Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with alpha-actinin.

Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.     

Microheterogeneity controls the rate of gelation of actin filament networks

Rapid sol-gel transitions of the actin cytoskeleton are required for many key cellular processes, including cell spreading and cell locomotion. Actin monomers assemble into semiflexible polymers that rapidly intertwine into a network, a process that in vitro takes approximately 1 min for an actin concentration of 1 mg/ml. The same actin filament network, however, takes approximately 1 h to exhibit a steady-state elasticity. We hypothesize that the slow gelation of F-actin is due to the slow establishment of a homogeneous meshwork. Using a novel method, time-resolved multiple particle tracking, which monitors the range of thermally excited displacements of microspheres imbedded in the network, we show that the increase in elasticity in a polymerizing solution of actin parallels the progressive decline of the network microheterogeneity. The rates of gelation and network homogenization slightly decrease with actin concentration and in the presence of the F-actin cross-linking proteins alpha-actinin and fascin, whereas the rate of actin polymerization increases dramatically with actin concentration. Our measurements show that the slow spatial homogenization of the actin filament network, not actin polymerization or the formation of polymer overlaps, is the rate-limiting step in the establishment of an elastic actin network and suggest that a new activity of F-actin binding proteins may be required for the rapid formation of a homogeneous stiff gel.     

The filamentous actin cross-linking/bundling activity of mammalian formins

Formins are multidomain proteins that regulate actin filament dynamics and are defined by the formin homology 2 domain. Biochemical assays suggest that mammalian formins display actin-filament nucleation, severing, and bundling activities. Whether formins can cross-link actin filaments into viscoelastic arrays and the effectiveness of formins' bundling activity compared with that of important filamentous actin (F-actin) cross-linking/bundling proteins are unknown. Here, we used rigorous in vitro rheologic assays to deconvolve the dynamic cross-linking activity from the bundling activity of formin FRL1 and the closely related mDia1 and mDia2. In addition, we compared these formins with the canonical F-actin bundling protein fascin and cross-linking/bundling proteins alpha-actinin and filamin. We found that FRL1 and mDia2, but not mDia1, can help F-actin form highly elastic networks. FRL1 and mDia2 mediate the formation of highly elastic F-actin networks as effectively and rapidly as alpha-actinin and filamin but only past a relatively high actin-to-formin molar ratio of 50:1. Past that threshold molar ratio, the mechanical properties of F-actin/formin networks are independent of formin concentration, similar to fascin. Moreover, unlike those for alpha-actinin and filamin but similar to those for fascin, F-actin/formin networks show no strain-induced hardening. mDia1 cannot bundle F-actin but can weakly cross-link filaments at high concentrations. Point mutagenesis reveals that reducing the barbed-end binding activity of FRL1 and mDia2 greatly enhances the rate of formation of F-actin gels but does not significantly affect the mechanical properties of the resulting networks at steady state. Together, these results suggest that the mechanical behaviors of FRL1 and mDia2 are fundamentally different from those of cross-linking/bundling proteins alpha-actinin and filamin but qualitatively similar to the mechanical behavior of the bundling protein fascin, albeit with a dramatically increased (>10-fold) threshold concentration for transition to bundling, which nevertheless leads to much stiffer F-actin networks than fascin.     

Mechanics and multiple-particle tracking microheterogeneity of alpha-actinin-cross-linked actin filament networks

Cell morphology is controlled by the actin cytoskeleton organization and mechanical properties, which are regulated by the available contents in actin and actin regulatory proteins. Using rheometry and the recently developed multiple-particle tracking method, we compare the mechanical properties and microheterogeneity of actin filament networks containing the F-actin cross-linking protein alpha-actinin. The elasticity of F-actin/alpha-actinin networks increases with actin concentration more rapidly for a fixed molar ratio of actin to alpha-actinin than in the absence of alpha-actinin, for networks of fixed alpha-actinin concentration and of fixed actin concentration, but more slowly than theoretically predicted for a homogeneous cross-linked semiflexible polymer network. These rheological measurements are complemented by multiple-particle tracking of fluorescent microspheres imbedded in the networks. The distribution of the mean squared displacements of these microspheres becomes progressively more asymmetric and wider for increasing concentration in alpha-actinin and, to a lesser extent, for increasing actin concentration, which suggests that F-actin networks become progressively heterogeneous for increasing protein content. This may explain the slower-than-predicted rise in elasticity of F-actin/alpha-actinin networks. Together these in vitro results suggest that actin and alpha-actinin provides the cell with an unsuspected range of regulatory pathways to modulate its cytoskeleton's micromechanics and local organization in vivo.     

The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.     

Functional synergy of actin filament cross-linking proteins

The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.     

Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.     

The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin

Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive.     

