Filament Assembly from Profilin-Actin

Abstract

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 μm under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 μm under physiological conditions. The PAcovpolymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure. Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 μm under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 μm under physiological conditions. The PAcovpolymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure. Profilin is a remarkable actin-binding protein. This small (15-kDa), ubiquitous, essential protein binds monomeric (G) actin in a 1:1 molar ratio (1Carlsson L. Nyström L.-E. Sundkvist I. Markey F. Lindberg U. J. Mol. Biol. 1977; 115: 465-483Crossref PubMed Scopus (530) Google Scholar). Although the profilin-actin complex is unable to spontaneously nucleate actin filaments, it can productively associate with the barbed ends specifically (2Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (237) Google Scholar, 3Pring M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Crossref PubMed Scopus (106) Google Scholar). This unique property confers a dual function to profilin, depending on the capping of barbed ends. When barbed ends are blocked by capping proteins, profilin sequesters G-actin. In contrast, in motile regions of the cell where uncapped barbed ends are actively elongating, the profilin-actin complex can actively participate in filament growth. The profilin-MgATP-actin complex (PA) 1The abbreviations used are: PA, profilin-MgATP-actin complex; NBD, 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole, EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; S1, myosin subfragment-1. can therefore be considered as an end-specific quasipolymerizable actin monomer. Recent results (4Didry D. Carlier M.-F. Pantaloni D. J. Biol. Chem. 1998; 273: 25602-25611Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) show that this property of profilin is used to enhance the processivity of treadmilling (i.e. steady-state barbed end assembly)in the presence of actin depolymerizing factor. In the mechanism that was proposed based on thermodynamic data (5Pantaloni D. Carlier M.-F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 6Perelroizen I. Didry D. Christensen H. Chua N.-H. Carlier M.-F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar), F-actin assembly from profilin-actin is possible only because of its coupling to ATP hydrolysis, as follows. Profilin-actin associates with the barbed end; the interaction of profilin with actin is weakened once the actin-bound ATP has been hydrolyzed; profilin then dissociates from that end, thus promoting the incorporation of one actin subunit in the filament and regenerating a free barbed end available for further growth from PA. Profilin is reused at each cycle and works as a catalyzer of assembly. While polymerization of actin alone proceeds in a manner uncoupled from ATP hydrolysis (7Pollard T.D. Weeds A.G. FEBS Lett. 1984; 170: 94-98Crossref PubMed Scopus (89) Google Scholar, 8Carlier M.-F. Pantaloni D. Korn E. J. Biol. Chem. 1984; 259: 9983-9986Abstract Full Text PDF PubMed Google Scholar), these two reactions are proposed to occur in a compulsory order when actin filaments grow from profilin-actin units. In this paper, the proposed mechanism of barbed end growth from profilin-actin is challenged by kinetic experiments. Understanding the kinetics of filament assembly from profilin-actin in depth also has implications concerning the structure of the filament. In the atomic model of the actin filament proposed by Holmes et al. (9Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1322) Google Scholar) and refined by Lorenz et al. (10Lorenz M. Popp D. Holmes K.C. J. Mol. Biol. 1993; 234: 826-836Crossref PubMed Scopus (445) Google Scholar), the profilin interaction area on actin is exposed at the barbed end, accounting for the association of profilin-actin to a growing barbed end, but not to a pointed end. Hence, within Holmes' model, it is anticipated that profilin can cap the barbed ends. On the other hand, examination of the actin-actin contacts in the crystals of the profilin-actin complex (11Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C.W. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (600) Google Scholar) shows evidence for a nonhelical ribbon structure of the profilin-actin complex, in which extensive actin-actin contacts, however different from those present in the Lorenz et al. atomic model of the actin filament (10Lorenz M. Popp D. Holmes K.C. J. Mol. Biol. 1993; 234: 826-836Crossref PubMed Scopus (445) Google Scholar) are involved. It was proposed (12Schutt C.E. Rozycki M.D. Myslik J.C. J. Struct. Biol. 1995; 115: 186-198Crossref PubMed Scopus (20) Google Scholar, 13Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (185) Google Scholar) that barbed end growth from profilin-actin could involve the transient extension of profilin-ATP-actin ribbons. The transition from ribbon to filament would be coupled to ATP hydrolysis and dissociation of profilin. According to this model, the profilin-actin ribbon would in only in the and the of the actin in the filament from the ribbon would be different from the Holmes atomic In the present the rate parameters for barbed end from profilin-actin a to under which profilin-actin would spontaneous self-assembly, with the to the profilin-actin The EDC-cross-linked profilin-actin complex to into filaments, which the same helical structure and same thermodynamic as F-actin filaments. Profilin-actin filaments therefore a to the structure of the actin filament and the of actin with other actin-binding was from J.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and as T.D. PubMed Scopus Google Scholar) in 0.2 was by of with 0.2 and μm for and used was by the of and was by the of was Weber A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). from and molar concentration was as J. Biol. Chem. Full Text PDF PubMed Google Scholar) and by by at a by of μm from with in Profilin was from by I. L. Didry D. Carlier M.-F. Biochemistry. PubMed Scopus Google Scholar). and of myosin by Pantaloni D. Carlier M.-F. Biochemistry. PubMed Scopus Google Scholar). at a at the following and and and nm. at at a with and the of filament growth measured used to at the barbed end Cooper J.A. J. Biol. 1996; PubMed Scopus Google Scholar), at the pointed end. in the presence of profilin profilin-actin complex, was with and and the of assembly was The actin that the spontaneous could be The rate of barbed end growth at a concentration of and in the presence of different of profilin was turbidimetrically. as with In is the concentration of and are the of free and and and are the barbed end association rate The of the was in because at actin the critical The as the concentration of profilin was the in the of to barbed end assembly as was by profilin. The concentration of was as where the dissociation constant for of in in μm a critical concentration as M.-F. J. Didry D. Chua N.-H. Pantaloni D. J. Biol. PubMed Scopus Google Scholar), from which the polymerization of μm F-actin to an of units at and to the of as a function of PA. of the data within this to the of the of in the of the in the presence of of PA, by of the to the data at different of profilin. The rate of barbed end growth was also measured at different of and in the in the presence of of profilin (i.e. of an of μm profilin of μm profilin I. Didry D. Christensen H. Chua N.-H. Carlier M.-F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google The for actin and profilin-actin used to the of for actin profilin-actin and of the critical for as in to be that was that of the actin in the was to profilin with that a small of was it would to spontaneous polymerization at actin in the presence of an of profilin. where the spontaneous polymerization measured in the of was from the The of of actin to of profilin to was as T.D. J. Biol. PubMed Scopus Google Scholar, Didry D. Carlier M.-F. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). actin was at 0.2 profilin was in 0.2 to was with and for at and 0.2 then the was to and the was to for it was by The of the was The at the end of the was on a in 0.2 0.2 profilin was in the a with one of with the of free actin and profilin-actin complex was with The was to a in 0.2 0.2 actin was in the and profilin-actin complex was then with 30% to the used for of complex M. Schutt C.E. Lindberg U. PubMed Scopus Google Scholar). The complex was to μm by a in a and at for The complex at this was at as by and to the of The of profilin-actin to with following of the of the profilin-actin complex that from was on This by within cycle of polymerization was as a of the of as follows. The of was with 0.2 and μm for and then with and at The was at for The which of was and for with a The was at for No was The of profilin-actin of profilin-actin complex to of the actin. This was used within a of of present the in the a and of profilin-actin complex a that in a in the different experiments. The concentration of the profilin-actin complex was by the as a Profilin-actin complex in was into PAcov by with and as for and was to by the of and at different of the polymerization of the on to the for the with and observed in a at a on for in to the of diffraction pattern in the presence of the and layer with a a of The filaments and to a to and a than the The is to nm. The of the the filaments was from the which in characterized by the presence of layer lines and which can be by the PAcov complex in was from ATP by Korn Biochemistry. PubMed Scopus Google Scholar), at and into with μm at for at in the in the of and to and of myosin was measured in the presence of and at in a The was by the of of the from the at into and for as M.