Control of actin polymerization in live and permeabilized fibroblasts

We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.     

End versus side branching by Arp2/3 complex

We investigate the issue of end versus side branching of actin filaments by Arp2/3 complex, using a combination of analytic theory, polymerization assays, and quantitative modeling. The analytic theory shows that the effect of capping protein on the initial stages of actin polymerization in the presence of Arp2/3 complex depends strongly on whether new Arp2/3 complex-induced branches grow from the sides or ends of existing filaments. Motivated by these results, we measure and quantitatively model the kinetics of actin polymerization in the presence of activated Arp2/3 complex, for a range of concentrations of capping protein. Our model includes the most important types of events involving actin and actin-binding proteins, and can be adjusted to include end branching, side branching, or both. The side-branching model gives a better fit to the experimental data than the end-branching model. An end-plus-side model including both types of branching gives a moderate improvement in the quality of the fit. Another side-branching model, based on aging of subunits' capacity for branch formation, gives a significantly better fit than the end-plus-side model. We discuss implications for actin polymerization in cells.     

Growth velocities of branched actin networks

The growth of an actin network against an obstacle that stimulates branching locally is studied using several variants of a kinetic rate model based on the orientation-dependent number density of filaments. The model emphasizes the effects of branching and capping on the density of free filament ends. The variants differ in their treatment of side versus end branching and dimensionality, and assume that new branches are generated by existing branches (autocatalytic behavior) or independently of existing branches (nucleation behavior). In autocatalytic models, the network growth velocity is rigorously independent of the opposing force exerted by the obstacle, and the network density is proportional to the force. The dependence of the growth velocity on the branching and capping rates is evaluated by a numerical solution of the rate equations. In side-branching models, the growth velocity drops gradually to zero with decreasing branching rate, while in end-branching models the drop is abrupt. As the capping rate goes to zero, it is found that the behavior of the velocity is sensitive to the thickness of the branching region. Experiments are proposed for using these results to shed light on the nature of the branching process.     

A biomimetic motility assay provides insight into the mechanism of actin-based motility

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.     

The effect of branching on the critical concentration and average filament length of actin

The dependences of the steady-state critical concentration and average filament length of actin solutions, on the filament branching and capping rates, are calculated using a rate methodology based on the total number of actin filaments. The methodology generalizes calculations of the "treadmilling" actin concentration at which an average filament has net zero growth rate. The predictions of the rate methodology are validated by comparison with stochastic-growth simulations that track the positions of all filament subunits over time. For side branching, the critical concentration drops proportionally to the square root of the branching rate; for end branching the drop is linear. The polymerization response to branching has a maximum as a function of the capping-protein concentration. The average filament length drops with increasing branching, because the critical concentration drops. Even small rates of filament uncapping have a large impact on the average filament length in vitro. The potential significance of these phenomena for cell behavior is evaluated.     

Effect of capping protein on the kinetics of actin polymerization

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (2) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin dimer. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. D., & Pollard, T. D. (1984) J. Cell Biol. 99, 217-225; Isenberg, G. A., Aebi, U., & Pollard, T. D. (1980) Nature (London) 288, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.     

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.     

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.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Optimal orientation in branched cytoskeletal networks

Actin cytoskeletal protrusions in crawling cells, or lamellipodia, exhibit various morphological properties such as two characteristic peaks in the distribution of filament orientation with respect to the leading edge. To understand these properties, using the dendritic nucleation model as a basis for cytoskeletal restructuring, a kinetic-population model with orientational-dependent branching (birth) and capping (death) is constructed and analyzed. Optimizing for growth yields a relation between the branch angle and filament orientation that explains the two characteristic peaks. The model also exhibits a subdominant population that allows for more accurate modeling of recent measurements of filamentous actin density along the leading edge of lamellipodia in keratocytes. Finally, we explore the relationship between orientational and spatial organization of filamentous actin in lamellipodia and address recent observations of a prevalence of overlapping filaments to branched filaments-a finding that is claimed to be in contradiction with the dendritic nucleation model.     