Assembly-disassembly of actin bundles in starfish oocytes: an analysis of actin-associated proteins in the isolated cortex

One rapid response of starfish oocytes to the maturation-inducing hormone, 1-methyladenine (1-MA), is the formation of transient actin-filled spikes on the cell surface. The presence and distribution of G- and F-actin and several actin-associated proteins were examined in cortices isolated from oocytes before, during, and after spike formation by using antibodies and the F-actin-specific stain, NBD-phallacidin. Before 1-MA addition, staining with antiactin and NBD-phallacidin indicates that most of the actin in the cortex is either G-actin or oligomeric actin, but rather little is F-actin. Application of the hormone results in the conversion and redistribution of this cortical actin into large bundles of F-actin which form the cores of spikes. When the spikes recede, F-actin disappears, and the amount of all forms of actin bound in the cortex appears to decrease. Antibodies to sea urchin egg myosin, fascin and a 220-kDa protein were used to examine these actin-associated proteins during the times that the organization of actin changes. Myosin and the 220-kDa protein are bound to the cortex and uniformly distributed before 1-MA application while fascin appears to be unbound. When spikes appear after 1-MA addition, fascin and the 220-kDa protein are localized coincidently with the spikes, whereas myosin remains uniformly distributed throughout the cortex and is excluded from the spikes. After spike resorption, fascin and the 220-kDa protein appear to lose their cortical binding while myosin retains its localization unchanged. These results indicate that actin, fascin and the 220-kDa protein undergo major organizational changes in the cortex in response to 1-MA.     

Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP’s main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP’s flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP’s effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP’s ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP’s network stiffening activity may be tuned by the local concentration of monomeric actin.     

The assembly of MreB, a prokaryotic homolog of actin

MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the well-studied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of approximately 3 nm, which is approximately 100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.     

A mechanochemical model of actin filaments

In eukaryotic cells, actin filaments are involved in important processes such as motility, division, cell shape regulation, contractility, and mechanosensation. Actin filaments are polymerized chains of monomers, which themselves undergo a range of chemical events such as ATP hydrolysis, polymerization, and depolymerization. When forces are applied to F-actin, in addition to filament mechanical deformations, the applied force must also influence chemical events in the filament. We develop an intermediate-scale model of actin filaments that combines actin chemistry with filament-level deformations. The model is able to compute mechanical responses of F-actin during bending and stretching. The model also describes the interplay between ATP hydrolysis and filament deformations, including possible force-induced chemical state changes of actin monomers in the filament. The model can also be used to model the action of several actin-associated proteins, and for large-scale simulation of F-actin networks. All together, our model shows that mechanics and chemistry must be considered together to understand cytoskeletal dynamics in living cells.     

Preliminarily investigating the polymorphism of self-organized actin filament in vitro by atomic force microscope

Actin is a major structural component of eukaryoticcytoskeleton and exists in monomer G-actin and filamen-tous F-actin. G-actin consists of 375 amino acid residueswith molecular weight 43 kD and is a highly conservedprotein expressed in most living organi…     

Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation

We elucidated the mechanism by which gelsolin, a Ca2+-dependent regulatory protein from lung macrophages, controls the network structure of actin filaments. In the presence of micromolar Ca2+, gelsolin bound Ca2+. The Ca2+-gelsolin complex reduced the apparent viscosity and flow birefringence of F-actin and the lengths of actin filaments viewed in the electron microscope. However, concentrations of gelsolin causing these alterations did not effect proportionate changes in the turbidity of actin filament solutions or in the quantity of nonsedimentable actin as determined by a radioassay. From these findings, we conclude that gelsolin shortens actin filaments without net depolymerization. Such an effect on the distribution of actin filament lengths led to the prediction that low concentrations of gelsolin would increase the critical concentration of actin-binding protein required for incipient gelation of actin filaments in the presence of Ca2+, providing an efficient mechanism for controlling actin network structure. We verified the prediction experimentally, and we estimated that the Ca2+-gelsolin complex effectively breaks the bond between actin monomers in filaments with a stoichiometry of 1:1. The effect of Ca2+-gelsolin complex on actin solation was rapid, independent of temperature between 0 degrees and 37 degrees C, and reversed by reducing the free Ca2+ concentration.     

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.     

The critical concentration of actin in the presence of ATP increases with the number concentration of filaments and approaches the critical concentration of actin.ADP

F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.     

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.     

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Some perspectives on the viscosity of actin filaments

Measurements of the dynamic viscosity of various actin filament preparations under conditions of low and controlled shear: (a) confirm the shear rate dependence of F-actin viscosities and show that this dependence obeys the power law relationship observed for entangled synthetic polymers; (b) permit estimation of the extent to which shear artifact amplifies changes in the apparent viscosity of F-actin measured in a falling ball viscometer; (c) show that gel-filtration chromatography of actin and the addition of cytochalasin B to F-actin bring about small (20-40%) changes in the viscosity of the F-actin solutions. These variations are consistent with alterations in the actin-binding protein concentrations required for incipient gelation, a parameter inversely related to average filament length. Therefore: (a) changes in the viscosity of F-actin can be magnified by use of the falling ball viscometer, and may exaggerate their biological importance; (b) chromatography of actin may not be required to obtain meaningful information about the rheology of actin filaments; (c) changes in actin filament length can satisfactorily explain alterations in F-actin viscosity exerted by cytochalasin B and by chromatography, obviating the need to postulate specific interfilament interactions.