-F. Pantaloni D. Korn E. J. Biol. Chem. 1984; 259: 9983-9986Abstract Full Text PDF PubMed Google Scholar). PAcov was at μm in the presence of and μm In myosin to a glass as L. A. F. A. J. 1995; Full Text PDF PubMed Scopus Google Scholar). The filaments to in 0.2 with on a a with of filaments with a profilin binds actin FEBS Lett. 1984; PubMed Scopus Google Scholar), the the in of actin as a of polymerization is in its which has to actin polymerization in the of M.-F. J. Didry D. Chua N.-H. Pantaloni D. J. Biol. PubMed Scopus Google Scholar), was The rate of barbed end assembly from was measured at a concentration and in the presence of of profilin. a shows that when filament barbed ends from profilin the growth in a of growth was observed at by profilin. as under and that profilin binds with an dissociation constant of 0.2 μm and that the complex does not participate in barbed end assembly. F-actin was also used to barbed end growth in the presence of μm in the presence of μm profilin. in the of profilin, barbed end but when profilin was with G-actin. of the F-actin was observed within In in the presence of profilin as a protein. In it does not cap the barbed ends. In contrast, when barbed ends from profilin only a of growth. The data are with the that profilin binds with an dissociation constant of and the profilin-MgATP-actin complex associates productively with the barbed ends of actin filaments. by profilin, filaments from profilin-actin units at a rate lower than from No further was observed at concentration of profilin as as capping of the barbed ends by profilin. In in the presence of profilin as a protein. In contrast, the complex of profilin with in barbed end assembly. The different of and are in with the of works (5Pantaloni D. Carlier M.-F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 6Perelroizen I. Didry D. Christensen H. Chua N.-H. Carlier M.-F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar) that the of profilin-actin in barbed end growth ATP hydrolysis to be coupled to a that was for but not for The hydrolysis of ATP polymerization of was measured at a concentration 1:1 complex μm was the of to enhance the rate of polymerization by Filament assembly a by a in light in than ATP was in a manner that from a that is coupled to hydrolysis requires the that the rate of be at concentration by the rate of ATP The rate for profilin-MgATP-actin association to and dissociation from the barbed ends can be from the of the of the rate of filament growth on profilin-actin concentration 8Carlier M.-F. Pantaloni D. Korn E. J. Biol. Chem. 1984; 259: 9983-9986Abstract Full Text PDF PubMed Google Scholar). The for and from growth rate The for the association rate for in with and T.D. J. Biol. PubMed Scopus Google Scholar), and for The rate for and profilin-MgATP-actin dissociation from the barbed ends from the of the of 0.2 and 0.3 for actin and In other when barbed ends are actively from profilin-actin as as from at a rate order of lower than the rate of dissociation of In the present kinetic data a model for barbed end growth from to which actin incorporation and profilin from the barbed end would be coupled to as in In this the dissociation rate constant measured in a of growth is the dissociation rate constant of D. The for profilin-actin would then be a with an and the critical concentration would be order of than the observed of 0.3 In a of of from and that the association of profilin-actin units to barbed ends was by this to be ATP hydrolysis, this to be of the barbed end growth from profilin-actin and was not observed when the from was data with profilin and A. profilin and in When observed in filaments from profilin-actin units a structure to those from actin. No profilin was in the of filaments. to nonhelical of filaments that be at the barbed ends in of growth. ATP hydrolysis is to be uncoupled from F-actin assembly (7Pollard T.D. Weeds A.G. FEBS Lett. 1984; 170: 94-98Crossref PubMed Scopus (89) Google Scholar, M.-F. Pantaloni D. Korn J. Biol. Chem. Full Text PDF PubMed Google Scholar), profilin bound to in a structure (12Schutt C.E. Rozycki M.D. Myslik J.C. J. Struct. Biol. 1995; 115: 186-198Crossref PubMed Scopus (20) Google Scholar). assembly at of profilin-actin observed in the of the No was the and the regions of the filaments from The that the profilin-actin ribbon at the of the growing filament was small to be by be a of that binds to filaments and the in ATP hydrolysis on F-actin Carlier M.