Single Molecule Kinetic Analysis of Actin Filament Capping

We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 × 106 m-1 s-1 and dissociation of 0.0003 s-1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), PI(4,5)P2, and PI(3,4,5)P3, prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 × 105 m-1 s-1) and a faster dissociation rate constant (0.004 s-1). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament. We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 × 106 m-1 s-1 and dissociation of 0.0003 s-1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), PI(4,5)P2, and PI(3,4,5)P3, prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 × 105 m-1 s-1) and a faster dissociation rate constant (0.004 s-1). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament. The motility of animal cells depends on the coordinated assembly of actin filaments at the leading edge. Arp2/3 complex promotes actin polymerization by nucleating filaments that grow as branches from a mother filament (1Svitkina T.M. Borisy G.G. J. Cell Biol. 1999; 145: 1009-1026Crossref PubMed Scopus (898) Google Scholar, 2Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1018) Google Scholar). Capping protein (CP, 3The abbreviations used are: CPcapping proteinTIRFtotal internal reflection fluorescencePPIpolyphosphoinositidePIphosphatidylinositolPIP2phosphatidylinositol 4,5-bisphosphateOGOregon Green 488MmCPMus musculus CPSpCPS. pombe CPHMMhidden Markov modelMILmaximum interval likelihoodddH2Odouble distilled H2O. 3The abbreviations used are: CPcapping proteinTIRFtotal internal reflection fluorescencePPIpolyphosphoinositidePIphosphatidylinositolPIP2phosphatidylinositol 4,5-bisphosphateOGOregon Green 488MmCPMus musculus CPSpCPS. pombe CPHMMhidden Markov modelMILmaximum interval likelihoodddH2Odouble distilled H2O. called CapZ in muscle) appears to play an important role in generating the zone of short, highly branched actin filaments at the leading edge. CP is a heterodimer of structurally similar α and β subunits that form a mushroom-like structure with one flexible and one immobile COOH-terminal “tentacle” that bind the two terminal subunits of an actin filament (3Yamashita A. Maeda K. Maeda Y. EMBO J. 2003; 22: 1529-1538Crossref PubMed Scopus (123) Google Scholar). CP binds the barbed end of actin filaments tightly with a Kd of 0.1–1 nm. Given an association rate constant of 3–4 μm-1 s-1 and a cytoplasmic concentration of 1 μm (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar), CP should terminate the growth of actin filaments with a half-time of about 0.2 s after their nucleation. Terminating growth quickly keeps the filaments short, so that they do not buckle under the force of polymerization as they push the membrane forward. capping protein total internal reflection fluorescence polyphosphoinositide phosphatidylinositol phosphatidylinositol 4,5-bisphosphate Oregon Green 488 Mus musculus CP S. pombe CP hidden Markov model maximum interval likelihood double distilled H2O. capping protein total internal reflection fluorescence polyphosphoinositide phosphatidylinositol phosphatidylinositol 4,5-bisphosphate Oregon Green 488 Mus musculus CP S. pombe CP hidden Markov model maximum interval likelihood double distilled H2O. Vertebrate CP dissociates from barbed ends with a half-time of 30 min (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar), far too slow for simple, spontaneous dissociation to be involved with the conversion of the network of short, branched filaments at the leading edge to longer, unbranched filaments toward the cell body. As CP prevents both dissociation of subunits from the barbed end and end-to-end filament annealing, some mechanism must accelerate the removal of CP from filament ends. Both lipids and proteins, such as Ena/VASP (5Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 6Svitkina T.M. Bulanova E.A. Chaga O.Y. Vignjevic D.M. Kojima S. Vasiliev J.M. Borisy G.G. J. Cell Biol. 2003; 160: 409-421Crossref PubMed Scopus (576) Google Scholar, 7Barzik M. Kotova T.I. Higgs H.N. Hazelwood L. Hanein D. Gertler F.B. Schafer D.A. J. Biol. Chem. 2005; 280: 28653-28662Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) and CARMIL (8Uruno T. Remmert K. Hammer J.A. J. Biol. Chem. 2006; 281: 10635-10650Abstract Full Text Full Text PDF PubMed Scopus Google Scholar), inhibit the of CP with actin bind CP and block with filaments both in A. Cell. Full Text PDF PubMed Scopus Google Scholar) and in Cooper J.A. PubMed Scopus Google Scholar). of to of CP, and actin in the rate of polymerization (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar). to the that PPIs not inhibit association of CP with barbed ends but dissociate CP from actin two to the growth of actin filament branches toward the of the membrane at the leading edge J. J. Cell Biol. PubMed Scopus Google Scholar, I.V. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). would be the for PPIs to block of CP with filament ends. of three residues a site the affinity of CP for and barbed ends K. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the binding on CP for the barbed end and dissociation of CP from a barbed end by would an the of from a used total internal reflection fluorescence to capping and of filaments in We association and dissociation rate constants for CP in with measured in bulk assays. We that PI(4,5)P2, and CP and prevent binding to barbed ends. none of these PPIs the dissociation of CP from yeast CP Cooper J.A. J. Cell Biol. PubMed Scopus Google Scholar), that PPIs do not inhibit binding of fission yeast CP to filament ends, to yeast and of an to an for an site by the surface of homology models of that in their to bind We actin from muscle one of and Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). with T.D. J. Cell Biol. PubMed Scopus Google Scholar) with Oregon Green 488 and with by and Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). and 0.2 1 and at × for at mouse capping protein and subunits on a single and from S. Cooper J.A. J. Cell Biol. PubMed Scopus Google Scholar). We fission yeast capping protein subunits and on a single Pollard T.D. Biol. Cell. 2005; PubMed Scopus Google Scholar). Capping proteins CP 1 in and at We the of a by a of concentrations with actin and the rate of actin assembly from Cooper J.A. PubMed Scopus Google Scholar). less than from muscle PubMed Scopus Google Scholar) with Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and at in 1 and We protein concentrations from the m-1 K. PubMed Scopus Google m-1 S. Cooper J.A. J. Cell Biol. PubMed Scopus Google m-1 Pollard T.D. Biol. Cell. 2005; PubMed Scopus Google m-1 S. PubMed Scopus Google Scholar). total actin concentration from the × m-1 and actin from m-1 Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). total actin concentration from the × m-1 and actin from m-1 T. K. J. PubMed Scopus Google Scholar). from in under and at CP to an of to a concentration of at for to and for min in a and an 1 min to form and as cells Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We cell for min with 0.2 μm in a of for 1 min with in and in We and to the at the with 1 and on for min to form concentration of We of at the of a and on the of concentrations of CP and of concentrations of and at for the both with of 30 and a similar that of for when filaments with CP dissociation the ends with to filament because of on for a total of with and on for min by with for a concentration of at for and used μm as for in of and for s on maximum We used 1 min after from after to a of and for and used in from μm to by with on for of and with for a of and a concentration of μm and μm fluorescence the fluorescence with at and at nm. of and from the by a constant (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar). that the concentration of at the of the and the polymerization rate constant for the used a of the s of polymerization to fluorescence at s and this actin polymerization that a used to the fluorescence for a concentration of and for We these to and polymerization from the of a of polymerization rate constants of μm-1 s-1 and s-1 T.D. J. Cell Biol. PubMed Scopus Google Scholar), the concentration of ends as for 30 min with a of with for 30 min in and with with in with for 1 and with with for 1 with to 1 in in and with PubMed Scopus Google Scholar). to μm by with on for an with for 1 min to barbed ends. We the with for min to barbed ends. from to a concentration of μm with on for of of μm actin and of μm of a large and the for min the of of μm to and filaments in and to a and with an We an for Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We and with for of at the surface as Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We filaments and a for Pollard T.D. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). maximum a to the filament to a filament and total the filament at 1 along this and both ends and as a single with by We for filament and growth in a to subunits J. Biol. PubMed Scopus Google Scholar). CP association CP to filaments with a of the barbed end growth of a filament measured the from the in filament to the end of the filament The of this by the measured growth the and capping, CP dissociation CP from filaments with of with a of barbed end growth of the filament and the from the measured and and the measured growth rate as We the affinity of for from the in the association rate constant in the of that binding CP by the of to total CP, from the in the association rate in with the association as The affinity of CP for from × × hidden Markov of measured of filaments A. Chem. Scopus Google Scholar) growth at growth and these growth as of the hidden used on A. J. 1996; Full Text PDF PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar), for and to rate constants for capping and We a simple, model for capping protein binding to filament barbed ends of 1 interval likelihood of the of of a for the from an to a and a rate for the CP the of toward the and to be in the of CP the of capping and in both and the two to form a single rate similar to the rate for a bulk to rate less to than the rate The association and dissociation rate and from the and of the rate concentration in single from an three as and We CP the PubMed Scopus Google Scholar) and to T. J. 2003; PubMed Scopus Google Scholar) for and of homology models on the structure of (3Yamashita A. Maeda K. Maeda Y. EMBO J. 2003; 22: 1529-1538Crossref PubMed Scopus (123) Google Scholar). We surface with the D. S. J.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) at and and of T.D. D.M. J. Chem. PubMed Scopus Google Scholar) and for values of and of Capping and Capping by Capping the growth and capping of actin filaments by muscle with Oregon Green a of filaments in μm and after the of capping protein to the of filaments growth that by capping The of filaments in a an with As slow a in capping the min of the capping in these to the of to an The association rate on concentration a an association rate constant of 2.6 μm-1 s-1 for binding to filament barbed ends is similar to in bulk of μm-1 s-1 for CP (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar) and μm-1 s-1 for CP A. K. Maeda Y. Cooper J.A. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in capping one two growth of a filaments because of surface binding The of these of this Capping protein rate and of CP from filament barbed ends, from CP, Schafer (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google of CP from filament barbed ends, from in a capping protein from filament barbed ends when CP We the of growth as dissociation of filaments at a constant rate but a and The of filaments a dissociation rate constant of s-1 of The of the dissociation and association rate constants a Kd of similar to with bulk of for capping protein (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar, A. K. Maeda Y. Cooper J.A. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of on Capping and by Capping bind capping proteins and inhibit capping of barbed ends in bulk Cooper J.A. PubMed Scopus Google Scholar). of actin filaments that μm concentrations of of three different PPIs inhibit the rate of capping a of capping protein concentrations The of the capping rate on concentration the in and with μm but in in PI(4,5)P2, and in that bound to bind filaments and that these because of would dissociation constants for PPIs of to μm capping protein association and dissociation with association and dissociation association and dissociation of CP from polyphosphoinositide from the in by μm μm μm μm μm μm μm μm association and dissociation of CP from polyphosphoinositide from the in by in a such as to inhibit binding to CP J. Biol. Chem. Full Text PDF PubMed Google Scholar) concentrations the association of CP with barbed ends slower in and than at concentrations and slower in concentrations the affinity of CP for PPIs to CP association with filaments slower in than in under that the the to the play some role in We used bulk to the concentration of barbed ends for by actin in the polymerization rate on the concentration of ends, that of to bulk of actin filaments the concentration of ends Cooper J.A. PubMed Scopus Google Scholar, D.A. Cooper J.A. J. Cell Biol. PubMed Scopus Google Scholar). faster in and than in filaments The of the is that and dissociation of capping protein from barbed ends. dissociation would to be the of a after the PPIs to filaments in both the published and in none of the PPIs filaments in the and PI(4,5)P2, the of dissociation in in these assays. Given this the bulk and the that filaments bulk and that PPIs prevent by CP. with PPIs in bulk and with large force would filaments of in a network S. J. D. A. M. J. Biol. Chem. Full Text PDF Google Scholar). this the of PPIs on of and to a similar concentration of ends and The of μm on the rate of measured with actin in bulk filaments to be in than μm