Stimulation of Actin Polymerization by Vacuoles via Cdc42p-dependent Signaling

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction. We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction. The cytoskeleton plays an important role in regulating many cellular processes including cell division, motility, polarization, endocytosis, and exocytosis (1Moseley J.B. Goode B.L. Microbiol. Mol. Biol. Rev. 2006; 70: 605-645Crossref PubMed Scopus (293) Google Scholar, 2Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3343) Google Scholar, 3Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: f111-f116Crossref PubMed Scopus (360) Google Scholar, 4Glotzer M. Science. 2005; 307: 1735-1739Crossref PubMed Scopus (577) Google Scholar). Two general classification of actin regulatory functions have been established; those that depend on stable actin filaments, such as polarized organelle transport (5Hill K.L. Catlett N.L. Weisman L.S. J. Cell Biol. 1996; 135: 1535-1549Crossref PubMed Scopus (185) Google Scholar, 6Fagarasanu A. Fagarasanu M. Eitzen G.A. Aitchison J.D. Rachubinski R.A. Dev. Cell. 2006; 10: 587-600Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 7Alberts P. Rudge R. Irinopoulou T. Danglot L. Gauthier-Rouviere C. Galli T. Mol. Biol. Cell. 2006; 17: 1194-1203Crossref PubMed Scopus (80) Google Scholar), and those that depend on dynamic actin remodeling, such as cell motility (2Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3343) Google Scholar). There is a growing body of evidence that supports the need for dynamic actin monomerpolymer transitions during exocytosis and membrane fusion (8Malacombe M. Bader M.F. Gasman S. Biochim. Biophys. Acta. 2006; 1763: 1175-1183Crossref PubMed Scopus (144) Google Scholar, 9Yu H.-Y.E. Bement W.M. Nat. Cell Biol. 2007; 9: 149-159Crossref PubMed Scopus (55) Google Scholar). Traditionally, cortical actin has been viewed as a barrier structure that would only require disassembly to allow membrane contact prior to exocytosis (10Orci L. Gabbay K.H. Malaisee W.J. Science. 1972; 175: 1128-1130Crossref PubMed Scopus (288) Google Scholar, 11Aunis D. Bader M.F. J. Exp. Biol. 1988; 139: 253-266Crossref PubMed Google Scholar, 12Trifaro J.M. Vitale M.L. Trends Neurosci. 1993; 16: 466-472Abstract Full Text PDF PubMed Scopus (151) Google Scholar). This would seemingly be the case for neurotransmitter release at the synapse, where actin remodeling is controlled by scinderin, which severs actin in a calcium-dependent manner to promote exocytosis (13Zhang L. Marcu M.G. Nau-Staudt K. Trifaro J.M. Neuron. 1996; 17: 287-296Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Interestingly, scinderin is also reported to negatively regulate exocytosis via actin nucleating activity (14Marcu M.G. Zhang L. Elzagallaai A. Trifaro J.M. J. Biol. Chem. 1998; 273: 3661-3668Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Lejen T. Skolnik K. Rose S.D. Marcu M.G. Elzagallaai A. Trifaro J.M. J. Neurochem. 2001; 76: 768-777Crossref PubMed Scopus (15) Google Scholar). However, it remains feasible that both activities are needed for distinct subreactions of exocytosis. Indeed, several studies implicate a dual role for F-actin 3The abbreviations used are: F-actin, filamentous actin; A.P.A., actin polymerization activity; A.P.B., actin polymerization buffer; ATPreg, ATP-regenerating system; F.R.B., fusion reaction buffer; G-actin, globular actin; JP, jasplakinolide; Lat B, latrunculin B; rxn, reaction; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; MBP, maltose-binding protein; PI, phosphatidylinositol. as both a barrier that requires disassembly and a positive regulator to provide directional movement and force (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar, 17Bittner M.A. Holz R.W. Mol. Pharmacol. 