Involvement of the Conserved Adaptor Protein Alix in Actin Cytoskeleton Assembly

Abstract

The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with α-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of α-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly. The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with α-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of α-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly. Normal fibroblasts grow as a single layer, assume flattened and elongated cell shape, and align in parallel in monolayer culture. These morphological properties are determined by coordinated actin cytoskeleton assembly, integrin-mediated cell adhesion, and extracellular matrix assembly (1Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 793-805Crossref PubMed Scopus (1831) Google Scholar). Filamentous actin (F-actin) 3The abbreviations used are: F-actin, filamentous actin; siRNA, small interfering RNA; PRD, proline-rich domain; GST, glutathione S-transferase; GFP, green fluorescent protein; DTT, dithiothreitol; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; PMSF, phenylmethyl-sulfonyl fluoride; PIPES, 1,4-piperazinediethanesulfonic acid. 3The abbreviations used are: F-actin, filamentous actin; siRNA, small interfering RNA; PRD, proline-rich domain; GST, glutathione S-transferase; GFP, green fluorescent protein; DTT, dithiothreitol; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; PMSF, phenylmethyl-sulfonyl fluoride; PIPES, 1,4-piperazinediethanesulfonic acid.-based structures, such as lamellipodia and filopodia at the leading edge of the cell and actin bundles in the central and peripheral regions of the cell body, are assembled adjacent to the plasma membrane and anchored to extracellular matrix through integrin-mediated cell adhesions (2Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1308) Google Scholar, 2Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1308) Google Scholar, 4Brakebusch C. Fassler R. EMBO J. 2003; 22: 2324-2333Crossref PubMed Scopus (392) Google Scholar, 5Blystone S.D. Biochim. Biophys. Acta. 2004; 1692: 47-54Crossref PubMed Scopus (42) Google Scholar). Dysregulation of any of these processes may cause alterations in fibroblast morphology, as often observed in transformed fibroblasts (6Pawlak G. Helfman D.M. Curr. Opin. Genet. Dev. 2001; 11: 41-47Crossref PubMed Scopus (283) Google Scholar, 7Kahn P. Heller D. Shin S. Cytogenet. Cell Genet. 1983; 36: 605-611Crossref PubMed Scopus (6) Google Scholar, 8Labat-Robert J. Semin. Cancer Biol. 2002; 12: 187-195Crossref PubMed Scopus (99) Google Scholar). Critical regulators of these processes are often identified by their ability to alter fibroblast morphology when their expression levels are increased or decreased. Alix/AIP1 (ALG-2 interacting protein X or 1) is the mammalian ortholog of an evolutionally conserved family of adaptor proteins (9Sadoul R. Biol. Cell. 2006; 98: 69-77Crossref PubMed Scopus (53) Google Scholar). Our investigation of biological functions of human Alix, which was originally identified by us and named Hp95 (10Wu Y. Pan S. Luo W. Lin S.H. Kuang J. Oncogene. 2002; 21: 6801-6808Crossref PubMed Scopus (40) Google Scholar, 11Wu Y. Pan S. Che S. He G. Nelman-Gonzalez M. Weil M.M. Kuang J. Differentiation. 2001; 67: 139-153Crossref PubMed Scopus (42) Google Scholar), has revealed its critical roles in regulating fibroblast morphology. In immortalized mouse fibroblast NIH/3T3 cells that are less flattened and aligned than their nonimmortalized counterparts, overexpression of Alix promoted cell flattening and alignment, whereas reduction of Alix expression produced opposite effects (10Wu Y. Pan S. Luo W. Lin S.H. Kuang J. Oncogene. 2002; 21: 6801-6808Crossref PubMed Scopus (40) Google Scholar). Alix overexpression also promoted cell flattening and monolayer growth in malignant HeLa cells (11Wu Y. Pan S. Che S. He G. Nelman-Gonzalez M. Weil M.M. Kuang J. Differentiation. 2001; 67: 139-153Crossref PubMed Scopus (42) Google Scholar). Because cell morphology is determined by coordinated actin cytoskeleton assembly, integrin-mediated cell adhesions, and extracellular matrix assembly, these findings indicate that Alix plays structural or regulatory roles in some of these processes. Functional studies of Alix and its orthologs have generated the consensus that Alix is positively involved in apoptotic induction (9Sadoul R. Biol. Cell. 2006; 98: 69-77Crossref PubMed Scopus (53) Google Scholar, 10Wu Y. Pan S. Luo W. Lin S.H. Kuang J. Oncogene. 