Gelsolin, a Multifunctional Actin Regulatory Protein

Abstract

polyphosphoinositide 4,5-bisphosphate phosphatidylinositol 5-kinase The actin cytoskeleton is an essential scaffold for integrating membrane and intracellular functions. It is very dynamic and is remodeled in response to a variety of signals. Growth factor stimulation promotes actin assembly at the plasma membrane to generate movement, whereas apoptotic signals cause cytoskeletal destruction to elicit characteristic membrane blebbing and morphological changes. Gelsolin is a Ca2+- and polyphosphoinositide 4,5-bisphosphate (PIP2)1-regulated actin filament severing and capping protein that is implicated in actin remodeling in growing and in apoptotic cells (reviewed in Refs. 1Liu Y.T. Rozelle A.L. Yin H.L. Maruta H. Kohama K. G Proteins, Cytoskeleton and Cancer. R. G. Landes Company, Austin, TX1998: 19-35Google Scholar and2Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar). This review summarizes data supporting the role of gelsolin in cytoskeletal remodeling and phosphoinositide signaling and discusses the structural basis for the Ca2+ and PIP2regulation of severing and capping by gelsolin. Gelsolin is the most potent actin filament severing protein identified to date. Severing is the weakening of enough non-covalent bonds between actin molecules within a filament to break the filament in two. Gelsolin severs stoichiometrically and with close to 100% efficiency (3Selden L.A. Kinosian H.J. Newman J. Lincoln B. Hurwitz C. Gershman L.C. Estes J.E. Biophys. J. 1998; 75: 3101-3109Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Severing is initiated after gelsolin binds to the side of an actin filament. Gelsolin binds filaments rapidly but severs slowly (3Selden L.A. Kinosian H.J. Newman J. Lincoln B. Hurwitz C. Gershman L.C. Estes J.E. Biophys. J. 1998; 75: 3101-3109Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar); the delay may reflect the time required for structural rearrangement within gelsolin (see “Structural Basis for Ca2+ Regulation”) and in the filament (4McGough A. Chiu W. Way M. Biophys. J. 1998; 74: 764-772Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) prior to severing. Gelsolin changes actin conformation and kinks the actin filament (4McGough A. Chiu W. Way M. Biophys. J. 1998; 74: 764-772Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), suggesting a mechanical basis for severing. After severing, gelsolin remains attached to the barbed end of the filament as a cap. As a result, short actin filaments that cannot reanneal with each other or elongate at their barbed ends are generated. In this way, the actin network is disassembled. The importance of Ca2+-mediated actin severing has been clearly documented during platelet activation (5Hartwig J.H. J. Cell Biol. 1992; 118: 1421-1442Crossref PubMed Scopus (336) Google Scholar), and gelsolin is the only known Ca2+-dependent severing protein identified to date. Gelsolin severing can also have a constructive effect because it increases the number of filaments. Uncapping of gelsolin from these filaments generates many polymerization-competent ends from which actin can grow to rebuild the cytoskeleton to new specifications. Therefore, gelsolin can promote actin polymerization by severing followed by uncapping (mechanism B, as discussed in the Prologue (74Yin H.L. Stull J.T. J. Biol. Chem. 1999; 274: 32529-32530Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) of this series). Cells from gelsolin null mice exhibit a variety of motility and actin defects. Gelsolin null fibroblasts have pronounced actin stress fibers (6Witke W. Sharpe A.H. Hartwig J.H. Azuma T. Stossel T.P. Kwiatkowski D.J. Cell. 1995; 81: 41-51Abstract Full Text PDF PubMed Scopus (379) Google Scholar), and this phenotype is consistent with an inability to sever and remodel actin filaments. They do not ruffle in response to growth factor (7Azuma T. Witke W. Stossel T.P. Hartwig J.H. Kwiatkowski D.J. EMBO J. 1998; 17: 1362-1370Crossref PubMed Scopus (235) Google Scholar), and they exhibit defective chemotaxis and wound healing. The rate of clotting is reduced (6Witke W. Sharpe A.H. Hartwig J.H. Azuma T. Stossel T.P. Kwiatkowski D.J. Cell. 1995; 81: 41-51Abstract Full Text PDF PubMed Scopus (379) Google Scholar), as would be consistent with the requirement of actin severing for platelet activation (5Hartwig J.H. J. Cell Biol. 1992; 118: 1421-1442Crossref PubMed Scopus (336) Google Scholar). Neurite retraction is defective (8Lu M. Witke W. Kwiatkowski D.J. Kosik K.S. J. Cell Biol. 1997; 138: 1279-1287Crossref PubMed Scopus (118) Google Scholar), and neurons are more susceptible to glutamate-induced excito-toxicity (9Endres M. Fink K. Zhu J. Stagliano N.E. Bondala V. Geddes J.W. Azuma T. Mattson M.P. Kwiatkowski D.J. Moscowitz M.A. J. Clin. Invest. 