2005; 67: 1089-1098Crossref PubMed Scopus (26) Google Scholar, 18Lang T. Wacker I. Wunderlich I. Rohrbach A. Giese G. Soldati T. Almers W. Biophys. J. 2001; 78: 2863-2877Abstract Full Text Full Text PDF Scopus (190) Google Scholar, 19Bernstein B.W. DeWitt M. Bamburg J.R. Brain Res. Mol. Brain Res. 1998; 53: 236-251Crossref PubMed Scopus (70) Google Scholar). We have proposed that F-actin stabilizes docked vesicle and provides localized force, which would be needed to overcome the energy barrier of membrane fusion (20Eitzen G. Biochim. Biophys. Acta. 2003; 1641: 175-181Crossref PubMed Scopus (191) Google Scholar). Rho GTPases are well known for their role in governing actin polymerization (21Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5272) Google Scholar). Cdc42p triggers actin polymerization via direct (22Higgs H.N. Pollard T.D. J. Cell Biol. 2000; 150: 1311-1320Crossref PubMed Scopus (420) Google Scholar, 23Rohatgi R. Ho H.Y. Kirschner M.W. J. Cell Biol. 2000; 150: 1299-1310Crossref PubMed Scopus (504) Google Scholar) or indirect (24Lechler T. Shevchenko A. Li R. J. Cell Biol. 2000; 148: 363-373Crossref PubMed Scopus (177) Google Scholar, 25Evangelista M. Klebl B.M. Tong A.H. Webb B.A. Leeuw T. Leberer E. Whiteway M. Thomas D.Y. Boone C. J. Cell Biol. 2000; 148: 353-362Crossref PubMed Scopus (197) Google Scholar) activation of the WASp ·WIP complex, which subsequently activates actin polymerization via Arp2/3 (26Higgs H.N. Pollard T.D. Annu. Rev. Biochem. 2001; 70: 649-676Crossref PubMed Scopus (550) Google Scholar). We have shown that membrane fusion is inhibited by antibodies to Las17p (yeast WASp ortholog) (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar) or deletion of VRP1 (yeast WIP ortholog) (27Tedrick K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar), using an established in vitro assay of vacuole membrane fusion (28Wickner W. EMBO J. 2002; 21: 1241-1247Crossref PubMed Scopus (136) Google Scholar). Cdc42p is located on the vacuole membrane and is needed for homotypic vacuole fusion (29Eitzen G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar, 30Muller O. Johnson D.I. Mayer A. EMBO J. 2001; 20: 5657-5665Crossref PubMed Scopus (65) Google Scholar), which suggests that actin polymerization may be a subreaction of the fusion mechanism. Indeed, both of the actin ligands jasplakinolide and latrunculin, which stabilize and destabilize F-actin, respectively, inhibit vacuole fusion implicating the need for cycles of actin polymerization and depolymerization. However, final membrane is only inhibited by F-actin via latrunculin, actin polymerization is the final process needed (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar). Here, we used assays to demonstrate that purified vacuole membranes actin polymerization in We used a pyrene-actin polymerization assay to that vacuole membranes actin polymerization via a mechanism. We used an assay to that F-actin is during of vacuoles in conditions that support in vitro membrane fusion. We also that polymerization-deficient actin is an of vacuole fusion, and F-actin directly with that have Our results suggest that actin polymerization is a subreaction of the membrane fusion mechanism. and used in this are in Rho overexpression were using G. C. B. Thomas D.Y. Whiteway M. 2004; PubMed Scopus (151) Google Scholar) as under were at in yeast or yeast when growing Rho overexpression was from strain to by for at with using a of of to of at the in were by at for at were was purified from by chromatography by chromatography as previously J. Pharmacol. 2006; PubMed Google Scholar). was to using a used in this in a were from the actin binding jasplakinolide and latrunculin were in at was in and and were in at was in at and were and used as previously (27Tedrick K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar, G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar). and were purified as from as previously (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar, G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar). was as a in was as a and were from yeast by on as previously A. Cell 17: Scopus Google Scholar). fusion was as previously A. Cell 17: Scopus Google Scholar). fusion of vacuoles from of fusion and to in fusion reaction ATPreg, and To assay the of actin polymerization, vacuoles were preincubated at in used for in vitro vacuole fusion. of vacuoles in of was where Vacuoles were at from in and to polymerization assay actin polymerization activity we used an established pyrene-actin polymerization assay Pollard T.D. PubMed Scopus Google Scholar). of a in was with of actin polymerization actin in polymerization was to which polymerization because of a of the for polymerization Pollard T.D. PubMed Scopus Google Scholar). were using a with a to of the pyrene-actin was for were and for polymerization activity was from polymerization by the of for of reaction were the of a reaction and by the of examine the of yeast actin on of purified yeast actin were for changes in the of actin by size-exclusion this assay vacuoles in were at for the and were