when by fluorescence The filaments in these measured μm The of the filament by actin μm by and μm by the We used with μm actin and to the concentrations of barbed ends capping protein the rate of of both and and the concentrations of ends μm with on the rate of with PPIs the rate and the concentration of barbed ends by by PI(4,5)P2, and by PI(3,4,5)P3, a of the in ends and with PI(4,5)P2, the rate a of the in ends and As these that longer, to than and that of barbed ends is because of filament and of CP by PPIs than the in bulk polymerization assays similar in and and because the concentration not in at 1 We the by of filaments with We filament with a of by capping by We these polymerization with a of to barbed end min and the filaments with filaments a of ends, and barbed ends, filaments ends and barbed ends. these the of and filament in and capping of filaments an capping the of filaments to concentrations of not not not the filaments min of the in barbed ends in bulk is to be to of Capping with from muscle in the of fission yeast capping protein of and and The capping end by dissociation of from barbed ends by to by so not association because capping protein binding to filaments is a Markov used growth of filaments and hidden Markov to the of filament at along with the association and dissociation of at filament barbed ends. We the of filaments to growth and their but by subunits from the because of filament We the of the growth for filament to the growth rate at growth the for the and the and in the rate of We used the T. Google Scholar) to to rate on the and of We of growth capping from filaments to the and of growth for The interval used these along with an of the association and dissociation rate constants to the hidden capping of filament maximum interval likelihood used the of and of from to to the of these two these rate the interval to the by the to rate We this the rate constants The rate by to the concentration of an association rate constant of μm-1 s-1 and a dissociation rate constant of s-1 for a binding affinity of nm. dissociation in for min with and capping protein by with We dissociation from the of filament growth as in and a single dissociation rate constant of s-1 of capping μm not the rate of association of with actin filaments We the of on dissociation by filaments with and of the with actin and μm The of the of by the of two as a dissociation of s with an of and a slow dissociation of min with an of that filament at the of the but and from The dissociation filaments that not the As a slow single dissociation in the slow dissociation the dissociation rate constant for in the of capping not fission yeast capping protein dissociation when measured and pombe capping protein rate and dissociation rate is for the slow of the double Markov dissociation Markov association rate is dissociation Markov association rate is rate with with μm with μm with μm with μm dissociation rate is for the slow of the double Markov dissociation Markov association rate is dissociation Markov association rate is in a to in of of PPIs on pombe CP an of to on We homology models of capping proteins from on the structure of CP (3Yamashita A. Maeda K. Maeda Y. EMBO J. 2003; 22: 1529-1538Crossref PubMed Scopus (123) Google Scholar) and a large of on the β and that is in fission yeast large and in yeast not large basic patch as a site on of the CP J. Biol. 2006; PubMed Scopus Google Scholar) and on the surface that with the barbed end of an actin filament (3Yamashita A. Maeda K. Maeda Y. EMBO J. 2003; 22: 1529-1538Crossref PubMed Scopus (123) Google Scholar, A. K. Maeda Y. Cooper J.A. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is a to to basic patch is to but from a of basic residues in the of fission yeast CP. of three of the basic residues in the the affinity of CP for about K. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). observing capping and of actin filaments with association and dissociation rate constants for mouse CP similar to bulk polymerization assays (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar). polymerization of bulk to a model of capping from filament annealing, and end so these must be in bulk capping We agree with Schafer (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar) that PPIs to filaments in a bulk polymerization the PPIs not actin filaments in of assays. for an to these We that filaments in bulk barbed ends. the of CP, these barbed ends PPIs the capping the of by and not in the bulk (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar), because they with filaments of subunits D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). from