1999; 10: 161-178Google Scholar). Neutrophil extravasation is compromised (6Witke W. Sharpe A.H. Hartwig J.H. Azuma T. Stossel T.P. Kwiatkowski D.J. Cell. 1995; 81: 41-51Abstract Full Text PDF PubMed Scopus (379) Google Scholar). These findings establish the importance of gelsolin in maintaining motility and actin dynamics. Despite multiple cellular pathology, the null animals (in a mixed strain background) are without gross phenotypic defects. This may reflect the existence of potent compensatory mechanisms. However, the compensation is incomplete and varies with the genetic background of the knockout animals. Gelsolin null animals in a pure strain mouse background are non-viable at perinatal and early postnatal stages (2Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar), indicating that gelsolin is necessary for survival. Membrane ruffling is a functional readout for a coordinated series of membrane and cytoskeletal events, and it is activated by the small GTPase, Rac. Gelsolin null fibroblasts have increased Rac expression (7Azuma T. Witke W. Stossel T.P. Hartwig J.H. Kwiatkowski D.J. EMBO J. 1998; 17: 1362-1370Crossref PubMed Scopus (235) Google Scholar), and Rac·GTP dissociates gelsolin-actin complexes (equivalent to uncapping) in cell extracts but not purified gelsolin-actin complexes (10Arcaro A. J. Biol. Chem. 1998; 273: 805-813Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These results suggest that gelsolin is a downstream effector of Rac, but there are additional steps between Rac and gelsolin activation/inactivation. A number of studies suggest that linkage through the type I phosphatidylinositol 5-kinases (PIP5KIs), the major enzymes that synthesize PIP2 (reviewed in Refs. 11Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar and 12Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), is an attractive possibility. PIP5KIs coimmunoprecipitate with Rac (13Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) and also Rho (14Chong L.D. Traynor-Kaplan A. Bokoch G.M. Schwartz M.A. Cell. 1994; 79: 507-513Abstract Full Text PDF PubMed Scopus (594) Google Scholar), a small GTPase that promotes stress fiber formation. PIP5KIs may thus be incorporated into signaling complexes that are targeted to the plasma membrane through Rac·GTP or Rho·GTP. This increases the local concentration of PIP2 in membrane microdomains to selectively activate downstream cascades (reviewed in Refs. 12Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar and15Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar). PIP2 has a pivotal role in the phosphoinositide cycle that drives signaling, cytoskeletal organization, and membrane trafficking (reviewed in Ref. 15Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar). Numerous cytoskeletal proteins are affected by PIP2 in vitro. They include gelsolin family proteins (16Janmey P.A. Stossel T.P. Nature. 1987; 325: 362-364Crossref PubMed Scopus (496) Google Scholar), profilin (17Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (639) Google Scholar), capping protein (18Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (337) Google Scholar), ADF/cofilin (19Yonezawa N. Nishida E. Iida K. Yahara I. Sakai H. J. Biol. Chem. 1990; 265: 8382-8386Abstract Full Text PDF PubMed Google Scholar), α-actinin (20Fukami K. Furuhashi K. Inagaki M. Endo T. Hatano S. Takenawa T. Nature. 1992; 359: 150-152Crossref PubMed Scopus (304) Google Scholar), vinculin (21Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (457) Google Scholar), ezrin/radixin/moesin (22Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (511) Google Scholar), and WASp family proteins (23Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (555) Google Scholar). The latter four proteins are activated by PIP2, whereas the first four are inactivated by PIP2. Ezrin/radixin/moesin, ADF/cofilin, and WASp are reviewed in this series (24Higgs H.N. Pollard T.D. J. Biol. Chem. 1999; 274: 32531-32534Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 25Tsukita S. Yonemura S. J. Biol. Chem. 1999; 274: 34507-34510Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 26Carlier M.