Tropomodulin caps the pointed ends of actin filaments

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.     

Electron microscopic determination of the actin filament end at which cytochalasin B blocks monomer addition using the acrosomal actin bundle from horseshoe crab sperm

G-actin freed from exogenous ATP was added to the pieces of isolated acrosomal actin bundles from horseshoe crab sperm to form filaments as reported earlier (Tilney, L.G., Bonder, E.M., & DeRosier, D.J. (1981) J. Cell Biol. 90, 485-494). The growth of a filament was far more rapid at one end (the preferred end) than the other end. These ends were shown to correspond to the barbed and pointed ends, respectively, by decoration of the filaments with myosin subfragment 1. Cytochalasin B inhibited the monomer addition at the preferred end. This technique is useful in determining the ends to which actin filament end-binding proteins from nonmuscle cells bind, which are considered to regulate the actin polymerization in the cells.     

Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.     

How capping protein binds the barbed end of the actin filament

Cytoskeletal filaments are often capped at one end, regulating assembly and cellular location. The actin filament is a right-handed, two-strand long-pitch helix. The ends of the two protofilaments are staggered in relation to each other, suggesting that capping could result from one protein binding simultaneously to the ends of both protofilaments. Capping protein (CP), a ubiquitous alpha/beta heterodimer in eukaryotes, tightly caps (K(d) approximately 0.1-1 nM) the barbed end of the actin filament (the end favored for polymerization), preventing actin subunit addition and loss. CP is critical for actin assembly and actin-based motility in vivo and is an essential component of the dendritic nucleation model for actin polymerization at the leading edge of cells. However, the mechanism by which CP caps actin filaments is not well understood. The X-ray crystal structure of CP has inspired a model where the C termini ( approximately 30 amino acids) of the alpha and beta subunits of CP are mobile extensions ("tentacles"), and these regions are responsible for high-affinity binding to, and functional capping of, the barbed end. We tested the tentacle model in vitro with recombinant mutant CPs. Loss of both tentacles causes a complete loss of capping activity. The alpha tentacle contributes more to capping affinity and kinetics; its removal reduces capping affinity by 5000-fold and the on-rate of capping by 20-fold. In contrast, removal of the beta tentacle reduced the affinity by only 300-fold and did not affect the on-rate. These two regions are not close to each other in the three-dimensional structure, suggesting CP uses two independent actin binding tentacles to cap the barbed end. CP with either tentacle alone can cap, as can the isolated beta tentacle alone, suggesting that the individual tentacles interact with more than one actin subunit at a subunit interface at the barbed end.     

Polymerization and depolymerization of actin with nucleotide states at filament ends

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.     

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G- actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G- actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.     

Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms

Actin binding proteins control actin assembly and disassembly by altering the critical concentration and by changing the kinetics of polymerization. All of these control mechanisms in some way or the other make use of the energy of hydrolysis of actin-bound ATP. Capping of barbed filament ends increases the critical concentration as long as ATP hydrolysis maintains a difference in the actin monomer binding constants of the two ends. A further increase in the critical concentration on adding a second cap, tropomodulin, to the other, pointed filament end also requires ATP hydrolysis as described by the model presented here. Changes in the critical concentration are amplified into much larger changes of the monomer pool by actin sequestering proteins, provided their actin binding equilibrium constants fall within a relatively narrow range around the values for the two critical concentrations of actin. Cofilin greatly speeds up treadmilling, which requires ATP hydroysis, by increasing the rate constant of depolymerization. Profilin increases the rate of elongation at the barbed filament end, coupled to a lowering of the critical concentration, only if ATP hydrolysis makes profilin binding to the barbed end independent of its binding constant for actin monomers.     

Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by alpha- and beta-isoactins

The polymerization of scallop β-like actin is significantly slower than that of skeletal muscle α-actin. To reveal which steps of polymerization contribute to this difference, we estimated the efficiency of nucleation of the two actins, the rates of filament elongation at spontaneous and gelsolin-nucleated polymerization and the turnover rates of the filament subunits at steady-state. Scallop actin nucleated nearly twice less efficient than rabbit actin. In actin filaments with free ends, when dynamics at the barbed ends overrides that at the pointed ends, the relative association rate constants of α- and β-actin were similar, whereas the relative dissociation rate constant of β-ATP-actin subunits was 2- to 3-fold higher than that of α-actin. The 2- to 3-fold faster polymerization of skeletal muscle versus scallop Ca-actin was preserved with gelsolin-capped actin filaments when only polymerization at the pointed end is possible. With gelsolin-induced polymerization, the rate constants of dissociation of ATP-actin subunits from the pointed ends were similar, while the association rate constant of β-actin to the pointed filament ends was twice lower than that of α-actin. This difference may be of physiological relevance for functional intracellular sorting of actin isoforms.     

ATP hydrolysis by the gelsolin-actin complex and at the pointed ends of gelsolin-capped filaments

To obtain kinetic information about the pointed ends of actin filaments, experiments were carried out in the presence of gelsolin which blocks all events at the kinetically dominant barbed ends. The 1:2 gelsolin-actin complex retains 1 mol/mol of actin-bound ATP, but it neither hydrolyzes the ATP nor exchanges it with ATP free in solution at a significant rate. On the other hand, the actin filaments with their barbed ends capped with gelsolin hydrolyze ATP relatively rapidly at steady state, apparently as a result of the continued interaction of ATP-G-actin with the pointed ends of the filaments. ATP hydrolysis during spontaneous polymerization of actin in the presence of relatively high concentrations of gelsolin lags behind filament elongation so that filaments consisting of as much as 50% ATP-actin subunits are transiently formed. Probably for this reason, during polymerization the actin monomer concentration transiently reaches a concentration lower than the final steady-state critical concentration of the pointed end. At steady state, however, there is no evidence for an ATP cap at the pointed ends of gelsolin-capped filaments, which differs from the barbed ends which do have an ATP cap in the absence of gelsolin. As there is no reason presently to think that gelsolin has any effect on events at the pointed ends of filaments, the properties of the pointed ends deduced from these experiments with gelsolin-capped filaments are presumably equally applicable to the pointed ends of filaments in which the barbed ends are free.     

Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process

We have re-examined the Ca(++)-dependent interaction of an intestinal microvillar 95- kdalton protein (MV-95K) and actin using the isolated acrosomal process bundles from limulus sperm. Making use of the processes as nuclei for assembling actin filaments, we quantitatively and qualitatively examined MV-95K’s effect on filament assembly and on F- actin, both in the presence and in the absence of Ca(++). The acrosomal processes are particularly advantageous for this approach because they nucleate large numbers of filaments, they are extremely stable, and their morphology can be used to determine the polarity of any nucleated filaments. When filament nucleation was initiated in the presence of MV-95K and the absence of Ca(++), there was biased filament assembly from the bundle ends. The calculated elongation rates from both the barbed and pointed filament ends were virtually indistinguishable from control preparations. In the presence of Ca(++), MV-95K completely inhibited filament assembly from the barbed filament end without affecting the initial rate of assembly from the pointed filament end. The inhibition of assembly results from MV-95K binding to and capping the barbed filament end, thereby preventing monomer addition. This indicates that, while MV-95K is a potent nucleator of actin assembly, it is also a potent inhibitor of actin filament elongation. To examine the effects of MV-95K on F-actin in the presence of Ca(++), we developed an assay where MV-95K is added to filaments previously assembled from acrosomal processes without causing filament breakage during mixing. These results clearly demonstrated that rapid filament shortening by MV-95K results through a mechanism of disrupting intrafilament monomer-monomer interactions. Finally, we show that tropomyosin-containing actin filaments are insensitive to cutting, but not to capping, by MV-95K in the presence of Ca(++).     

Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin.