the actin concentration and capping rate that of the filaments at μm at μm and of filaments and μm at the that Schafer (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar) in their this to in bulk to for must filament with CP from removal of CP from filament ends. assays to the that Ena/VASP (5Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 7Barzik M. Kotova T.I. Higgs H.N. Hazelwood L. Hanein D. Gertler F.B. Schafer D.A. J. Biol. Chem. 2005; 280: 28653-28662Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) and CARMIL (8Uruno T. Remmert K. Hammer J.A. J. Biol. Chem. 2006; 281: 10635-10650Abstract Full Text Full Text PDF PubMed Scopus Google Scholar) capping protein from so the of these important CP should be at a single filament We that and inhibit capping in (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar, Cooper J.A. PubMed Scopus Google Scholar) at the single filament but Schafer (4Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar), a of capping by a of with and that in capping, the dissociation constants for binding and in the of μm in 1 and about in K. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) investigated by PPIs microscopy. filaments and actin the and the of filaments at μm the concentration that to filaments in their bulk of filaments after the half-time for CP in the of of filaments min after CP on their dissociation rate constant of × of filaments should of filaments might be a of CP in the to barbed ends. faster than the CP dissociation half-time of min at μm As filament because of of filaments and in assays filaments in the of filament must be to growth because of filament than at concentrations of is that the CP and of filaments in their after the to an surface that block filament growth CP dissociation because of binding of filament ends to the concentrations of lipids as an barbed ends to grow as they As filaments to their cells in the of K. D. Cooper J.A. J. Biol. Chem. 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PubMed Scopus Google Scholar). and actin filaments PubMed Scopus Google Scholar), but for CP. with CP the barbed end the of CP some filaments filament but not filament capping protein a of these be because of spontaneous by on Capping pombe CP the the to binds the barbed end of actin filaments slower and dissociates faster than mouse CP and is the CP that is not by a to the of these binding S. pombe CP a at than mouse CP so from actin and PPIs to the both the along the actin-binding A. S. A. Maeda Y. EMBO J. 2006; PubMed Scopus Google Scholar) surface large and on the actin-binding on than The of these is an is one of the as a site by a of CP structure J. Biol. 2006; PubMed Scopus Google Scholar). the of this basic patch in S. pombe CP the of binding of to this basic patch would sterically actin binding to the barbed end of an actin filament would binding of to this for their to dissociate from actin of mouse CP K. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) an site by three and the of three residues the affinity of CP for in a fluorescence and in an the affinity of the CP for filament barbed ends of and barbed ends for CP at this these in fission yeast and less to the of binding of the large basic patch and by J. Biol. 2006; PubMed Scopus Google Scholar) would be (3Yamashita A. Maeda K. Maeda Y. EMBO J. 2003; 22: 1529-1538Crossref PubMed Scopus (123) Google Scholar) site residues and of the β bind two in the structure of CP. Given the of these residues the from the actin-binding A. S. A. Maeda Y. EMBO J. 2006; PubMed Scopus Google Scholar), binding at this site would to to CP two of these three residues in both yeast and for the of binding in S. pombe CP. of one of these to on binding K. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the site be as a is about the phosphatidylinositol and the actin in Both fission and yeast in fission yeast and in the of these to actin and in actin patch but not patch actin in at the Given that the of with actin and that the but not the of actin is that than on CP in The association rate constant for with the of actin patch and s Pollard T.D. J. Cell Biol. 2005; PubMed Scopus Google Scholar) and about one CP for two Arp2/3 Pollard T.D. 2005; PubMed Scopus Google Scholar). about of barbed ends at in a cytoplasmic concentration of μm Pollard T.D. 2005; PubMed Scopus Google Scholar) and an association rate constant of μm-1 the half-time for capping would be is a too slow to of the filaments the growth so the in the The half-time for dissociation of from filaments is to barbed ends from polymerization the and some mechanism must for the with