Regulation of actin dynamics in rapidly moving cells: a quantitative analysis

We develop a mathematical model that describes key details of actin dynamics in protrusion associated with cell motility. The model is based on the dendritic-nucleation hypothesis for lamellipodial protrusion in nonmuscle cells such as keratocytes. We consider a set of partial differential equations for diffusion and reactions of sequestered actin complexes, nucleation, and growth by polymerization of barbed ends of actin filaments, as well as capping and depolymerization of the filaments. The mechanical aspect of protrusion is based on an elastic polymerization ratchet mechanism. An output of the model is a relationship between the protrusion velocity and the number of filament barbed ends pushing the membrane. Significantly, this relationship has a local maximum: too many barbed ends deplete the available monomer pool, too few are insufficient to generate protrusive force, so motility is stalled at either extreme. Our results suggest that to achieve rapid motility, some tuning of parameters affecting actin dynamics must be operating in the cell.     

Visualization and force measurement of branching by Arp2/3 complex and N-WASP in actin filament

To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.     

New proposed mechanism of actin-polymerization-driven motility

We present the first numerical simulation of actin-driven propulsion by elastic filaments. Specifically, we use a Brownian dynamics formulation of the dendritic nucleation model of actin-driven propulsion. We show that the model leads to a self-assembled network that exerts forces on a disk and pushes it with an average speed. This simulation approach is the first to observe a speed that varies nonmonotonically with the concentration of branching proteins (Arp2/3), capping protein, and depolymerization rate, in accord with experimental observations. Our results suggest a new interpretation of the origin of motility. When we estimate the speed that this mechanism would produce in a system with realistic rate constants and concentrations as well as fluid flow, we obtain a value that is within an order-of-magnitude of the polymerization speed deduced from experiments.     

Control of actin assembly and disassembly at filament ends

The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.     

Growth of branched actin networks against obstacles

A method for simulating the growth of branched actin networks against obstacles has been developed. The method is based on simple stochastic events, including addition or removal of monomers at filament ends, capping of filament ends, nucleation of branches from existing filaments, and detachment of branches; the network structure for several different models of the branching process has also been studied. The models differ with regard to their inclusion of effects such as preferred branch orientations, filament uncapping at the obstacle, and preferential branching at filament ends. The actin ultrastructure near the membrane in lamellipodia is reasonably well produced if preferential branching in the direction of the obstacle or barbed-end uncapping effects are included. Uncapping effects cause the structures to have a few very long filaments that are similar to those seen in pathogen-induced "actin tails." The dependence of the growth velocity, branch spacing, and network density on the rate parameters for the various processes is quite different among the branching models. An analytic theory of the growth velocity and branch spacing of the network is described. Experiments are suggested that could distinguish among some of the branching models.     

Exogenous nucleation sites fail to induce detectable polymerization of actin in living cells

Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.     

Processive capping by formin suggests a force-driven mechanism of actin polymerization

Regulation of actin polymerization is essential for cell functioning. Here, we predict a novel phenomenon-the force-driven polymerization of actin filaments mediated by proteins of the formin family. Formins localize to the barbed ends of actin filaments, but, in contrast to the standard capping proteins, allow for actin polymerization in the barbed direction. First, we show that the mechanism of such "leaky capping" can be understood in terms of the elasticity of the formin molecules. Second, we demonstrate that if a pulling force acts on the filament end via the leaky cap, the elastic stresses can drive actin polymerization. We estimate that a moderate pulling force of approximately 3.4 pN is sufficient to reduce the critical actin concentration required for barbed end polymerization by an order of magnitude. Furthermore, the pulling force increases the polymerization rate. The suggested mechanism of force-driven polymerization could be a key element in a variety of cellular mechanosensing devices.     

The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins

The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.     

Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4- mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on- rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.