by to and of of F-actin and were by and on for of the was to a at in and were and the of actin was by of of and vacuoles in were for the and were by in by and in of were by of of antibodies to in binding was in using were for at with were in with of by and of and was as previously N. R.A. Wickner W. EMBO J. 2004; PubMed Scopus Google Scholar). binding of F-actin and was by of GST, or or with yeast in for at or at to allow for actin and SNARE were by for actin in and in were with (27Tedrick K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar), and vacuole was while were growing in or for in conditions in This has previously been shown to vacuole fusion in vivo Weisman L.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (80) Google Scholar). were using a with a a and We have shown that actin which F-actin or inhibit vacuole membrane fusion (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar). results that both actin and actin was needed for membrane fusion. However, this with the that actin is a barrier requiring only depolymerization. Our was to directly examine vacuole-associated actin polymerization activity. To the of a to actin polymerization we used which undergoes a when F-actin A. Cell 17: Scopus Google Scholar). yeast which vacuole membrane fusion (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar, K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar), also stimulated the polymerization of and actin ligands or this activity on their known pyrene-actin polymerization we as the of of purified vacuoles actin vacuoles purified from yeast showed of pyrene-actin polymerization, vacuoles purified from a VRP1 deletion strain were of this activity of actin polymerization increased activity in both both the to of vacuoles with the of vacuoles and recovered the of vacuoles is the yeast WIP G. N. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which with WASp the of actin polymerization via the Arp2/3 (26Higgs H.N. Pollard T.D. Annu. Rev. Biochem. 2001; 70: 649-676Crossref PubMed Scopus (550) Google Scholar). We have previously shown that vacuoles from the strain membrane fusion in vitro (27Tedrick K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar), and results suggest this may be because of the of actin polymerization activity. We a vacuole to the needed to actin We found that polymerization increased with vacuole with activity at vacuole of and The was with vacuoles that were preincubated for in a reaction used for in vitro membrane fusion A. Cell 17: Scopus Google Scholar). This showed a were significantly increased with vacuoles and was also which showed actin were for assay conditions revealed that of vacuoles increased polymerization activity and to we further the of vacuole on their to actin Vacuoles were for in F.R.B., and for of actin activity of activity in stimulated a in activity, which was also with activity only of suggest that purified vacuoles actin polymerization activity which is reduced during while activity. We this by vacuoles in the of reaction and activity activity was in the of an reaction We also membrane fusion for conditions and with membrane fusion that or reduced membrane fusion on vacuoles could be directly using the pyrene-actin assay because of during we an assay on which would examine the remodeling of yeast actin by this assay, vacuoles were in membrane fusion reaction at membranes were and actin by the of were to a for and the of actin was by F-actin the well from actin and nucleating actin A. Mol. Cell. Biochem. PubMed Google Scholar). was in to to changes in This showed that actin was with of F-actin, the of F-actin This well with results which showed that vacuoles were in pyrene-actin polymerization of were with vacuoles purified from the VRP1 deletion and F-actin in the presence of the of showed reduced F-actin results suggest that F-actin is an subreaction of in vitro vacuole membrane fusion. to the was and However, at and of and with F-actin of the further conditions and for vacuole-associated actin polymerization activity, we for vacuoles preincubated in a of conditions We found that polymerization activity with and was significantly when was However, polymerization activity was significantly on the presence of because the were when only and This from assay results which showed that F-actin However, this is to because would provide of which has been previously shown to polymerization activity A. M. B. A. E. A. G. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). is that by the of were for polymerization activity. of vacuoles in the presence of to reduced which supports the need for an to be in the presence of M.A. Holz R.W. Mol. Pharmacol. 2005; 67: 1089-1098Crossref PubMed Scopus (26) Google Scholar) and be to and by including actin polymerization and vacuole fusion (22Higgs H.N. Pollard T.D. J. Cell Biol. 2000; 150: 1311-1320Crossref PubMed Scopus (420) Google Scholar, A. D. S. A. Wickner W. A. Mol. Biol. Cell. 2000; PubMed Scopus Google Scholar, R.A. Wickner W. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). of vacuoles with the increased actin polymerization activity that the of during vacuole fusion has a role in actin However, membrane were also because of vacuoles with activity We also and Rho GTPases were for actin of the or the Rho and was by with and activity was reduced only by results suggest that during membrane fusion an actin is on the vacuole membrane with that be from the to activity. polymerization activity is by Rho GTPases because with reduced activity. of by Rho are known to be of actin remodeling (1Moseley J.B. Goode B.L. Microbiol. Mol. Biol. Rev. 2006; 70: 605-645Crossref PubMed Scopus (293) Google Scholar, A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5272) Google Scholar). We have previously shown that both Cdc42p and are on the vacuole membrane and are needed for vacuole fusion (29Eitzen G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar). The that both vacuole fusion (29Eitzen G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar) and polymerization suggests that Cdc42p regulates actin polymerization for fusion. of vacuoles with antibodies Cdc42p showed of polymerization activity as activity This suggests that Cdc42p and regulate actin To further examine the of actin remodeling, we polymerization assays using vacuoles from dominant-active or Rho Vacuoles purified from a strain showed in activity while dominant-active increased this activity This a role for Cdc42p in actin of also an in vacuole-associated actin polymerization activity we a role for directly actin polymerization is needed for vacuole fusion we vacuole in a polymerization-deficient actin mutant strain J. Pharmacol. 2006; PubMed Google Scholar, Biochem. Cell Biol. 2007; PubMed Scopus Google this strain has which is of a membrane fusion of in vivo vacuole fusion via Weisman L.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (80) Google Scholar) the fusion of vacuoles in the polymerization-deficient strain We also purified and polymerization-deficient actin in vitro vacuole fusion actin on fusion to mutant actin inhibited fusion at the results support the need for actin polymerization during membrane fusion. we to that the of fusion antibodies to were used to from vacuoles that were for and in fusion reaction in the presence of to stabilize F-actin, or in the presence of Lat to destabilize and Rho and However, the SNARE showed increased actin or when and reduced when Lat The also showed were by this was the reaction between the and actin has been previously reported in which may have a role in of the to membranes for the of J. J. M. L.S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). We also and directly to and and were in on or glutathione and with purified yeast actin would have to this was in of the showed an with which increased an that would actin polymerization and This with the results from GST, and with However, an with F-actin was under conditions it is that actin with that are of a SNARE complex, and the interaction with may be its functions M.M. Zhang M. Science. 2001; PubMed Scopus Google Scholar, R. M. C. EMBO J. 2004; PubMed Scopus Google Scholar). Interestingly, the of both and which are to M.M. Zhang M. Science. 