Mechanical properties of actin

We used a cone and plate rheometer to evaluate the mechanical properties of actin over a wide range of oscillation frequencies and shear rates. Remarkably, both filamentous and nonfilamentous actin behaved as viscoelastic solids in both oscillatory and shear type experiments, providing that they were given ample time to equilibrate. Actin was purified by gel filtration from rabbit skeletal muscle and Acanthamoeba. Nonfilamentous actin in 2 different buffers had similar properties. In a low ionic strength buffer the absence of filaments was confirmed by electron microscopy, ultracentrifugation, and the fluorescence of pyrene-labeled actin. In 0.6 M KI, actin was monomeric by gel filtration. Filamentous actin had similar properties in 2 mM MgCl2 with either 50 mM KC1 or 500 mM KC1. Under all 4 of these conditions, actin required about 1000 min at 25 degrees C for the rheological properties to equilibrate. Under conditions where the oscillation of the rheometer did not affect the mechanical properties, all of the actin preparations had dynamic viscosities that were inverse functions of the frequency and dynamic elasticites that leveled off at low frequencies as expected for viscoelastic solids. For filamentous actin, the values of these parameters were about 2 times higher than for nonfilamentous actin. In shear experiments, both filamentous and nonfilamentous actin exhibited shear rate-dependent yield stresses. When filamentous and nonfilamentous actin structures were disrupted by transient shearing, the dynamic elasticity recovered to 90% in 30 min. Ovalbumin in the low ionic strength buffer also behaved as a viscoelastic material with elasticity and viscosity about 10 times lower than nonfilamentous actin, while cytochrome c behaved as a Newtonian fluid with a viscosity of 0.02 poise.     

The cortical actin cytoskeleton of unactivated zebrafish eggs: Spatial organization and distribution of filamentous actin,nonfilamentous actin,and myosin-II

Actin and nonmuscle myosin heavy chain (myosin-II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS-polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase-I, or anti-actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin-II in whole mounts and egg fragments. Myosin-II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin-II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin-II provides insight into possible functions of these proteins before and after fertilization. © 1996 Wiley-Liss, Inc.     

Viscoelasticity of F-actin measured with magnetic microparticles

Dispersed submicroscopic magnetic particles were used to probe viscoelasticity for cytoplasm and purified components of cytoplasm. An externally applied magnetic field exerted force on particles in cells, in filamentous actin (F-actin) solutions, or in F-actin gels formed by the addition of the actin gelation factor, actin-binding protein (ABP). The particle response to magnetic torque can be related to the viscoelastic properties of the fluids. We compared data obtained on F-actin by the magnetic particle method with data obtained on F-actin by means of a sliding plane viscoelastometer. F-actin solutions had a significant elasticity, which increased by 20-fold when gels were formed by ABP addition. Both methods gave consistent results, but the dispersed magnetic particles indicated quantitatively greater rigidity than the viscoelastometer (two and six times greater for F-actin solutions and for F-actin plus ABP gels, respectively). These differences may be due to the fact that, compared with traditional microrheometers, dispersed particle measurements are less affected by long-range heterogeneity or domain-like structure. The magnetometric method was used to examine the mechanical properties of cytoplasm within intact macrophages; the application of the same magnetometric technique to both cells and well-defined, purified protein systems is a first step toward interpreting the results obtained for living cells in molecular terms. The magnetic particle probe system is an effective nonoptical technique for determining the motile and mechanical properties of cells in vitro and in vivo.     

The viscoelastic properties of actin solutions

The viscoelastic properties in actin solutions were investigated by measuring their elastic modulus and viscous modulus using a rheometer. The polymerization/gelation process of actin solutions was accompanied by an increase of both parameters, indicating the formation of a protein network. High shear rotational motion destroyed this network which, however, would reanneal if left undisturbed. At 25 °C under low ionic strength conditions, the viscoelastic moduli of a Spudich-Watt globular (G) actin preparation increased with time, while G-actin, purified by gel filtration maintained low viscoelastic moduli. The rigidity of the filamentous (F) actin network in a solution of Spudich-Watt actin, measured by the elastic modulus, was somewhat lower than that of gel-filtration-purified actin at the same protein concentration. The crosslink density of these F-actin networks was estimated, using models from rubber elasticity theory. The calculated density was 1 crosslink/50 actin monomers for the purified actin and 1 crosslink/120 actin monomers for Spudich-Watt actin. The results are consistent with the idea that a small amount of regulatory factor(s), which could be removed by the gel filtration step, modulates the structure of an actin network.     

