Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.     

Acrosomal reaction of the Thyone sperm. III. The relationship between actin assembly and water influx during the extension of the acrosomal process

In an attempt to investigate the role of water influx in the extension of the acrosomal process of Thyone sperm, we induced the acrosomal reaction in sea water whose osmolarity varied from 50 to 150% of that of sea water. (a) Video sequences of the elongation of the acrosomal processes were made; plots of the length of the acrosomal process as a function of (time)1/2 produced a straight line except at the beginning of elongation and at the end in both hypotonic and hypertonic sea water (up to 1.33 times the osmolarity of sea water), although the rate of elongation was fastest in hypotonic sea water and was progressively slower as the tonicity was raised. (b) Close examination of the video sequences revealed that regardless of the tonicity of the sea water, the morphology of the acrosomal processes were similar. (c) From thin sections of fixed sperm, the amount of actin polymerization that takes place is roughly coupled to the length of the acrosomal process formed so that sperm with short processes only polymerize a portion of the actin that must be present in those sperm. From these facts we conclude that the influx of water and the release of actin monomers from their storage form in the profilactin (so that these monomers can polymerize) are coupled. The exact role of water influx, why it occurs, and whether it could contribute to the extension of the acrosomal process by a hydrostatic pressure mechanism is discussed.     

Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation

Thyone sperm were induced to undergo the acrosomal reaction with a calcium ionophore A23187 in sea water containing 50 mM excess CaCl2, and the extension of the acrosomal process was recorded with high- resolution, differential interference contrast video microscopy at 60 fields/sec. The length of the acrosomal process was measured at 0.25-s intervals on nine sperm. When the data were plotted as (length)2 vs. time, the points fell exactly on a straight line except for the initial and very final stages of elongation. Cytochalasin B alters the rate of elongation of the acrosomal process in a dose-dependent way, inhibiting the elongation completely at high concentrations (20 micrograms/ml). However, no inhibition was observed unless excess Ca++ was added to sea water. The concentration of actin in the periacrosomal cup of the unreacted sperm is as high as 160 mg/ml; we calculate this concentration from the number and lengths of the actin filaments in a fully reacted sperm, and the volume of the periacrosomal cup in the unreacted sperm. These results are consistent with the hypothesis proposed earlier that monomers add to the ends of the actin filaments situated at the tip of the growing acrosomal process (the preferred end for monomer addition), and that the rate of elongation of the process is limited by diffusion of monomers from the sperm head (periacrosomal cup) to the tip of the elongating process. During the extension of the acrosomal process, a few blebs distributed along its lengths move out with the process. These blebs maintain a constant distance from the tip of the growing process. At maximum length, the straight acrosomal process slackens into a bow, and numerous new blebs appear. A few seconds later, the process suddenly straightens out again and sometimes actually contracts. The behavior of the blebs indicates that membrane is inserted at the base of the growing acrosomal process, and that membrane assembly and water uptake must be coupled to actin assembly during elongation. We discuss how the dynamic balance of forces seems to determine the shape of the growing acrosomal process, and how actin assembly may be controlled during the acrosomal reaction.     

A moving boundary model of acrosomal elongation

A sperm penetrates an egg by extending a long, actin-filled tube known as the acrosomal process. This simple example of biomotility is one of the most dramatic. In Thyone, a 90 m process can extend in less than 10 s. Experiments have shown that actin monomers stored in the base of the sperm are transported to the growing tip of the acrosomal process where they add to the ends of the existing filaments.The force that drives the elongation of the acrosomal process has not yet been identified although the most frequently discussed candidate is the actin polymerization reaction. Developing what we believe are realistic moving boundary models of diffusion limited actin fiber polymerization, we show that actin filament growth occurs too slowly to drive acrosomal elongation. We thus believe that other forces, such as osmotically driven water flow, must play an important role in causing the elongation. We conjecture that actin polymerization merely follows to give the appropriate shape to the growing structure and to stabilize the structure once water flow ceases.Work partially supported by the United States Department of Energy     

Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined

The polarity of the actin filaments which assemble from the nucleating body or actomere of Thyone and Pisaster sperm was determined using myosin subfragment 1 decoration. The polarity was found to be unidirectional with the arrowheads pointing towards the cell center. When polymerization is induced at low temperature with concentrations of actin near the critical concentration for polymerization, elongation of filaments occurs preferentially off the apical end. If the sperm are induced to undergo the acrosomal reaction with an ionophore, the polarity of the actin filaments attached to the actomere is the same as that already described, but the filaments which polymerize parallel to, but peripheral to, those extending from the actomere are randomly polarized. These randomly polarized filaments appear to result from spontaneous nucleation. When sperm are induced to undergo the acrosomal reaction with eggs, the polarity of the actin filaments is also unidirectional with the arrowheads pointing towards the cell center. From these results we conclude: (a) that the actomere, by nucleating the polymerization of actin filaments, controls the polarity of the actin filaments in the acrosomal process, (b) that the actomere recognizes a surface of the actin monomer that is different from that surface recognized by the dense material attached to membranes, and (c) that egg myosin could not act to pull the sperm into the egg. Included is a discussion of how the observation that monomers add largely to one end of a decorated filament in vitro relates to these in vivo observations.     

A mechanical model for elongation of the acrosomal process in Thyone sperm

We present a mechanical model for the elongation of the acrosomal process in Thyone sperm based upon osmotically driven hydrostatic forces.     

Protein fluxes along the filopodium as a framework for understanding the growth-retraction dynamics: The interplay between diffusion and active transport

We present a picture of filopodial growth and retraction from physics perspective, where we emphasize the significance of the role played by protein fluxes due to spatially extended nature of the filopodium. We review a series of works, which used stochastic simulations and mean field analytical modeling to find the concentration profile of G-actin inside a filopodium, which, in turn, determines the stationary filopodial length. In addition to extensively reviewing the prior works, we also report some new results on the role of active transport in regulating the length of filopodia. We model a filopodium where delivery of actin monomers toward the tip can occur both through passive diffusion and active transport by myosin motors. We found that the concentration profile of G-actin along the filopodium is rather non-trivial, containing a narrow minimum near the base followed by a broad maximum. For efficient enough actin transport, this non-monotonous shape is expected to occur under a broad set of conditions. We also raise the issue of slow approach to the stationary length and the possibility of multiple steady-state solutions.Key words: filopodia, active transport, molecular motors, stochastic process, mean-field theory     

Protein fluxes along the filopodium as a framework for understanding the growth-retraction dynamics

We present a picture of filopodial growth and retraction from physics perspective, where we emphasize the significance of the role played by protein fluxes due to spatially extended nature of the filopodium. We review a series of works, which used stochastic simulations and mean field analytical modeling to find the concentration profile of G-actin inside a filopodium, which, in turn, determines the stationary filopodial length. In addition to extensively reviewing the prior works, we also report some new results on the role of active transport in regulating the length of filopodia. We model a filopodium where delivery of actin monomers towards the tip can occur both through passive diffusion and active transport by myosin motors. We found that the concentration profile of G-actin along the filopodium is rather non-trivial, containing a narrow minimum near the base followed by a broad maximum. For efficient enough actin transport, this non-monotonous shape is expected to occur under a broad set of conditions. We also raise the issue of slow approach to the stationary length and the possibility of multiple steady state solutions.     

Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm.

When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize.     

Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy

Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope. The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (20 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) suspended in sea water containing 50 mM excess CaCl2. Before activation of the sperm, the acrosomal region appears as a 1.1-microM diameter sphere, slightly less dense than the rest of the sperm head. Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After approximately 1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material. In this paper, we also consider the meaning of the enhanced DIC image seen in the high-resolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope.     

Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm.

Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding inophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaCl at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the ammount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomer.     

polymerization of actin: v.a new organelle, the actomere, that initiates the assembly of actin filaments in thyone sperm

Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding ionophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaCl at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the amount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomere.     

Isolation and localization of a spectrin-like protein from echinoderm sperm

Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 microns long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca(++)-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.     

Actin filaments elongate from their membrane-associated ends

In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end “preferred” for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle.     