-F. J. Biol. Chem. 263: Full Text PDF PubMed Google Scholar), was used to profilin would be to to was in the presence of different of profilin. of the of the profilin bound to In to the incorporation of profilin into the of of a profilin-actin and to the of the actin-actin contacts that can be when profilin bound to actin, a of the profilin-actin complex was and The profilin-actin complex was for its ATP was bound to the complex following on in profilin the property to enhance the rate of in the was with profilin-ATP-actin 1:1 complex in with μm free in the and the in of with the of for bound ATP was of the kinetics on the and in in the presence of μm free the rate of and for the and complex, a rate constant of was measured for on actin under the same conditions. therefore was and on and The that the profilin-actin complex is by also that the property of profilin of is not in the In the complex is to the PAcov was to the of The of spontaneous polymerization of actin PAcov and actin and PAcov The observed when actin and PAcov are could not be as the of the for actin alone and which that PAcov can with The of PAcov on the of filament growth at the barbed and pointed ends this PAcov on the rate of growth at the pointed ends and the rate of assembly by the in of in with the data in capping of barbed ends by these results that with The kinetics of spontaneous polymerization of PAcov at different was by The polymerization in in a by an of a nucleation-growth process to the polymerization of actin The was for than for This is of the of filaments from than from Korn J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the by the polymerization process of with the that of the PAcov the of as observed for F-actin filaments. The of at the end of the polymerization process with the concentration of PAcov in the of PAcov at different in the μm at for when the was The concentration of PAcov present in the was 0.2 μm for these the in μm PAcov was to be at a than the one measured for F-actin M.-F. J. Didry D. Chua N.-H. Pantaloni D. J. Biol. PubMed Scopus Google Scholar), that the of the PAcov that light was than the of the actin filament. critical concentration was from of the of PAcov present in the of by of a of μm by at in that PAcov with a critical concentration of 0.2 The ATP bound to actin in the PAcov complex was the polymerization of When μm PAcov was in the presence of was in the of the at the end of the polymerization μm in the that In the polymerization of PAcov with the polymerization of The structure of the PAcov was by of of PAcov at a of filaments. The ends of the that the filaments that the in a The kinetics of of these was in and at a concentration of PAcov for the different of and growth to be of the at different of the polymerization process in that the only filaments which with each other in of filaments on the at the end of the 6 when the in was than of the with The of the PAcov filaments layer lines at of 36 and 6 to the and layer lines of These results that PAcov into helical filaments that have the same helical as of the observed at of assembly show an results from the in of filaments to profilin, which is a protein. filaments that a each in of can the filaments and J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The exposed at the of profilin also contacts the PAcov filaments and the of was extensive at ionic the from the of actin, in a the actin from the at the end of the PAcov and was with 30% and the that this at the same rate and to the same as actin and did not not of phalloidin to the PAcov filaments was by the in of W. 1992; PubMed Scopus Google J. Biochemistry. 1996; PubMed Scopus Google Scholar). The for of to μm F-actin that the of phalloidin is in the two to the of profilin to actin the and its The of myosin to filaments and PAcov filaments in the of ATP was in in a in 6 show that binds to the was lower than the 1:1 for to of was bound PAcov In the filaments from by to the by The myosin to the PAcov filament with No diffraction pattern could be from the not In an ionic is to the of by into filaments. These two reactions can be by light Pantaloni D. Carlier M.-F. Biochemistry. PubMed Scopus Google Scholar, Carlier M.