Depolymerization of Actin Filaments by Profilin

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1–5 μm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10–100 μm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, ∼40 μm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization. Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1–5 μm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10–100 μm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, ∼40 μm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization. Profilin is a multifunctional actin regulatory protein that binds actin monomers with a 1:1 stoichiometry. Profilin-actin complexes fail to form nuclei and, in the absence of preformed actin filaments, will not assemble into actin filaments. However, profilin-actin complexes can readily add to free barbed ends of actin filaments at rates similar to free actin monomers (1Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Google Scholar). Therefore, profilin is widely regarded as a protein that promotes actin filament assembly (1Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Google Scholar, 2Pring M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar, 3Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Google Scholar, 4Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Google Scholar, 5Baum B. Perrimon N. Nat. Cell Biol. 2001; 3: 883-890Google Scholar, 6Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google Scholar, 7Hopmann R. Miller K.G. Mol. Biol. Cell. 2003; 14: 118-128Google Scholar). The in vivo effects of profilin on the actin filament cytoskeleton, however, have proved enigmatic and are not readily explained by the known functions of profilin. Genetic experiments in which both profilin isoforms in Dictyostelium discoideum were deleted result in an increase in F-actin of ∼70% (8Haugwitz M. Noegel A.A. Karakesisoglou J. Schleicher M. Cell. 1994; 79: 303-314Google Scholar). Microinjection of profilin in Swiss 3T3 cells causes extensive depletion of filamentous actin, but cortical F-actin is selectively preserved (9Hajkova L. Bjorkegren Sjogren C. Korenbaum E. Nordberg P. Karlsson R. Exp. Cell Res. 1997; 234: 66-77Google Scholar). Similarly, microinjection of profilin into normal rat kidney cells induces actin filament disassembly, selectively sparing actin in the cortex and contractile ring. Although monomer sequestration by profilin could be responsible for depletion of F-actin, the pattern of loss of actin was in contrast to the nonspecific depletion of filaments observed when other monomer sequestering agents, DNase I and vitamin D binding protein, were employed (10Cao L.G. Babcock G.G. Rubenstein P.A. Wang Y.L. J. Cell Biol. 1992; 117: 1023-1029Google Scholar), implying that monomer sequestration was not entirely responsible for these in vivo observations. Others have shown similar differential effects on actin filament populations, with reduced density of parallel actin bundles in Chinese hamster ovary cells overexpressing profilin (11Finkel T. Theriot J.A. Dise K.R. Tomaselli G.F. Goldschmidt-Clermont P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1510-1514Google Scholar). Another unique characteristic of profilin is its ability to bind to poly-l-proline. A number of actin regulatory proteins contain profilin binding sequences of the type XPPPPP (where X = G, A, P, or S) (12Purich D.L. Southwick F.S. Biochem. Biophys. Res. Commun. 1997; 231: 686-691Google Scholar). For example, vasoactive stimulatory phosphoprotein (VASP) 1The abbreviations used are: VASP, vasoactive stimulatory phospho-protein; N-WASP, neuronal Wiskott-Aldrich syndrome protein; pyrenyllabeled actin, actin labeled on Cys-374 with N-(1-pyrene)iodoacetamide. contains 16–20 profilin binding sites, and N-WASP contains 12 profilin binding sites. These proteins localize to regions of high actin filament turnover and are capable of attracting concentrations of total profilin in the millimolar range (13Kang F. Laine R.O. Bubb M.R. Southwick F.S. Purich D.L. Biochemistry. 1997; 36: 8384-8392Google Scholar). Although the relationship between free and total profilin in vivo has not been determined, there are data that suggest that the cellular content of polymerization competent profilin-actin complex is limited (14Babcock G. Rubenstein P.A. Cell. Motil. Cytoskeleton. 