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.     

Random copolymerization of ATP-actin and ADP-actin

The equilibrium of the copolymerization of ATP-actin and ADP-actin was investigated by an analysis of the critical concentrations of mixtures of ATP-actin and ADP-actin. The molar ratio of bound ATP to bound ADP was controlled by the ratio of free ATP and ADP. The experiments were performed under conditions (100 mM KCl, l mM MgCl2, pH 7.5, 25 degrees C) where the ATP hydrolysis following binding of actin monomers to barbed filament ends was so slow that the distribution of ATP or ADP bound to the subunits near the ends of filaments was not affected by ATP hydrolysis. According to the analysis of the critical concentrations, the equilibrium constants for incorporation of ATP-actin or ADP-actin into filaments were independent of the type of nucleotide bound to contiguous subunits.     

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.     

Evidence for an ATP cap at the ends of actin filaments and its regulation of the F-actin steady state

The correlation between the time courses of actin polymerization under continuous sonication and the associated ATP hydrolysis has been studied. ATP hydrolysis was not mechanistically coupled to polymerization, i.e. not necessary for polymerization, but occurred on F-actin in a subsequent monomolecular reaction. Under sonication, polymerization was complete in 10 s while hydrolysis of ATP on the polymer required 200 s. A value of 0.023 s-1 was found for the first order rate constant of ATP hydrolysis on the polymer at 25 degrees C, pH 7.8, in the presence of 0.2 mM ATP, 0.1 mM CaCl2, and 1 mM MgCl2, independent of the F-actin concentration. The conversion of ATP X F-actin to ADP X F-actin was accompanied by an increase in fluorescence of a pyrenyl probe covalently attached to actin, consistent with a 2-fold greater fluorescence for ADP X F-actin than for ATP X F-actin, with a rate constant of 0.022 s-1. In contrast, the fluorescence of F-actin labeled with 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole did not change significantly when ATP or ADP was bound. The direct consequence of the uncoupling between polymerization and ATP hydrolysis is the formation of an ATP cap at the ends of the filaments, which maintains the stability of the polymer, while most of the filament contains bound ADP. The heterogeneity of the filament with respect to ATP and ADP results in a nonlinear relationship between the rate of elongation and the concentration of G-actin with a discontinuity at the critical concentration, where the rate of growth is zero. In this respect, F-actin in ATP behaves similarly to microtubules in GTP.     

Actin polymerization and ATP hydrolysis

This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of P_i. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-P_i subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, P_i release is reversible, which allows the regulation of filament dynamics by cellular P_i. The nature of the divalent metal ion — Ca²⁺ or Mg²⁺ — tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins.     

Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.     

Effect of ATP removal and inorganic phosphate on length redistribution of sheared actin filament populations. Evidence for a mechanism of end-to-end annealing

The effect of inorganic phosphate (Pi) on the depolymerization of F-actin has been measured. Pi inhibits disassembly of pyrene-labelled F-actin at steady-state induced either by dilution, or by shearing, suggesting that Pi decreases the off rate constant, k-, for dissociation. This effect of Pi is maximal at 20 mM, unlike the effect of Pi in reducing the critical concentration at the pointed end (maximal at 2 mM). This difference in concentration dependence for the two effects is interpreted as different affinities of Pi for the barbed and pointed ends, presumably as ADP-Pi-actin species. The contribution of ATP/ADP phase changes at filament ends (i.e. "dynamic instability") to length redistribution in sheared polymer steady-state actin filament populations was determined by (1) converting ATP to ADP in the system to prevent phase changes, or (2) adding 20 mM-Pi to the system to inhibit depolymerization. The observed absence of effect of these treatments on length redistribution excludes all mechanisms which involve phase change-driven disassembly or monomer exchange at filament ends, and appears to constrain the mechanism to one of end-to-end annealing under these conditions.     

Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis

Motile and morphogenetic cellular processes are driven by site-directed assembly of actin filaments. Formins, proteins characterized by formin homology domains FH1 and FH2, are initiators of actin assembly. How formins simply bind to filament barbed ends in rapid equilibrium or find free energy to become a processive motor of filament assembly remains enigmatic. Here we demonstrate that the FH1-FH2 domain accelerates hydrolysis of ATP coupled to profilin-actin polymerization and uses the derived free energy for processive polymerization, increasing 15-fold the rate constant for profilin-actin association to barbed ends. Profilin is required for and takes part in the processive function. Single filaments grow at least 10 microm long from formin bound beads without detaching. Transitory formin-associated processes are generated by poisoning of the processive cycle by barbed-end capping proteins. We successfully reconstitute formin-induced motility in vitro, demonstrating that this mechanism accounts for the puzzlingly rapid formin-induced actin processes observed in vivo.     

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).     

Filament assembly from profilin-actin

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 microM 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 microM under physiological conditions. The PAcov polymer 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.     

Nonlinear increase of elongation rate of actin filaments with actin monomer concentration

The nonlinear increase of the elongation rate of actin filaments above the critical monomer concentration was investigated by nucleated polymerization of actin. Significant deviations from linearity were observed when actin was polymerized in the presence of magnesium ions. When magnesium ions were replaced by potassium or calcium ions, no deviations from linearity could be detected. The nonlinearity was analyzed by two simple assembly mechanisms. In the first model, if the ATP hydrolysis by polymeric actin is approximately as fast as the incorporation of monomers into filaments, terminal subunits of lengthening filaments are expected to carry to some extent ADP. As ADP-containing subunits dissociate from the ends of actin filaments faster than ATP-containing subunits, the rate of elongation of actin filaments would be nonlinearly correlated with the monomer concentration. In the second model (conformational change model), actin monomers and filament subunits were assumed to occur in two conformations. The association and dissociation rates of actin molecules in the two conformations were thought to be different. The equilibrium distribution between the two conformations was assumed to be different for monomers and filament subunits. The ATP hydrolysis was thought to lag behind polymerization and conformational change. As under the experimental conditions the rate of ATP hydrolysis by polymeric actin was independent of the concentration of filament ends, the observed nonlinear increase of the rate of elongation with the monomer concentration above the critical monomer concentration was unlikely to be caused by ATP hydrolysis at the terminal subunits. The conformational change model turned out to be the simplest assembly mechanism by which all available experimental data could be explained.     

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.     

Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

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.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

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.     

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.     

Nematode sperm motility: nonpolar filament polymerization mediated by end-tracking motors

In nematode sperm cell motility, major sperm protein (MSP) filament assembly results in dynamic membrane protrusions in a manner that closely resembles actin-based motility in other eukaryotic cells. Paradoxically, whereas actin-based motility is driven by addition of ATP-bound actin subunits onto actin filament plus-ends located at the cell membrane, MSP dimers assemble from solution into nonpolar filaments that lack a nucleotide binding site. Thus, filament polarity and on-filament ATP hydrolysis, although essential for actin-based motility, appear to be unnecessary for membrane protrusions by MSP. As a potential resolution to this paradox, we propose a model for MSP filament assembly and force generation by MSP filament end-tracking proteins. In this model, ATP hydrolysis drives affinity-modulated, processive interactions between membrane-associated proteins and elongating filament ends. However, in contrast to the "actoclampin" model for actin filament end-tracking motors, ATP activates the tracking protein (or a soluble cofactor) rather than the MSP subunits themselves (in contrast to activation of actin subunits by ATP binding). The MSP end-tracking model predicts properties that are consistent with several key observations of MSP-based motility, including persistent membrane attachment, polymerization of filament ends at the membrane with depolymerization of free-filament ends away from the membrane, as well as a saturating dependence of polymerization rate on the concentration of non-MSP soluble cytoplasmic components.