2002; 21: 6801-6808Crossref PubMed Scopus (40) Google Scholar, 12Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13Trioulier Y. Torch S. Blot B. Cristina N. Chatellard-Causse C. Verna J.M. Sadoul R. J. Biol. Chem. 2004; 279: 2046-2052Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and sorting of endocytosed cell surface receptors into luminal vesicle of multivesicular bodies, also called late endosomes (14Odorizzi G. Katzmann D.J. Babst M. Audhya A. Emr S.D. J. Cell Sci. 2003; 116: 1893-1903Crossref PubMed Scopus (174) Google Scholar, 15Luhtala N. Odorizzi G. J. Cell Biol. 2004; 166: 717-729Crossref PubMed Scopus (163) Google Scholar, 16Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (530) Google Scholar). Structurally, Alix and its orthologs are characterized by an N-terminal Bro1 domain, a middle region, and a C-terminal proline-rich domain (PRD). The Bro1 domain, named after the first identified ortholog of this family, yeast Bro1, was originally predicted by computer program-based sequence analysis and thought to consist of the N-terminal 160 amino acid residues. Recent x-ray crystallography of bacterially produced fragments of Bro1 suggested that the N-terminal 380 residues (called broad Bro1 domain) are required to form a structurally stable domain for partner protein interaction (17Kim J. Sitaraman S. Hierro A. Beach B.M. Odorizzi G. Hurley J.H. Dev. Cell. 2005; 8: 937-947Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The middle region contains coiled-coil motif, potential sites for additional partner protein interaction (15Luhtala N. Odorizzi G. J. Cell Biol. 2004; 166: 717-729Crossref PubMed Scopus (163) Google Scholar). The PRD contains multiple polyproline motifs, which are potential docking sites for proteins containing SH3 domains. The apoptotic function of Alix requires its interaction with the calcium-binding protein ALG-2 at the PRD (12Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 18Missotten M. Nichols A. Rieger Sadoul R. Cell 1999; PubMed Scopus Google Scholar, J. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, H. Yamada C. H. H. Y. M. J. 2004; PubMed Scopus Google Scholar). The of Alix in sorting requires the at the broad Bro1 domain and the at the PRD B. A. S. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. A. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. B. D.M. He D.M. A. J. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). These functions of Alix not to to the of Alix fibroblast morphology. these functions of Alix, Alix with growth factor multiple actin and in of B. J. Cell Sci. 2003; 116: PubMed Scopus Google Scholar). the or the of these was not determined in this these the that Alix is a of the protein that coordinated actin cytoskeleton assembly, integrin-mediated cell adhesions, and extracellular matrix assembly. this we used nonimmortalized WI38 cells from human fibroblasts as the to in the protein Alix functions that cell morphology. Here we that Alix associates with actin cytoskeleton and promotes its assembly. of that and Alix generated by the Alix in studies as the (11Wu Y. Pan S. Che S. He G. Nelman-Gonzalez M. Weil M.M. Kuang J. Differentiation. 2001; 67: 139-153Crossref PubMed Scopus (42) Google Scholar). for and Alix are in with and and into the and sites of the proteins from these cells transformed with produced and at of with at for The cells by at for and in phosphate-buffered with DTT, PMSF, and of and The cells by and the cell by at for the the cell with at The with of and in the proteins the with glutathione in and in at Alix or Alix fragments by in and these proteins from their expression by with the and the These directly produced from the RNA by in by in and of with after from the of the and for by both and from the that the and their with the mouse cell from by and from the in in from The and protein The of determined by their at Cell and cells, from human from and cultured in in with that was with and WI38 cells not and the to used in this and green protein and by and The sequence was of was as J. W. A. 2001; PubMed Scopus Google Scholar). In WI38 cells in and for of cells was with of with from the The cells cultured or at after the at cells Cell observed after and by and at by of Cell and the and of WI38 cells from in and in with and of and and for Alix and and α-actinin, WI38 cells by in containing PMSF, and of and cell at for and protein of determined by a protein from of WI38 cell was by of of cell of with of of protein The with of and by in of The proteins by and the as we G. Kuang J. Oncogene. 