Growing evidence suggests that the nucleotide bound to actin filaments serves as a timer to control actin filament turnover during cell motility (Pollard, T. D., Blanchoin, L., and Mullins, R. D. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 545-576). We re-examined the hydrolysis of ATP by polymerized actin using mechanical quenched-flow methods to improve temporal resolution. The rate constant for ATP hydrolysis by polymerized Mg actin is 0.3 s(-1), 3-fold faster than that measured manually. The ATP hydrolysis rate is similar when Mg ATP actin elongates either the pointed end or the barbed end of filaments. Polymerized Ca actin hydrolyzes ATP at 0.05 s(-1). Mg ATP actin saturated with profilin can elongate barbed ends at >60 s(-1), 2 orders of magnitude faster than ATP hydrolysis (0.3 s(-1)). Given that profilin binds to a surface on actin that is buried in the Holmes model of the actin filament, we expect that profilin will block subunit addition at the barbed end of a filament. Profilin must move from this site at rates much faster than it dissociates from monomers (4 s(-1)). ATP hydrolysis is not required for this movement.     

Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and ARP2/3 complex.

The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 micrometer. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.     

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.     

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.     

Capping one end of an actin filament affects elongation at the other end

The rates of elongation at the free ends of actin filaments were compared to those of intact filaments, when the one end was masked with muscle beta-actinin or cytochalasin D, using fixed actoheavy meromyosin and Limulus acrosomal actin bundles as seeds. Experimental conditions were chosen so as to prevent spontaneous filament formation as far as possible. The rate of elongation at the barbed end of fixed actoheavy meromyosin was reduced to about one-fourth when the other pointed end was capped by beta-actinin, and that at the pointed end was reduced to one-third when the barbed end was blocked by cytochalasin D. Similar effects were also observed with the packed actin bundles of horseshoe crab sperm, although the decreases in elongation were less marked: 50-60% of the control both in the presence of beta-actinin and cytochalasin D. To explain the peculiar "end effect" described above, it is proposed that possible conformational changes at one end of an actin filament caused by the binding of a capping substance are transmitted successively to the other end so as to affect the elongation there.     

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.     

Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).     

Tropomodulins: life at the slow end

Dynamic exchange of actin monomers at filament ends is crucial for the functional architecture of many cytoskeletal-dependent processes. Recent evidence indicates that tropomodulins (Tmods), a conserved family of actin-capping proteins that bind to the pointed (slow-growing) end of actin filaments, regulate a variety of actin structures, including dynamic actin networks found in some motile cells. Actin structures that are more stable, such as sarcomeric thin filaments, require capping by Tmods to specify filament lengths and to provide filament stability. Here, we discuss the functional differences between the capping of pointed and barbed ends within the context of these actin-filament systems, and how Tmods uniquely contribute to their regulation and organization.     

How ATP hydrolysis controls filament assembly from profilin-actin: implication for formin processivity

Formins catalyze rapid filament growth from profilin-actin, by remaining processively bound to the elongating barbed end. The sequence of elementary reactions that describe filament assembly from profilin-actin at either free or formin-bound barbed ends is not fully understood. Specifically, the identity of the transitory complexes between profilin and actin terminal subunits is not known; and whether ATP hydrolysis is directly or indirectly coupled to profilin-actin assembly is not clear. We have analyzed the effect of profilin on actin assembly at free and FH1-FH2-bound barbed ends in the presence of ADP and non-hydrolyzable CrATP. Profilin blocked filament growth by capping the barbed ends in ADP and CrATP/ADP-Pi states, with a higher affinity when formin is bound. We confirm that, in contrast, profilin accelerates depolymerization of ADP-F-actin, more efficiently when FH1-FH2 is bound to barbed ends. To reconcile these data with effective barbed end assembly from profilin-MgATP-actin, the nature of nucleotide bound to both terminal and subterminal subunits must be considered. All data are accounted for quantitatively by a model in which a barbed end whose two terminal subunits consist of profilin-ATP-actin cannot grow until ATP has been hydrolyzed and Pi released from the penultimate subunit, thus promoting the release of profilin and allowing further elongation. Formin does not change the activity of profilin but simply uses it for its processive walk at barbed ends. Finally, if profilin release from actin is prevented by a chemical cross-link, formin processivity is abolished.