1993; 24: 179-188Google Scholar, 15Sanders M.C. Wang Y.L. J. Cell Biol. 1990; 110: 359-365Google Scholar), raising the possibility that high concentrations of actin-free profilin are attainable locally. Previous in vitro studies examining the effects of profilin on actin filament kinetics have used profilin concentrations in the range of 0.5–5 μm. Cognizant of the ability of cells to locally generate much higher concentrations of profilin, we have examined the effects of profilin concentrations an order of magnitude higher (10–100 μm, final concentrations) than previously studied. Using an experimental design that simplifies kinetic analysis, we have found that profilin accelerates the off-rate of the barbed end in a concentration-dependent fashion. We have further discovered that high concentrations of profilin can competitively dissociate the barbed-end capping protein, CapG. These findings add two hitherto-unappreciated functions for profilin that may help to account for the multiple and complex in vivo changes in actin filament concentration and morphology attributed to raising and lowering cell profilin content. Materials—Rabbit skeletal muscle Ca2+-actin was prepared from frozen muscle (Pel-Freez; Rogers AR) in buffer G (5.0 mm Tris-HCl, 0.2 mm ATP, 0.2 mm dithiothreitol, 0.1 mm CaCl2, and 0.01% sodium azide, pH 7.8). Actin labeled on Cys-374 with N-(1-pyrene)iodoacetamide (i.e. pyrenyl-labeled actin) was prepared with 0.7 to 0.95 mol of labeling compound/mol of protein using the method of Kouyama and Mihashi (16Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Google Scholar). Latrunculin A was purchased from Biomol (Plymouth Meeting, PA). Human profilin, gelsolin, and CapG were produced as recombinant proteins as described previously (13Kang F. Laine R.O. Bubb M.R. Southwick F.S. Purich D.L. Biochemistry. 1997; 36: 8384-8392Google Scholar, 17Dabiri G.A. Young C.L. Rosenbloom J. Southwick F.S. J. Biol. Chem. 1992; 267: 16545-16552Google Scholar). CapZ was purified from rabbit skeletal muscle to greater than as by using the method of Cell Motil. Cytoskeleton. its concentration is the concentrations are but the data from a concentrations are that not a capping protein or a of actin was to by the of and and were to final concentrations of and and the actin was to to an F-actin The was for capping that the actin was not to polymerization. rates were after to of the F-actin into buffer the of capping proteins with or latrunculin A. The of in these was S. Bubb M.R. J. Biol. Chem. 2001; Scholar). In capping proteins were at the of of the F-actin the of these were with the of capping protein at the of In both of the concentration of capping protein is the final concentration after actin concentration was as a function of after by the of in in a with at and at The of depolymerization was by a to data to the in This method is on an experimental by and a a of depolymerization is that the filaments are that the filament number not the used for the of actin was by of and to final concentrations of and the actin was to to F-actin was to in profilin and gelsolin or CapG. The were for to at and were in a with at and at of is an based on similar to previously used to the of profilin with both and F-actin M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google can be for other barbed-end capping protein, A is the actin monomer concentration, is the concentration of filaments of actin actin when is the concentration of capping protein, and the are the equilibrium for which the = may or may not be on the with is in the as to the is for profilin, the between the two for of a to F-actin is in (1Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Google Scholar, 3Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Google Scholar, 4Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Google Scholar, 6Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google Scholar). of the depolymerization data in of a competitive binding model be in the absence of a however, the be in the could be attributed to a from equilibrium than a of the the of barbed-end capping by profilin is in buffer J. A. Carlier M.F. J. Biol. Chem. 1999; 274: Scholar). both profilin and capping protein are are for protein with and equilibrium that are unique for protein example, and for profilin, and and for the other capping the for of capping protein from F-actin, is but this is not the for profilin H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google Scholar). the a for be by relatively binding to actin monomer or a for monomer binding by a capping protein may or may not be can be to these We that the from the filament is the of the and rates is to as in the of sequestration of actin by CapG is The concentrations of CapG used in capping experiments were in the of magnitude the equilibrium of CapG for actin monomers μm). The concentration of free profilin is to be much greater than the concentration of actin filaments. In the binding of profilin and CapG is to be in rapid equilibrium with the barbed