2005; PubMed Scopus Google Scholar). from and and and from cells cultured in a In some the at with of cells with with and for with in cells first for with in in PBS, by with PBS, for with in in cells with and by the with and after the cells with in for and in with a of the was used as the and used to and WI38 cells cultured for and the was by containing of of at cells with and with the of or Alix or Alix proteins of The at for with WI38 cell in of PMSF, and of and containing The with the and with of by and with or Alix to or at with of actin in of The with and and with of in parallel with actin by and with F-actin was Alix in WI38 cell or proteins the protein for this the by at for the of in the and was by with as (10Wu Y. Pan S. Luo W. Lin S.H. Kuang J. Oncogene. 2002; 21: 6801-6808Crossref PubMed Scopus (40) Google Scholar). by the when of and F-actin in WI38 Cell and in WI38 cell was a Cell Biol. 2004; PubMed Scopus Google Scholar, A. Y. J. A. PubMed Scopus Google Scholar). WI38 cells with or Alix after cells and cells into and cultured for an additional in with of actin of and and at for cells of the and for at in The was and used as the The was in of with and used as the F-actin of the and F-actin by and with that cortactin, or of in the Alix of and that of protein with of WI38 cell containing of proteins at and with The with of and by the that by and at with of and with an The was with and of and acid. The from and and acid was from The was from to an to for and and for and as as Alix for WI38 to investigation of the function of Alix in immortalized mouse fibroblast NIH/3T3 cells revealed roles of Alix in regulating fibroblast morphology. this to the for this we WI38 cells with Alix or as a and the of Alix knockdown WI38 cell morphology. of cell with generated by us that Alix expression by in Alix cells at and at after the whereas actin expression not In to Alix cells, cells levels of both Alix and actin of the morphology of the cells a revealed that Alix knockdown cells membrane and than cells, cell and Alix cells first and the of Alix knockdown cell morphology their of cell growth and not These that Alix is required for WI38 cells to typical fibroblast morphology. Alix with in WI38 the of Alix in the that regulates fibroblast morphology, we first determined the localization of Alix in WI38 cells in to F-actin-based structures with and and the A. H. Sci. S. A. PubMed Scopus Google Scholar, I. P. W. Sci. S. A. PubMed Scopus Google Scholar). These and Alix in of WI38 cells observed that of the F-actin-based structures as as which endosomes functions of Alix in In of the structures at the cell that the of both at the cell edge and actin bundles at the of the in cells that In cells, lamellipodia and stress we cultured WI38 cells which stress fiber assembly, Alix at stress These that Alix associates with actin cytoskeleton in WI38 Alix in WI38 Alix has functions in actin cytoskeleton assembly, we WI38 cells with or and cells with which both F-actin that has assembled into structures and F-actin in the M. D. M. R. Sci. S. A. PubMed Scopus Google Scholar, M.M. P. Cell PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar). the F-actin was in Alix knockdown cells than in The was by of the of Alix cells and the cells in the was observed in the protein of actin of WI38 cells with the F-actin the by as These that Alix knockdown inhibits F-actin in WI38 In of this when of and Alix knockdown WI38 cells into and the in cells was than that in Alix knockdown cells In to the of Alix knockdown the F-actin Alix knockdown cells also assembled less stress than cells, in central of the was most when cells which stress fiber assembly In and Alix knockdown cells of adhesions, as revealed by of middle the that Alix knockdown inhibits stress fiber assembly in WI38 cells through integrin-mediated cell Alix with the mechanism by which Alix associates with actin cytoskeleton and promotes its assembly, we determined Alix directly interacts with we first of in the of bacterially produced or and determined their interaction with F-actin by the F-actin J. J.H. S. J. J. Cell Biol. PubMed