-F. Ressad F. Pantaloni D. J. Biol. Chem. 1999; 274: 33827-33830Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The challenge will be to identify cytoskeletal proteins that are physiologically regulated by PIP2 and determine how they are differentially regulated. PIP2 involvement in cytoskeletal regulation is supported by experiments that manipulate PIP2 content in intact cells and in cell-free models. Microinjection of a monoclonal antibody to PIP2 prevents stress fiber and focal adhesion formation (21Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (457) Google Scholar). PIP5KI overexpression induces the formation of short actin bundles (27Shibasaki Y. Ishihara H. Kizuki N. Asano T. Oka Y. Yazaki Y. J. Biol. Chem. 1997; 272: 7578-7581Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and increases the movement of dynamic actin spots containing a number of actin regulatory proteins (28Schafer D.A. Welch M.D. Machesky L.M. Bridgman P.C. Meyer S.M. Cooper J.A. J. Cell Biol. 1998; 143: 1919-1930Crossref PubMed Scopus (152) Google Scholar). In contrast, overexpression of synaptojanin, the inositol polyphosphate 5-phosphatase that dephosphorylates PIP2, reduces actin stress fibers (29Sakisaka T. Itoh T. Miura K. Takenawa T. Mol. Cell. Biol. 1997; 17: 3841-3849Crossref PubMed Scopus (147) Google Scholar). Moreover, Hartwig et al. (30Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar) were able to reconstitute the entire pathway between thrombin stimulation, Rac activation, PIP2 synthesis, and barbed end nucleated actin assembly in permeabilized platelets. However, in neutrophils and other systems, the relation is less clear. Although Rac·GTP dissociates gelsolin-actin complexes (10Arcaro A. J. Biol. Chem. 1998; 273: 805-813Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and stimulates PIP2synthesis, it does not promote actin assembly in lysates (31Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Crossref PubMed Scopus (145) Google Scholar). Instead, Cdc42, which stimulates filopodia formation in cells, promotes de novo actin assembly in vitro, in a PIP2-dependent manner that is mediated through WASp and the Arp2/3 complex (32Rohatgi 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 (1076) Google Scholar). The range of responses and the contradictory effects of small GTPases and PIP2 on nucleated actin assembly in vivo andin vitro and in different types of cells may be reconciled by postulating that there are multiple pathways for actin assembly. Plasma membrane lysis may disrupt the critical coupling between Rac and actin polymerization much more than that between Cdc42 and actin. Gelsolin overexpression increases membrane ruffling and chemotaxis (33Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1991; 251: 1233-1236Crossref PubMed Scopus (259) Google Scholar, 34Sun H.-Q. Lin K.-M. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (84) Google Scholar), consistent with the role of gelsolin in dynamic actin remodeling. Surprisingly, CapG, a gelsolin relative that caps but does not sever actin, and the completely unrelated capping protein also increase cell motility when overexpressed (35Sun H.-Q. Kwiatkowska K. Wooten D.C. Yin H.L. J. Cell Biol. 1995; 129: 147-156Crossref PubMed Scopus (94) Google Scholar, 36Hug C. Jay P.Y. Reddy I. McNally J.G. Bridgman P.C. Elson E.L. Cooper J.A. Cell. 1995; 81: 591-600Abstract Full Text PDF PubMed Scopus (146) Google Scholar). A priori, pure capping proteins are expected to be less effective in promoting actin dynamics than severing/capping proteins, because they do not increase the number of actin filaments per se(compare mechanisms B (severing and uncapping) and C (uncapping only) in the Prologue to this minireview series (74Yin H.L. Stull J.T. J. Biol. Chem. 1999; 274: 32529-32530Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar)). These results indicate that capping/uncapping may be sufficient to increase actin dynamics. More detailed study will be required to distinguish between the contributions of severing and capping. Overexpression studies reveal that gelsolin may have other roles in addition to direct cytoskeletal regulation. Overexpressed gelsolin (34Sun H.