2001; PubMed Scopus Google Scholar, W. L. Zhang M. Mol. Biol. Cell. 2006; 17: PubMed Scopus Google Scholar). is known to actin and remodeling Trends Biochem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) and because both have the to with we that this may have an important role in actin remodeling needed for membrane fusion. of membrane fusion using yeast vacuoles have revealed a complex, (28Wickner W. EMBO J. 2002; 21: 1241-1247Crossref PubMed Scopus (136) Google Scholar). We have previously shown that in actin remodeling such as Las17p and the WASp ·WIP of are needed for vacuole fusion (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar, K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar), and that actin polymerization or inhibit vacuole fusion in membranes fusion actin polymerization this was a of the fusion To this we used a sensitive in vitro pyrene-actin polymerization assay J. J. M. L.S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and showed that vacuoles actin of vacuoles in that support membrane fusion also increased their to polymerization This activity requires the actin remodeling of the complex, which is with the vacuole membrane (16Eitzen G. Wang L. Thorngren N. Wickner W. J. Cell Biol. 2002; 158: 669-679Crossref PubMed Scopus (140) Google Scholar). of VRP1 (yeast in a of polymerization activity and fusion activity (27Tedrick K. Trischuk T. Lehner R. Eitzen G. Mol. Biol. Cell. 2004; 15: 4609-4621Crossref PubMed Google Scholar). with results polymerization-deficient actin vacuole fusion we that vacuoles actin polymerization, and that this activity is to the membrane fusion We of vacuole fusion to actin polymerization activity is to the vacuole fusion that the of membrane fusion such as (28Wickner W. EMBO J. 2002; 21: 1241-1247Crossref PubMed Scopus (136) Google Scholar) on polymerization activity This suggests that the polymerization is to the fusion at this of the is needed prior to and requires the activity of the and (28Wickner W. EMBO J. 2002; 21: 1241-1247Crossref PubMed Scopus (136) Google Scholar). polymerization activity was also on and and this to be to antibodies inhibit polymerization fusion requiring is the of the A. D. S. A. Wickner W. A. Mol. Biol. Cell. 2000; PubMed Scopus Google Scholar). the of via has also been shown to be for fusion R.A. Wickner W. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). the role in fusion we have shown that the vacuole-associated polymerization activity and the of polymerization activity may be to that during fusion. We also showed that actin polymerization is by was previously shown that Cdc42p has a role in vacuole fusion (29Eitzen G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar, 30Muller O. Johnson D.I. Mayer A. EMBO J. 2001; 20: 5657-5665Crossref PubMed Scopus (65) Google Scholar), and subsequently shown that actin remodeling is needed for membrane fusion (20Eitzen G. Biochim. Biophys. Acta. 2003; 1641: 175-181Crossref PubMed Scopus (191) Google Scholar). However, a direct between actin remodeling and Cdc42p was and this was both Cdc42p and are localized to the vacuole membrane and for fusion (29Eitzen G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar). we provide of evidence that directly Cdc42p to the of actin this activity was reduced when vacuoles were from a strain Cdc42p vacuoles with Cdc42p antibodies reduced activity Our results are by in vivo studies in which showed that exocytosis could be by dominant-active Cdc42p J. Cell 2001; PubMed Google Scholar). was also shown that polymerization via activation a positive role in and that latrunculin J. Cell 2001; PubMed Google Scholar). We of in vitro actin polymerization via dominant-active this may be by such as Cdc42p Interestingly, we polymerization when dominant-negative was We that Rho may for this because are many of such in M. A.H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. B.A. M.L. M. J. Cell Biol. 2000; 148: PubMed Scopus Google Scholar). We are Cdc42p and during the activation of actin polymerization and membrane fusion. To provide direct evidence that actin polymerization is needed for membrane fusion we a polymerization-deficient actin mutant This strain showed vacuole fusion defects in vivo and purified polymerization-deficient actin inhibited vacuole fusion in vitro actin also inhibited fusion, only at which suggest a is needed between the of actin needed to promote fusion as to which inhibit fusion. This could a with in fusion such that of actin would this from its Indeed, we found that vacuole and directly F-actin in a purified only actin during vacuole fusion with actin have been shown to a structure on docked and it is at this where membrane fusion (20Eitzen G. Biochim. Biophys. Acta. 2003; 1641: 175-181Crossref PubMed Scopus (191) Google Scholar, L. Wickner W. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Wickner W. J. Cell Biol. 2003; PubMed Scopus Google Scholar). of in the for was shown to depend on actin such that which to F-actin transitions this L. Wickner W. J. 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