-F. Pantaloni D. Biochemistry. Scopus Google Scholar). those ionic assembly process could be within when at to μm was to 1:1 complex in In the with 1:1 complex and the polymerization was in On the other hand, the of was by PAcov filaments. The rate of ATP hydrolysis, under different ionic of was lower than the ATPase rate measured in the presence of the same of F-actin in a The of actin and PAcov filaments myosin-coated glass in While actin filaments at a of the profilin-actin filaments and to the myosin-coated actin and PAcov as as actin filaments with an of and a PAcov at the same and the same motile as filaments. these data but not that the in the profilin-actin but to the presence of profilin the of the myosin which is for its along actin filaments. the that to and from PAcov that with PAcov as it does with G-actin. physiological ionic filaments actively from profilin-actin at a rate that is only lower than from These results are in with an M. T.D. J. Biol. PubMed Scopus Google Scholar), which and the as evidence for a capping of profilin at Profilin is not incorporated into the filaments, in the at with the from a data show that filament growth from coupled to ATP hydrolysis, is not coupled to for this is by the critical concentration for barbed end assembly from profilin-actin and of the to a of the dissociation rate which is with the presence of at the barbed ends in a of growth. In the proposed growth from profilin-actin and ATP hydrolysis are and a M.-F. Pantaloni D. J. Biol. Chem. 263: Full Text PDF PubMed Google Scholar) the of at the ends of barbed ends. The of the at profilin-actin concentration both and is within this to ATP hydrolysis, which profilin and filament growth. The rate of ATP hydrolysis would be under physiological ionic and would be with the measured for the association rate constant a of the association rate constant is M.-F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), then the ATPase rate would be at lower ionic a of of not This is in with results that the rate of hydrolysis filament a of in the presence of M.-F. Pantaloni D. Korn J. Biol. Chem. Full Text PDF PubMed Google Scholar). The profilin-actin complex to be considered as a with a critical concentration of 0.3 This in of of actin filaments in profilin J.A. U. A. PubMed Scopus Google Scholar). When filaments are at in the presence of profilin, the critical of free and profilin-actin can be from the of the of and F. of the of Scholar) by following where and are the critical for polymerization of and is the concentration of and is the dissociation constant of the profilin-actin the from μm to and from to 0.3 μm This is with results (5Pantaloni D. Carlier M.-F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (458) Google Scholar). The profilin does not cap the barbed ends to The profilin-actin complex a to the structure of the actin filament. The a of by EDC, and an of profilin is to be the of the of actin and of profilin in the According to Schutt et al. (12Schutt C.E. Rozycki M.D. Myslik J.C. J. Struct. Biol. 1995; 115: 186-198Crossref PubMed Scopus (20) Google Scholar), this to a of actin and of profilin in the profilin-actin The profilin-actin complex the of the profilin-actin its assembly the two current models of actin assembly of Holmes et al. (9Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1322) Google Scholar, M. Popp D. Holmes K.C. J. Mol. Biol. 1993; 234: 826-836Crossref PubMed Scopus (445) Google Scholar) and of Schutt et al. (11Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C.W. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (600) Google Scholar), two the model of the the profilin-actin complex was to be unable to to and profilin along the On the other hand, PAcov was to cap the barbed ends of actin filaments. the profilin-actin complex was to into a nonhelical ribbon The not the from The data show that the profilin-actin does not cap the actin filaments and with actin. the EDC-cross-linked profilin-actin complex into filaments that the same helical and thermodynamic as F-actin filaments, that the actin-actin contacts are in F-actin and in and that the of the profilin-actin by the does not F-actin assembly. This is also supported by the kinetics of phalloidin to F-actin and and by the of the myosin to the profilin-actin filament. In order to of the data within Holmes' model, that as as ATP is following profilin-actin association to the barbed end, the of profilin for actin by of When the two are the in the profilin to the filament the in the of the association of a to the barbed end. In this the to of by in and The in of profilin for actin following of ATP is a that has to be to the present data within Holmes' model, in to for the critical concentration for PAcov assembly. It would profilin does not cap the barbed ends. It that the of ATP a of and which does not in the atomic model from the structure of This to be challenged by further experiments. The in of profilin for actin also be by the of profilin following the this the of profilin-actin of to be in the PAcov complex, that profilin is not possible of which would be with Holmes' model, is that both the and the actin-actin that the F-actin filament are polymerization of the In the of the in of profilin for F-actin would not the profilin to bound to This also for the of profilin to cap the barbed but it that the of actin in the filament is different from both the Holmes and Schutt of the structure of the and are under to those