-Q. Lin K.-M. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (84) Google Scholar) and CapG (35Sun H.-Q. Kwiatkowska K. Wooten D.C. Yin H.L. J. Cell Biol. 1995; 129: 147-156Crossref PubMed Scopus (94) Google Scholar) modulate phospholipase Cγ and phospholipase Cβ activity in a biphasic manner both in vivo and in vitro. These effects depend on PIP2 binding (34Sun H.-Q. Lin K.-M. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (84) Google Scholar), suggesting that gelsolin enhances or competes with other PIP2-binding proteins for their common substrate. This potent effect may be achieved by altering the packing of PIP2 molecules within the membrane bilayer (37Flanagan L.A. Cunningham C.C. Chen J. Prestwich G.D. Kosik K.S. Janmey P.A. Biophys. J. 1997; 73: 1440-1447Abstract Full Text PDF PubMed Scopus (75) Google Scholar). In conclusion, these results suggest that as PIP2 content and availability change during signaling, cross-talk between PIP2-regulated proteins provides a selective mechanism for positive as well as negative regulation of phosphoinositide signaling. This is particularly relevant as more PIP2-regulated proteins are identified. Gelsolin coimmunoprecipitates with several PIP2-interacting proteins, and it alters the activity of phosphatidylinositol 3-kinase and phospholipase D as well (reviewed in Refs. 1Liu Y.T. Rozelle A.L. Yin H.L. Maruta H. Kohama K. G Proteins, Cytoskeleton and Cancer. R. G. Landes Company, Austin, TX1998: 19-35Google Scholar and 2Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar). Gelsolin is phosphorylated by c-Src in vitro, and phosphorylation is enhanced by PIP2 (38De Corte V. Gettesmans J. Vandekerckhove J. FEBS Lett. 1997; 401: 191-196Crossref PubMed Scopus (74) Google Scholar). The physiological significance of these associations and phosphorylation has not been determined. Gelsolin is a substrate for caspase-3 (39Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1040) Google Scholar, 40Kamada S. Kusano H. Fujita H. Ohtsu M. Koya R.C. Kuzumaki N. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8532-8537Crossref PubMed Scopus (95) Google Scholar), the effector caspase in both the death receptor and mitochondrial apoptotic pathways. Gelsolin cleaved by caspase-3 no longer requires Ca2+ to sever actin filaments (Fig.1) (see also “Structural Basis for Ca2+ Regulation”), and it dismantles the membrane cytoskeleton to cause blebbing, a hallmark of apoptosis. Overexpression of the Ca2+-independent severing N-half induces apoptosis, whereas gelsolin null neutrophils have a delayed onset of apoptosis (39Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1040) Google Scholar). These findings highlight the importance of inhibiting gelsolin severing to preserve the integrity of the cell and to selectively activate gelsolin under proliferative conditions. The pro-apoptotic role of gelsolin is supported by the finding that most cancer cells (which usually have reduced apoptosis) have significantly lower gelsolin expression (41Dong Y. Asch H.L. Medina D. Ip M. Guzman R. Asch B.B. Int. J. Cancer. 1999; 81: 930-938Crossref PubMed Scopus (23) Google Scholar) (reviewed in Ref. 2Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar). Furthermore, gelsolin overexpression, especially of a mutant form that is more sensitive to PIP2, suppresses Ras transformation (42Fujita H. Laham L.E. Janmey P.A. Kwiatkowski D.J. Stossel T.P. Banno Y. Nozawa Y. Mllauer L. Ishizaki A. Kuzumaki N. Eur. J. Biochem. 1995; 229: 615-620Crossref PubMed Google Scholar). Nevertheless, other data do not fit into this straightforward scheme. One group found that gelsolin overexpression protects against apoptosis (39Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1040) Google Scholar). The relation between gelsolin, apoptosis, and tumorigenesis probably reflects a complex balance between the multiple effector functions of gelsolin. The structural basis for gelsolin regulation by Ca2+is now beginning to be understood. Gelsolin has two tandem homologous halves, each of which contains a 3-fold segmental repeat (segments S1–S3 and S4–S6, respectively) (43Kwiatkowski D.P. Stossel T.P. Orkin S.H. Mole J.E. Colten H.R. Yin H.L. Nature. 