Kinetics of nucleation-controlled polymerization. A perturbation treatment for use with a secondary pathway

We present a perturbation method for analyzing nucleation-controlled polymerization augmented by a secondary pathway for polymer growth. With this method, the solution to the kinetic equations assumes a simple analytic closed form that can easily be used in fitting data. So long as the formation of polymers by the secondary pathway depends linearly on the concentration of monomers polymerized, the form of the solutions is the same. This permits the analysis of augmented growth models with a minimum number of modeling assumptions, and thus makes it readily possible to distinguish between a variety of secondary processes (heterogeneous nucleation, lateral growth, and fragmentation). In addition, the parameters of the homogeneous process, such as the homogeneous nucleus size, can be determined independent of the nature of the secondary mechanism. We describe applications of this method to the polymerization of actin, collagen, and sickle hemoglobin. We present an extensive analysis of data on actin polymerization (Wegner, A., and P. Savko, 1982, Biochemistry, 21:1909-1913) to illustrate the use of the method. Although our conclusions generally agree with theirs, we find that lateral growth describes the secondary pathway better than the fragmentation model originally proposed. We also show how this method can be used to study the degree of polymerization, the parentage of polymers, and the behavior of polymers in cycling experiments.     

Structure of actin-containing filaments from two types of non-muscle cells

Bundles of actin-containing filaments from the acrosomal process of horseshoe crab sperm and from sea urchin egg contain a second protein having a molecular weight of about 55,000. Electron micrographs of these filamentous bundles show features reminiscent of paracrystalline arrays of actin except that bundles from the sea urchin egg have distinctive transverse bands every 110 Å. From optical diffraction patterns of the micrographs, we deduced very similar models for both structures. The models consist of hexagonal arrays of actin filaments cross-linked by the second protein. The pattern of transverse bands in bundles derived from the sea urchin eggs is accounted for by postulating that the second protein is bonded to actin only at positions where cross-linking can occur, rather than being bonded to every actin. The helical symmetry of the actin requires that the bonding contacts involved in the cross-linking be slightly different at different positions along the length of the bundle. The technique of image reconstruction was used to obtain a three-dimensional map of the bundles from the acrosomal process.     

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.     

Emergence and maintenance of variable-length actin filaments in a limiting pool of building blocks

Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the length of single actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different lengths using the same set of molecular building blocks. Here, we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth-rate modulation by actin-binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneity. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning filamentous actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.     

A continuum model of protrusion of pseudopod in leukocytes.

The morphology of human leukocytes, the biochemistry of actin polymerization, and the theory of continuum mechanics are used to model the pseudopod protrusion process of leukocytes. In the proposed model, the pseudopod is considered as a porous solid of F-actin network, the pores of which are full of aqueous solution. G-actin is considered as a "solute" transported by convection and diffusion in the fluid phase. The pseudopod grows as actin filaments elongate at their barbed ends at the tip of the pseudopod. The driving force of extension is hypothesized as being provided by the actin polymerization. It is assumed that elongation of actin filaments, powered by chemical energy liberated from the polymerization reaction, does mechanical work against opposing pressure on the membrane. This also gives rise to a pressure drop in the fluid phase at the tip of the pseudopod, which is formulated by an equation relating the work done by actin polymerization to the local state of pressure. The pressure gradient along the pseudopod drives the fluid filtration through the porous pseudopod according to Darcy's Law, which in turn brings more actin monomers to the growing tip. The main cell body serves as a reservoir of G-actin. A modified first-order equation is used to describe the kinetics of polymerization. The rate of pseudopod growth is modulated by regulatory proteins. A one-dimensional moving boundary problem based on the proposed mechanism has been constructed and approximate solutions have been obtained. Comparison of the solutions with experimental data shows that the model is compatible with available observations. The model is also applicable to growth of other cellular systems such as elongation of acrosomal process in sperm cells.     

Under physiological conditions actin disassembles slowly from the nonpreferred end of an actin filament

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. Because of the taper of these cross-linked bundles of actin filaments, one can distinguish directly the preferred end for assembly from the nonpreferred end. Loss of growth with time from the nonpreferred end was directly assessed by electron microscopy and found to be dependent upon salt concentration. Under physiological conditions (100 mM KCl, 1 mM MgCl2) and excess ATP (0.5 mM), depolymerization of the newly assembled actin filaments at the nonpreferred end over an 8-h period was 0.024 micron/h. Thus, even after 8 h, 63% of the bundles retained significant growth on their nonpreferred ends, the average length being 0.21 micron +/- 0.04. However, in the presence of 1.2 mM CaCl2, disassembly of actin monomers from the nonpreferred end increased substantially. By 8 h, only 7% of the bundles retained any actin growth on the nonpreferred ends, and the depolymerization rate off the nonpreferred end was 0.087 micron/h. From these results we conclude that, in the absence of other cellular factors, disassembly of actin subunits from actin filaments (subunit exchange) is too slow to influence most of the motile events that occur in cells. We discuss how this relates to treadmilling.