end with of and as in based on M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar, 4Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Google Scholar, 6Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google Scholar), the elongation for of profilin-actin is to be to The for profilin-actin complex is competitive binding between profilin and CapG for the barbed end in a for the concentration of free barbed as a function of the concentrations of profilin and CapG and the total concentration of filaments, = This result is into the and the of depolymerization is for of and the of actin from the barbed end in the or absence of profilin, the concentration of filaments, and the for profilin and CapG from the barbed end. that for profilin and actin, the that of a this is to for Profilin the of F-actin with the of M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar), and the experimental of H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google for actin, profilin accelerates actin filament depolymerization concentrations of profilin in of and a acceleration in depolymerization by profilin was observed The concentration of actin monomer at the in the depolymerization data is monomer sequestering function of profilin be In we that the of H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google be explained by monomer sequestration by the effects of profilin with that of monomer sequestering latrunculin A. At high latrunculin A had effect on the of depolymerization. monomer sequestration by latrunculin A not the acceleration of depolymerization by profilin. the when the capping protein in is profilin. depolymerization is accelerated by a factor of when barbed-end saturating concentrations of profilin are of profilin on the depolymerization for filaments by CapG. of the depolymerization of 0.1 pyrenyl-labeled F-actin on profilin concentration in or absence of CapG. the same was in of latrunculin A, and the are with Profilin binds to pyrenyl-labeled actin monomers than to monomers A.A. J. Biol. Chem. Scholar), a factor in the of depolymerization data that are based on the of the off-rate of pyrenyl-labeled from However, a polymerization in the of profilin, the effect of of actin on the depolymerization is that the for and are but that with actin J.A. T.D. J. Res. Cell Motil. Scholar), actin can dissociate when are In the at the barbed end might not bind profilin and dissociate at the from In that of from a pyrenyl-labeled filament be at the of and the between observed rates in the and absence of profilin be of be profilin to pyrenyl-labeled and the effect of be increase to pyrenyl-labeled actin in the depolymerization similar to in that the magnitude of by a in is not with the experiments using at at a to the acceleration of depolymerization by profilin not Profilin CapG from the of F-actin, and the with a actin depolymerization as C.L. A. Southwick F.S. J. Biol. Chem. 1994; Scholar), but of profilin can this effect of latrunculin A that this result is not to monomer sequestration. At profilin, depolymerization data with or CapG are The that the depolymerization rates with or CapG at saturating concentrations of profilin is with competitive binding between CapG and profilin for the barbed end of actin filaments. these data are with the that profilin and CapG form a at the barbed end that has the same as profilin at the same end. in and the We the differences between this result and that of to in the experimental method that of data not at the of after the is and in the The in this is in Profilin on the of in by CapZ or but on the of These contrast to the with profilin has no effect on the depolymerization of filaments by gelsolin, at higher concentrations of profilin CapG and gelsolin are of the same of proteins that filaments by similar F.S. J. Biol. Chem. Scholar). The depolymerization for the in is relatively a result that not with of filament that that filament be at in the of gelsolin L. Biol. Scholar). these data are as depolymerization of filaments of similar and there are data in the that this for low of actin to gelsolin P.A. J. T. J. Biol. Chem. Scholar). In this by profilin be to result in an increase in the of depolymerization as a function of This is not observed at final concentration of profilin μm). these is not to explain the experimental differences between CapG and The off-rate of gelsolin is for is in the range of N. A. Eur. J. Biochem. Scholar), data suggest that is profilin has no to compete with ends the capping protein from the under these with Although the of the in of based on the of for gelsolin, the observed may be explained by (1Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Google a M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google an equilibrium of profilin for the barbed end high that the of profilin not or D. Carlier M.F. Cell. 1993; 75: 1007-1014Google or independent In contrast, the for CapG can be as in C.L. Southwick F.S. Weber A. Biochemistry. 