1986; 323: 455-458Crossref PubMed Scopus (373) Google Scholar, 44Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) (Fig. 1). The N- and C-halves are connected by a long linker, which is cleaved by caspase-3 (39Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1040) Google Scholar, 40Kamada S. Kusano H. Fujita H. Ohtsu M. Koya R.C. Kuzumaki N. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8532-8537Crossref PubMed Scopus (95) Google Scholar)in vivo and in vitro, and by many other proteasesin vitro. The isolated C-half binds a single actin molecule only when Ca2+ is above 10−6m. 2K.-M. Lin, M. Mejillano, and H. L. Yin, submitted for publication. The isolated N-half binds two actin molecules to sever and cap, even in the absence of Ca2+. Because severing by full-length gelsolin requires 10−6m Ca2+, the C-half must act as a regulatory domain to inhibit severing by the N-half. In addition, the C-half potentiates severing by the N-half, possibly through cooperative binding to the filament (3Selden L.A. Kinosian H.J. Newman J. Lincoln B. Hurwitz C. Gershman L.C. Estes J.E. Biophys. J. 1998; 75: 3101-3109Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). There are two published high resolution structures of gelsolin, and a third is on its way. We will discuss results from the first two structures here and review results from the third structure later under “Structural Basis for Ca2+ Regulation.” The S1-actin-Ca2+ structure (46McLaughlin P.J. Gooch J.T. Mannherz H.-G. Weeds A.G.A. Nature. 1993; 364: 685-692Crossref PubMed Scopus (498) Google Scholar) shows how a single gelsolin segment binds actin. Because of the similarities between segments (43Kwiatkowski D.P. Stossel T.P. Orkin S.H. Mole J.E. Colten H.R. Yin H.L. Nature. 1986; 323: 455-458Crossref PubMed Scopus (373) Google Scholar,44Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), it can be used as a template for modeling how the other segments bind actin. The full-length gelsolin crystal formed in the absence of Ca2+ (gelsolin/EGTA) shows that inactive gelsolin has a compact quarternary structure in the absence of Ca2+ (44Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Its two halves are held together by a C-terminal S6 tail, which latches onto S2 (Figs. 1 and 2 A) (the tail latch, see “Structural Basis for Ca2+ Regulation.” Within each half, the first and third segments (S1 and S3, S4 and S6, respectively, for the N- and C-halves) are joined into a 10-strand β-sheet that is sterically incompatible with actin binding (Fig.2 B). This explains why neither S1 nor S4 binds actin in the absence of Ca2+. It also predicts that Ca2+must induce major conformational changes in each half and in the relation between the halves to accommodate actin binding. A recent cryoelectron microscopic study of a gelsolin construct missing S1 attached to an actin filament (4McGough A. Chiu W. Way M. Biophys. J. 1998; 74: 764-772Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) hints at the extent of the change that is required. The reconstructed image shows that gelsolin S2–S6 binds to actin molecules in neighboring filament strands (via S2 and S4). The distances between the S2 and S4 and the S1 and S2 actin-binding sites on the filament indicate that there must be large scale conformational changes before S1, S2, and S4 can simultaneously bind actin. The convoluted linker probably unwinds, and parts of the S1 or S2 core domain may have to unravel to extend the linker between S1 and S2 (as proposed in Refs. 44Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar and 47McGough A. Curr. Opin. Struct. Biol. 1998; 8: 166-176Crossref PubMed Scopus (109) Google Scholar). A model for how this sequence of events may occur is shown in Fig.3. The questions of how gelsolin is clamped in the inactive configuration under submicromolar Ca2+ conditions and how Ca2+ switches gelsolin on are clearly important because they pertain to proliferative and apoptotic signaling. Although gelsolin was first identified as a Ca2+-regulated protein that binds two Ca2+ with 10−6m K d (48Yin H.L. Zaner K.S. Stossel T.P. J. Biol. Chem. 1980; 255: 9494-9500Abstract Full Text PDF PubMed Google Scholar), subsequent studies find that gelsolin interaction with Ca2+ is considerably more complex. Isolated gelsolin domains have at least three Ca2+-binding sites, with submicromolar and micromolar K d values (49Pope B. Maciver S. Weeds A. Biochemistry. 