Scopus (78) Google Scholar). in of the in the with F-actin, whereas the in the of these revealed that residues and of Alix, These that an N-terminal region of Alix directly interacts with F-actin. the region in Alix that interacts with F-actin, we produced Alix that the N-terminal Bro1 domain the C-terminal domain or both of in and their interaction with F-actin by the F-actin and F-actin as as F-actin These that the N-terminal Bro1 domain and the C-terminal PRD of Alix may a docking for F-actin. this we produced and PRD the adjacent residues of Alix by in and determined their interaction with F-actin by the was produced for a of or with F-actin that both the Bro1 domain and the PRD with F-actin. to Alix interacts with as we Alix from with and by the small of was by not and of as These that the Bro1 domain and the PRD of Alix with as at a than with F-actin. Alix with F-actin in a and into the of we of F-actin in the of bacterially produced by was used in this less than and The first of a of and of Because we determined that of was for F-actin not actin was in at in the amount of that to F-actin increased in parallel with the amount of the that Alix binds to F-actin in a The of of of and a of in the amount of that to F-actin a at that actin was in at and this actin whereas was in this actin at with F-actin, that the of may of Alix an actin is an abundant the and interaction of Alix with F-actin in cell-free the that Alix is an abundant protein as this we of proteins from WI38 cells or in parallel with of middle region of Alix with and the amount of Alix in the was used as a was from Alix by was this and at not a for WI38 cell was observed that of generated than the Alix in the proteins the and Alix into we that Alix for of the proteins in WI38 cell lysates, Alix into the of abundant a of has to with a of partner proteins that and the of Alix in WI38 cell that associates with F-actin, we actin in WI38 cell for the to and determined the of Alix that with F-actin. of Alix in WI38 cell with actin whereas as a was in the not These that actin is a partner protein of Alix in WI38 this we of WI38 cells with a of multiple and the by and in actin was the in the Alix the and Alix. In of the identified Alix that not by the and of the which is of their or in the Alix the actin as the was than in the Alix and its by which not actin in the Alix not The also a protein that with Alix. analysis of this protein its Alix. These that actin is a partner protein of Alix in WI38 Alix with Alix is an adaptor its interaction with F-actin and localization at lamellipodia the that Alix interacts with an protein at is of the proteins at lamellipodia promotes and ARP2/3 actin J. 2004; PubMed Scopus Google Scholar, Curr. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). A. A. H. J. Cell Sci. 2005; PubMed Scopus Google Scholar) in a that Alix knockdown in HeLa cells of endosomes and the actin cytoskeleton proteins such as and we the that Alix interacts with cortactin. this we first WI38 cells with and in Alix with at we determined interaction of Alix with by and protein of WI38 cell with or mouse was in the Alix not in the of WI38 cell with Alix by of the proteins with that with of the Alix fragments that residues in the C-terminal half of the middle region that this region contains a we determined the effects of Alix knockdown and localization by Alix knockdown and and stress fiber assembly as whereas not localization at the cell Alix knockdown cells was in to in the of expression in Alix knockdown cells we determined the of Alix knockdown the of in the and F-actin by Alix knockdown the of in the F-actin by these that Alix directly interacts with and that this interaction plays roles in to Alix with with stress the that Alix may with an protein that at stress fibers. this we Alix interaction with α-actinin, an protein that associates with stress Cell 2004; PubMed Scopus Google Scholar). of WI38 cell with or by Alix and α-actinin with in WI38 cell the region in Alix as we for that in the N-terminal of the middle region of Alix which F-actin the α-actinin than the that the contains for Alix with we determined the of Alix knockdown α-actinin with F-actin and stress fibers. Alix knockdown not α-actinin expression Alix knockdown the of α-actinin in the F-actin by as determined by of α-actinin in the and F-actin in of and cells when and Alix knockdown cells with α-actinin a of stress in cells, whereas as or bundles at the cell in Alix knockdown cells these that Alix interacts with α-actinin and suggested that this interaction promotes α-actinin interaction with F-actin and its roles in stress fiber assembly. that Alix promotes cell flattening and alignment in immortalized NIH/3T3 cells (10Wu Y. Pan S. Luo W. Lin S.H. Kuang J. Oncogene. 