1990; Scholar, from an of the and the equilibrium data not for the in than in to an on for of CapG of This is an order of magnitude of the for competitive binding is a for the of data for the of depolymerization of filaments with or CapG under of by profilin. a barbed-end capping protein, was to a with actin filaments similar to the with gelsolin, profilin was to augment the depolymerization The of CapZ from barbed ends is L. M. Mol. Biol. Cell. Scholar, Cooper J.A. J. Cell Biol. or of as for gelsolin, the data shown in be to competitive binding by profilin. In other depolymerization rates of filaments by CapZ and profilin were for to and these data not that saturating of profilin the of depolymerization to increase as a function of The of depolymerization were on when filaments were by gelsolin or by CapZ and In contrast to the when capping protein was to with F-actin, the capping protein was with F-actin at the of of profilin the depolymerization In this a with rapid depolymerization was by a to the same observed in the absence of profilin. For both gelsolin and higher of capping protein to actin the and of rapid depolymerization. The for these is that profilin actin the CapZ is but after this profilin depolymerization in that gelsolin and CapZ have similar effects when to F-actin at the same as profilin. for the depolymerization rates in the and absence of profilin are for gelsolin and The depolymerization for gelsolin and CapZ the gelsolin have after of the and capping of gelsolin, in ends from which can dissociate N. A. Eur. J. Biochem. Scholar). and a to the with of Profilin and CapG for the of data are of independent with actin In the data for profilin are for CapG or in however, there is F-actin in the at high concentrations of profilin to with the data are with competitive binding in that profilin concentration the capping effect of lowering the concentration and the of the data are to the concentration both from the loss of capping by CapG and by effects of profilin on the concentration of barbed ends D. Carlier M.F. Cell. 1993; 75: 1007-1014Google Scholar). the of the concentration to profilin nor the of these effects on the of ends have been previously concentrations of profilin capping by CapG and CapG concentrations acceleration of depolymerization by profilin of CapG are that of the data in the or absence of profilin, with competitive of the relatively of profilin, at with profilin is to but the data are with the of competitive a data for at profilin or at CapG can be relatively well using a of binding The a range of experimental is based on that are in the used to these data the of actin from the barbed end in the or absence of profilin These are in with M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar, 4Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Google Scholar, 6Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google the acceleration of depolymerization by profilin was to be greater than for actin M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar). The concentration of filaments, was to this the to by this The for profilin and CapG from the barbed end were and of CapG were used to the data in and with an higher for the CapG used in We not have an for the between but that the to the data relatively in and that data were with the of competitive of Actin by of profilin the of cells have been to range from in cells as cells K. J. T. Goldschmidt-Clermont P.J. Biol. 1997; to in cells as F.S. Young C.L. J. Cell Biol. 1990; 110: Scholar). Furthermore, studies of of P. 2001; Scholar, J. Cell Biol. 2002; as well as P.J. J. 2001; Scholar, T. C. 2001; have the of proteins that can attract and N-WASP to regions of rapid actin filament turnover. of the multiple profilin binding sites on and N-WASP, these protein complexes can attract concentrations of profilin that the millimolar range (13Kang F. Laine R.O. Bubb M.R. Southwick F.S. Purich D.L. Biochemistry. 