1995; 34: 1583-1588Crossref PubMed Scopus (65) Google Scholar). On binding actin, intermolecular and intramolecular Ca2+-binding sites are created (46McLaughlin P.J. Gooch J.T. Mannherz H.-G. Weeds A.G.A. Nature. 1993; 364: 685-692Crossref PubMed Scopus (498) Google Scholar,50Weeds A.G. Gooch J. McLaughlin P. Pope B. Bengtsdotter M. Karlsson R. FEBS Lett. 1995; 360: 227-230Crossref PubMed Scopus (25) Google Scholar), 3R. C. Robinson, M. Mejillano, V. Le, L. D. Burtnick, H. L. Yin, and S. Choe, submitted for publication. so gelsolin can potentially bind even more Ca2+ ions. Paradoxically, a recent study finds that gelsolin binds only two Ca2+ ions in the presence of actin, and they bind cooperatively (52Gremm D. Wegner A. Eur. J. Biochem. 1999; 262: 330-334Crossref PubMed Scopus (16) Google Scholar). The challenge will be to determine which of the currently identified Ca2+-binding sites are physiologically relevant and how their occupancy alters gelsolin conformation. At 37 °C, half-maximal severing is observed at 2 × 10−6m Ca2+,2 which is well within the physiological range encountered during surface receptor stimulation. A small decrease in pH also reduces the Ca2+requirement for severing significantly (53Kinosian H.J. Newman J. Lincoln B. Selden L.A. Gershman L.C. Estes J.E. Biophys. J. 1998; 75: 3092-3100Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 54Lamb J.A. Allen P.G. Tuan B.Y. Janmey P.A. J. Biol. Chem. 1993; 268: 8999-9004Abstract Full Text PDF PubMed Google Scholar, 55Lin K.-M. Wenegieme E.F. Lu P.-J. Chen C.-S. Yin H.L. J. Biol. Chem. 1997; 272: 20443-20450Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), suggesting that gelsolin can integrate these signals to generate fine-tuned responses in cells. Biochemical and physical studies indicate that Ca2+ opens up gelsolin by inducing a conformational change in the C-half to expose actin-binding sites on the N-half. The gelsolin/EGTA crystal structure shows that the C terminus of gelsolin has a tail extension that contains an unstructured strand capped with a short terminal helix (Fig. 1). The tail helix is in close contact with the actin binding helix of S2 (Fig. 2 A) and may act as a latch to inhibit actin binding by the N-half in the absence of Ca2+. This is called the tail latch hypothesis. The importance of the S6 tail in Ca2+ regulation is now supported by deletion studies. Deletion of the tail helix decreases the Ca2+ concentration for half-maximal activation of severing from 2 × 10−6m to 10−7m,2 whereas deletion of the entire tail abolishes the Ca2+ requirement for severing altogether (57Kwiatkowski D.P. Janmey P.A. Yin H.L. J. Cell Biol. 1989; 108: 1717-1726Crossref PubMed Scopus (150) Google Scholar).2 Furthermore, tail helix deletion abolishes the change in intrinsic tryptophan fluorescence observed at 10−6m Ca2+ but not that at submicromolar Ca2+ (53Kinosian H.J. Newman J. Lincoln B. Selden L.A. Gershman L.C. Estes J.E. Biophys. J. 1998; 75: 3092-3100Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 56Pope B.J. Gooch J.T. Weeds A.G. Biochemistry. 1997; 36: 15848-15855Crossref PubMed Scopus (54) Google Scholar).2 Therefore, the tail latch is the major switch that releases the final constraint on the N-half to initiate the severing cascade. Gelsolin is unique among the gelsolin family proteins (see “The Gelsolin Superfamily”) in relegating Ca2+ regulation of its N-half to the C-half through its tail. It may have evolved the unique tail latch mechanism to achieve stringent regulation of severing and capping and to permit dispensing with Ca2+ regulation entirely during apoptosis simply by cleaving the severing half from the regulatory half. S4–S6 has remained a black box for many years, even though it is the primary Ca2+-activated switch for gelsolin. The Ca2+activation model can now be refined considerably because a crystal structure of the C-half complexed with actin and Ca2+ has just become available.3 As discussed above, in the absence of Ca2+, S4 and S6 are melded together into an extended β-sheet (44Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) (Fig. 2 B). In the presence of Ca2+, the β-sheet is broken; S4 and S6 are completely separated along their interface. S6 swings away from S4 and forms new contacts with S5. Actin inserts into the space vacated