by Cdc42 and found that merlin and moesin proteins that the FERM domain the of actin The merlin FERM domain caused a in the of actin polymerization to Arp2/3 complex, N-WASP, and Cdc42 FERM domain and FL moesin also in as FL merlin FERM domain binding N-WASP FL proteins in the pull-down their effect appears in to the of the actin polymerization proteins that binding to N-WASP in the pull-down were able to inhibit N-WASP-mediated actin The C of moesin and merlin which bind N-WASP, the of The C terminus of the ERMs contains the The that effect of region in that F-actin binding not to the and it is the FERM domain binding to N-WASP that whether with the WH1 domain a protein. This protein the WH1 and is capable of binding of the to the VCA, which is by Cdc42, Arp2/3 complex activation J.A. Lim 2000; PubMed Scopus Google Scholar). full-length N-WASP which was and was by the concentration of FERM merlin and FL moesin We also the ability of merlin and moesin to inhibit domain function and found that N-WASP that are able to Arp2/3 complex the WH1 domain are not This also that merlin and moesin have effect on other Cdc42, the Arp2/3 complex, and actin in WIP and its related proteins and also bind the WH1 domain and WIP inhibits N-WASP actin polymerization in a (13.Martinez-Quiles N. Rohatgi R. Anton I.M. Medina M. Saville S.P. Miki H. Yamaguchi H. Takenawa T. Hartwig J.H. Geha R.S. Ramesh N. Nat. Cell Biol. 2001; 3: 484-491Crossref PubMed Scopus (227) Google Scholar, M. Miki H. S. T. H. S. Takenawa T. Biochem. 2002; PubMed Scopus Google Scholar, H.Y. Rohatgi R. Ma L. Kirschner M.W. Sci. S. A. 2001; PubMed Scopus Google Scholar). In merlin and moesin inhibit N-WASP function in actin assembly in vitro through binding of their FERM to the WH1 domain of N-WASP. These results the of proteins to WIP binding the WH1 domain and N-WASP activity. The S. cell actin polymerization to and between cells. The Shigella protein cell N-WASP to the Arp2/3 complex and an actin tail C. V. Li R. D. M.F. J. Cell Biol. 1999; PubMed Scopus Google Scholar). This is on the of N-WASP as N-WASP-deficient cells Shigella not tail and motility (28.Snapper S.B. Takeshima F. Anton I. Liu C.H. Thomas S.M. Nguyen D. Dudley D. Fraser H. Purich D. Lopez-Ilasaca M. Klein C. Davidson L. Bronson R. Mulligan R.C. Southwick F. Geha R. Goldberg M.B. Rosen F.S. Hartwig J.H. Alt F.W. Nat. Cell Biol. 2001; 3: 897-904Crossref PubMed Scopus (270) Google Scholar, S. S. K. T. J. R. 2001; PubMed Scopus Google Scholar) a to the effect of ERMs on N-WASP function in vivo. Ezrin, a member of the ERM family, is for Shigella into HeLa cells. its FERM which a negative effect on a in Shigella A. N. M. J. P. P. J. Cell Sci. 1999; Google Scholar). We the in vivo of the by the effect of exogenous ERMs on actin tail ERMs were merlin and moesin FERM in binding and N-WASP in the of N-WASP and actin-binding in merlin, that effect of direct actin binding on tail be was as to expression constructs and between the various FERM with to N-WASP binding and 1 and We used FL, N-term, C-term, and active and of active radixin is a that the protein the be and is of radixin constructs into fibroblasts and with cells were and for to the of Shigella caused by FERM domain A. N. M. J. P. P. J. Cell Sci. 1999; Google which also in were to whether tail other radixin constructs not alter Shigella and were for their effect on tail We found that constitutively active radixin actin tail by such effect FL, radixin, which is in the conformation in vivo, not inhibit tail results that bind N-WASP suggesting that the ERM is the able to bind N-WASP and thereby inhibit tail The radixin C-term, which binds actin effect despite with F-actin in the that the is through N-WASP that the active tail by N-WASP, it with N-WASP and a increase in tail with N-WASP has been to have effect on Shigella tail V. F. I. R. G. D. M. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar). Thus the is a of ERM of N-WASP and not an effect of active Rho, Rac, and Cdc42 is for tail J. V. A. P. M.F. C. J. Cell Sci. 1999; PubMed Google Scholar, T. Takeshima F. F. Alt F.W. S.B. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google the that active ERM is not by the of these GTPases. these results that constitutively active radixin inhibits N-WASP Shigella tail through their in vivo. These results the using the actin assembly The in vitro assays ability to FERM domain function the negative effect in vivo was FL ERM was able to inhibit N-WASP in vitro FL ERM not have a effect in vivo. This can be explained by the that GST fusion ERMs can be ERMs in vivo. ERM binding and N-WASP in vitro, in vivo, is effect on tail to the of N-WASP to bind FL ERMs they are We were able to by using the active FL ERM, which was found to inhibit tail of also to the effect of the FERM domain on tail the FERM not inhibit N-WASP to the of Shigella is through the of its WH1 and with S. S. K. T. J. R. 2001; PubMed Scopus Google Scholar). the FERM of bind the WH1 domain of N-WASP, examined whether active radixin disrupted the of N-WASP, thereby tail However, N-WASP was able to localize to the of in cells with active This to be the mechanism of ERM is that FERM domain binding a N-WASP with binding of N-WASP was previously to be regulated by the between the and has that negatively N-WASP function in vivo, that N-WASP is to of through the WH1 domain (13.Martinez-Quiles N. Rohatgi R. Anton I.M. Medina M. Saville S.P. Miki H. Yamaguchi H. Takenawa T. Hartwig J.H. Geha R.S. Ramesh N. Nat. Cell Biol. 2001; 3: 484-491Crossref PubMed Scopus (227) Google Scholar, 14.Ho H.Y. Rohatgi R. Lebensohn A.M. Le M. Li J. Gygi S.P. Kirschner M.W. Cell. 2004; 118: 203-216Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). report the of a protein to WIP as an N-WASP that of N-WASP function may also be through domain the that the WH1 domain may as a negative for N-WASP. in syndrome to the WH1 domain of suggesting that loss of the by domain may be the mechanism of the effect of merlin and ERMs on the actin cytoskeleton was to either binding to actin or to an effect on Rho GTPase report that direct binding between the FERM domain of and N-WASP can regulate actin assembly independent of their actin binding and GTPase Thus that merlin and ERM proteins can directly regulate N-WASP provides a novel mechanism by which these proteins effect actin reorganization and also an of N-WASP regulation. We for critical of the with