1997; 36: 8384-8392Google Scholar). profilin functions in the cells in vitro actin kinetics to be at profilin concentrations to be found in the with high concentrations of profilin, however, are profilin concentrations of actin under these conditions. at assembly and experiments using filaments to monomer to barbed filament ends in rapid of the nuclei at these high profilin concentrations μm). that the actin off-rate in the of saturating concentrations of profilin is to that observed in the absence of profilin. This to the of for profilin-actin to that for actin This result was by but could not be using A. actin M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar). We that could not be the on the barbed end of F-actin were depolymerization and However, and that the at a of A. A. Biochemistry. Scholar), higher than the in the or absence of profilin, and the barbed-end be to be under both elongation and depolymerization conditions. In the for the to depolymerization was that the profilin concentration was not high or that the is not for A. The experimental data that profilin can increase depolymerization rates was by H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google for in which free profilin is to the barbed end of filaments, the of profilin-actin from the barbed end has been at to the of actin from filaments M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Google Scholar, 4Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Google Scholar, 6Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Google Scholar). is that the in vivo function of profilin is and profilin may have that augment barbed-end filament both filament assembly and The that a in profilin the of capping protein in R. Miller K.G. Mol. Biol. Cell. 2003; 14: 118-128Google can be as of actin polymerization by profilin. However, in a profilin promotes filament at free barbed filament the absence of profilin may the effects of the loss of capping the of the by filaments are by profilin depolymerization by both and the actin data for filaments by CapG are with between profilin and CapG for the filament with competitive the data for with and CapG at by profilin. other this method of actin filaments the concentration of free actin at effects could explain competitive binding at the barbed end. The binding for gelsolin and of which high to this of gelsolin, from however, both profilin and gelsolin these binding sites of the P.J. 1993; Scholar, U. 1993; Scholar). The effect of gelsolin or CapG can be greater to that of profilin has effects on actin that could binding by gelsolin and CapG S. Bubb M.R. J. Biol. Chem. 2001; Scholar). to competitive binding could be However, the simple of a complex of profilin, CapG and actin at the barbed end is a complex be to similar when profilin to filaments and when CapG to profilin-actin filaments, but this is not the as in by the at saturating concentrations of profilin or of CapG. the data are with a simple model of competitive CapG may with than on an actin filament capping F.S. J. Biol. Chem. Scholar), that the range of with actin monomer and filaments are much greater than in this The model is to be an of the was not observed for CapZ or This is to be the of the rates of both these capping This differential effect of profilin on capping protein function may help to explain the in of actin filament to raising and lowering the profilin concentration in vivo (9Hajkova L. Bjorkegren Sjogren C. Korenbaum E. Nordberg P. Karlsson R. Exp. Cell Res. 1997; 234: 66-77Google Scholar, L.G. Babcock G.G. Rubenstein P.A. Wang Y.L. J. Cell Biol. 1992; 117: 1023-1029Google Scholar, T. Theriot J.A. Dise K.R. Tomaselli G.F. Goldschmidt-Clermont P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1510-1514Google Scholar). The of showing rapid depolymerization of actin by profilin when filaments are into gelsolin or CapZ is At concentrations of filaments and free capping of in L. M. Mol. Biol. Cell. Scholar), the for capping of a filament is than a and no than can be to dissociate capping in the of saturating profilin. the other CapZ gelsolin with filaments in the free concentration of capping proteins may be of magnitude than observed in the and capping of free ends be to filament depolymerization by free profilin. depolymerization of actin may be to extensive of filament P.A. S. J. J. Biochemistry. and, in with actin may actin P.A. S. J. D. A. E. M. J. Biol. Chem. 1994; Scholar), and high concentrations of free profilin could facilitate of actin filament The filament could be depolymerization and to other sites of filament the that filament assembly between filament H.J. Selden L.A. Estes J.E. Gershman L.C. Biochemistry. 1993; Scholar, L.G. Wang Y.L. J. Cell Biol. 1990; Scholar). be in that after profilin in the cell could with other actin regulatory proteins to uncap not but gelsolin and For example, proteins that are for profilin, as VASP, may increase the concentration of profilin to facilitate This is a the function to proteins J.E. M. G.A. Cooper J.A. G.G. Cell. 2002; Scholar). the by profilin not could actin but could to increase actin filament elongation and the of