by S6 and creates an intermolecular Ca2+-binding site coordinated by S4 and actin. These results confirm that that there are large scale domain rearrangements during Ca2+ activation. Although there is ample evidence for reversible gelsolin-actin association in cells (reviewed in Ref. 1Liu Y.T. Rozelle A.L. Yin H.L. Maruta H. Kohama K. G Proteins, Cytoskeleton and Cancer. R. G. Landes Company, Austin, TX1998: 19-35Google Scholar), gelsolin uncapping after severing cannot be achieved simply by reducing Ca2+. This is because a Ca2+ molecule is trapped between gelsolin S1 and actin, and it is inaccessible to EGTA (46McLaughlin P.J. Gooch J.T. Mannherz H.-G. Weeds A.G.A. Nature. 1993; 364: 685-692Crossref PubMed Scopus (498) Google Scholar). Phosphoinositides, particularly PIP2 (16Janmey P.A. Stossel T.P. Nature. 1987; 325: 362-364Crossref PubMed Scopus (496) Google Scholar, 58Lu P.J. Wang D.S. Lin K.M. Yin H.L. Chen C.S. Am. Chem. Soc. Symp. Ser. 1999; 718: 38-54Google Scholar), are the only known agents that inhibit gelsolin severing and dissociate gelsolin from actin in vitro (59Janmey P.A. Iida K. Yin H.L. Stossel T.P. J. Biol. Chem. 1987; 262: 12228-12236Abstract Full Text PDF PubMed Google Scholar). Gelsolin binds PIP2 with micromolar affinity (55Lin K.-M. Wenegieme E.F. Lu P.-J. Chen C.-S. Yin H.L. J. Biol. Chem. 1997; 272: 20443-20450Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Binding is enhanced by Ca2+ and by low pH (55Lin K.-M. Wenegieme E.F. Lu P.-J. Chen C.-S. Yin H.L. J. Biol. Chem. 1997; 272: 20443-20450Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Gelsolin prefers PIP2 to phosphatidylinositol-3,4,5-P3 (58Lu P.J. Wang D.S. Lin K.M. Yin H.L. Chen C.S. Am. Chem. Soc. Symp. Ser. 1999; 718: 38-54Google Scholar, 60Lu P.J. Hsu A.-L. Wang D.-S. Yan H. Yin H.L. Chen C.S. Biochemistry. 1998; 37: 5738-5745Crossref PubMed Scopus (73) Google Scholar,61Hartwig J.H. Kung S. Kovacsovics T. Janmey P.A. Cantley L.C. Stossel T.P. Toker A. J. Biol. Chem. 1996; 271: 32986-32993Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and does not bind inositol trisphosphate (17Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (639) Google Scholar). Therefore, gelsolin binding is stereoselective and enhanced by the diacylglycerol chain. This requirement is unlike the situation with many pleckstrin homology proteins, which bind phosphoinositides and the equivalent inositol phosphates with similar affinity (62Rameh L.E. Arvidsson A.-K. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Gelsolin can simultaneously bind between one and three PIP2molecules within lipid vesicles, and binding is highly dependent on the physical characteristics of the bilayer (63Tuominen E.K.J. Holopainen J.M. Chen J. Prestwich G.D. Bachiller P.R. Kinnunen P.K.J. Janmey P.A. Eur. J. Biochem. 1999; 262: 1-9Crossref PubMed Scopus (85) Google Scholar). Therefore, gelsolin regulation by PIP2 does not depend simply on the absolute PIP2 mass but is rather dictated by a complex relation between lipid bilayer composition and geometry. This, together with the ability of gelsolin to change its affinity for PIP2 in response to Ca2+ and pH (55Lin K.-M. Wenegieme E.F. Lu P.-J. Chen C.-S. Yin H.L. J. Biol. Chem. 1997; 272: 20443-20450Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and to induce changes in PIP2 packing within the membrane (37Flanagan L.A. Cunningham C.C. Chen J. Prestwich G.D. Kosik K.S. Janmey P.A. Biophys. J. 1997; 73: 1440-1447Abstract Full Text PDF PubMed Scopus (75) Google Scholar), hints at the large number of signals that are sensed and integrated by gelsolin to carry out its functions. In addition, we are just beginning to appreciate the active role gelsolin may have in modulating phosphoinositide signaling (see “Effects of Gelsolin Overexpression on Cell Motility and Signaling”). PIP2 inhibits the gelsolin N-half, and the N-half PIP2 binding sequences are mapped to a common flat, solvent-exposed surface in the linker between S1–S2 and the beginning of S2 in the gelsolin/EGTA crystal (Fig. 1). These sites have an acidic amino acid consensus that does not resemble the pleckstrin homology domain (64Janmey P.A. Lamb J. Allen P.G. Matsudaira P.T. J. Biol. Chem. 1992; 267: 11818-11823Abstract Full Text PDF PubMed Google Scholar, 65Yu F.-X. Sun H.