2002; 21: 6801-6808Crossref PubMed Scopus (40) Google Scholar). the first to the mechanism by which Alix regulates cell morphology. WI38 cells as the which requires Alix expression for of typical fibroblast morphology, we have that Alix associates with both F-actin and the F-actin-based structures lamellipodia and stress fibers. Alix expression by both decreases F-actin content and inhibits F-actin assembly into In addition, Alix interacts with F-actin in a and in a cell-free and actin is the most abundant partner protein of Alix in WI38 cell Alix also binds cortactin, which the ARP2/3 complex-mediated initiation of actin and α-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown the amount of and α-actinin in the F-actin and lamellipodial localization of cortactin. These findings establish involvement of Alix in actin cytoskeleton assembly and at of the by which Alix regulates fibroblast morphology. findings a for the that Alix knockdown in HeLa cells an of endosomes and the proteins involved in such as and A. A. H. J. Cell Sci. 2005; PubMed Scopus Google Scholar). Alix knockdown the cell by which both F-actin-based structures such as lamellipodia and stress and F-actin and F-actin in the M. D. M. R. Sci. S. A. PubMed Scopus Google Scholar, M.M. P. Cell PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar) that Alix plays roles in actin or studies have that actin is at lamellipodial or endosomes by Curr. Opin. Cell Biol. 2003; PubMed Scopus Google Scholar). binds the and the complex-mediated actin polymerization. not lamellipodial at the plasma membrane and of endosomes in the Curr. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) also the of F-actin that assembled into stress and of F-actin-based structures Curr. Opin. Biol. 2002; 12: PubMed Scopus Google Scholar, Rev. 2001; PubMed Scopus Google Scholar). these the of Alix knockdown lamellipodial localization of may or to the effects both F-actin In to F-actin Alix knockdown cells also and stress in the central region of the cells this to the of Alix knockdown the F-actin Alix localization at stress and Alix with the F-actin protein α-actinin the that Alix has an function in stress fiber assembly. studies have that key in stress fiber assembly are of F-actin by α-actinin and with F-actin bundles with bundles Cell 2004; PubMed Scopus Google Scholar). Because Alix directly interacts with α-actinin and promotes its with F-actin, Alix may a in the F-actin in stress fiber assembly. Alix has in processes by a of partner of these studies the of these proteins in the Alix in any cell or of Alix to structures or In this we the first analysis of the in the Alix in mammalian cells and that actin is a Alix partner in WI38 was with of the of the Alix with F-actin in WI38 These findings indicate that of actin cytoskeleton assembly is a biological function of Alix in mammalian The yeast ortholog of Alix, Bro1, was originally identified by increased In to this the cells and cell Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). the characterized roles of Bro1 in functions of Bro1 or proteins for these morphological Because assembly of cytoskeleton by F-actin proteins has to yeast cells from stress and cell and H. EMBO J. 12: PubMed Scopus Google Scholar), is that the of Alix in regulating the actin cytoskeleton is evolutionally conserved and that Bro1 these through in the actin Alix is structurally characterized by an N-terminal Bro1 domain, a middle region, and a C-terminal PRD (14Odorizzi G. Katzmann D.J. Babst M. Audhya A. Emr S.D. J. Cell Sci. 2003; 116: 1893-1903Crossref PubMed Scopus (174) Google Scholar). in studies have that the PRD interacts with multiple that are involved in apoptotic induction sorting and H. Yamada C. H. H. Y. M. J. 2004; PubMed Scopus Google Scholar, B. A. S. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. B. D.M. He D.M. A. J. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). region adjacent to the PRD interacts with proteins and B. A. S. 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