-Q. Janmey P.A. Yin H.L. J. Biol. Chem. 1992; 267: 14616-14621Abstract Full Text PDF PubMed Google Scholar). PIP2 induces a conformational change (55Lin K.-M. Wenegieme E.F. Lu P.-J. Chen C.-S. Yin H.L. J. Biol. Chem. 1997; 272: 20443-20450Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,66Xian W. Vegners R. Janmey P.A. Braunlin W.H. Biophys. J. 1995; 69: 2695-2702Abstract Full Text PDF PubMed Scopus (40) Google Scholar) that may interfere with the local rearrangements required to permit S2 and S1 to bind two actin molecules simultaneously (Fig. 3). Structural information about gelsolin complexed with PIP2will be required to determine whether this is how PIP2inhibits gelsolin and if PIP2 binds to other gelsolin domains as well. Many members of the gelsolin family that have three or six gelsolin repeats have been identified (reviewed in Refs. 1Liu Y.T. Rozelle A.L. Yin H.L. Maruta H. Kohama K. G Proteins, Cytoskeleton and Cancer. R. G. Landes Company, Austin, TX1998: 19-35Google Scholar and 2Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar). They have distinct as well as overlapping patterns of tissue expression, which is consistent with specialized function (67Arai M. Kwiatkowski D.J. Dev. Dyn. 1999; 215: 297-307Crossref PubMed Scopus (31) Google Scholar). Except for gelsolin, Ca2+ regulation and actin binding are not segregated into the two halves of the molecule. For example, villin, a six-domain protein, has a Ca2+-dependent N-half (68Matsudaira P. Jakes R. Walker J.E. Nature. 1985; 315: 248-250Crossref PubMed Scopus (33) Google Scholar), and CapG, a three-domain protein, has built-in Ca2+regulation in its actin-binding segment (69Yu F.-X. Johnston P.A. Sudhof T.C. Yin H.L. Science. 1990; 250: 1413-1415Crossref PubMed Scopus (172) Google Scholar). Novel members with long N-terminal extensions have also been discovered. For example, flightless I has an N-terminal extension of 16 tandem leucine-rich repeats (70Campbell H.D. Schimansky T. Claudianos C. Ozsarac N. Kasprzak A.B. Cotsell J.N. Young I.G. de Couet H.G. Miklos G.L.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11386-11390Crossref PubMed Scopus (121) Google Scholar), which can potentially mediate heterologous protein-protein interactions. Its binding partners include a family of novel proteins with coiled coil repeats (71Liu Y.T. Yin H.L. J. Biol. Chem. 1998; 273: 7920-7927Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 72Fong K.S. de Couet H.G. Genomics. 1999; 58: 146-157Crossref PubMed Scopus (46) Google Scholar) and possibly Ras (73Goshima M. Kariya K. Yamawaki-Kataoka Y. Okada T. Shibatohge M. Shima F. Fujimoto E. Kataoka T. Biochem. Biophys. Res. Commun. 1999; 257: 111-116Crossref PubMed Scopus (53) Google Scholar). Through these interactions, flightless I may link the actin cytoskeleton to intracellular and membrane structures to integrate signaling responses. This review has focused on the intracellular functions of gelsolin and emphasizes progress made to obtain a mechanistic model of how gelsolin regulates the actin cytoskeleton in response to Ca2+ and phosphoinositide signaling. With a clear understanding of how actin regulatory proteins work individually, we are now poised to delineate how they cooperate to orchestrate actin dynamics within living cells. Some progress has already been made. One study shows that gelsolin and capping protein coordinately cap barbed ends during platelet activation (73Goshima M. Kariya K. Yamawaki-Kataoka Y. Okada T. Shibatohge M. Shima F. Fujimoto E. Kataoka T. Biochem. Biophys. Res. Commun. 1999; 257: 111-116Crossref PubMed Scopus (53) Google Scholar). Another study finds that filaments capped by gelsolin and Arp2/3 at the barbed and pointed ends, respectively, can still undergo rapid turnover in the presence of ADF/cofilin, and new uncapped barbed ends are generated (51Barkalow K. Witke W. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1996; 134: 389-399Crossref PubMed Scopus (131) Google Scholar). Therefore, gelsolin severing and capping can theoretically promotede novo nucleation by Arp2/3 by increasing the number of pointed filament ends and increasing the actin monomer pool (45Ressard F. Didry D. Egile C. Pantaloni D. Carlier M.F. J. Biol. Chem. 1999; 274: 20970-20977Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These findings extend the scope of the involvement of gelsolin in regulating actin dynamics.