Mechanical properties of brain tubulin and microtubules

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.     

Viscoelasticity of F-actin and F-actin/gelsolin complexes

Actin is the major protein of eukaryote peripheral cytoplasm where its mechanical effects could determine cell shape and motility. The mechanical properties of purified F-actin, whether it is a viscoelastic fluid or an elastic solid, have been a subject of controversy. Mainstream polymer theory predicts that filaments as long as those found in purified F-actin are so interpenetrated as to appear immobile in measurements over a reasonable time with available instrumentation and that the fluidity of F-actin could only be manifest if the filaments were shortened. We show that the static and dynamic elastic moduli below a critical degree of shear strain are much higher than previously reported, consistent with extreme interpenetration, but that higher strain or treatment with very low concentrations of the F-actin severing protein gelsolin greatly diminish the moduli and cause F-actin to exhibit rheologic behavior expected for independent semidilute rods, and defined by the dimensions of the filaments, including shear rate independent viscosity below a critical shear rate. The findings show that shortening of actin filaments sufficiently to permit reasonable measurements brings out their viscoelastic fluid properties. Since gelsolin shortens F-actin, it is likely that the effect of high strain is also to fragment a population of long actin filaments. We confirmed recent findings that the viscosity of F-actin is inversely proportional to the shear rate, consistent with an indeterminate fluid, but found that gelsolin abolishes this unusual shear rate dependence, indicating that it results from filament disruption during the viscosity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [³⁵S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.     

Mechanical pulse wave propagation in gel, normal and oedematous tissues.

The velocity, attenuation and frequency content of the mechanical pulse wave propagation in gels of various water contents, in normal tissues from various sites and in oedematous tissues from different patients were investigated. The properties of the propagated pulse wave depend on the water content of the gel and the viscoelastic properties of the tissues. From the dependence of the pulse wave propagation velocity on elasticity, viscosity and density, information may be obtained concerning the effects of oedema on the mechanical properties of tissue.     

How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper

Processes such as cell locomotion and morphogenesis depend on both the generation of force by cytoskeletal elements and the response of the cell to the resulting mechanical loads. Many widely accepted theoretical models of processes involving cell shape change are based on untested hypotheses about the interaction of these two components of cell shape change. I have quantified the mechanical responses of cytoplasm to various chemical environments and mechanical loading regimes to understand better the mechanisms of cell shape change and to address the validity of these models. Measurements of cell mechanical properties were made with strands of cytoplasm submerged in media containing detergent to permeabilize the plasma membrane, thus allowing control over intracellular milieu. Experiments were performed with equipment that generated sinusoidally varying length changes of isolated strands of cytoplasm from Physarum polycephalum. Results indicate that stiffness, elasticity, and viscosity of cytoplasm all increase with increasing concentration of Ca2+, Mg2+, and ATP, and decrease with increasing magnitude and rate of deformation. These results specifically challenge assumptions underlying mathematical models of morphogenetic events such as epithelial folding and cell division, and further suggest that gelation may depend on both actin cross-linking and actin polymerization.     

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.     

Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos

Cells are not directly accessible in vivo and therefore their mechanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vivo assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of thermally excited nanoparticles embedded within the cytoplasm of developing embryos reflect local viscoelastic properties. As a proof of principle, we probe local viscoelastic properties of the cytoplasm of developing Caenorhabditis elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior-posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of the mitotic spindle stems from an asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.     

Unique properties of eukaryote-type actin and profilin horizontally transferred to cyanobacteria

A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 μm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 μm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.     

Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells

We have previously isolated and crystallized a complex from calf spleen, containing actin and a smaller protein which we call profilin. In this paper we describe some properties of this complex, and show that association with profilin is sufficient to explain the persistent monomeric state of some of the actin in spleen extracts; moreover, spleen profilin will recombine with skeletal muscle actin to form a non-polymerizable complex resembling that isolated from spleen. Profilin is not restricted to spleen, but is found in a variety of other tissues and tissue-cultured cell lines. We propose that reversible association of actin with profilin in the cell may provide a mechanism for storage of monomeric actin and controlled turnover of microfilaments.     

Profilin tyrosine phosphorylation in poly-L-proline-binding regions inhibits binding to phosphoinositide 3-kinase in Phaseolus vulgaris

The profilin family consists of a group of ubiquitous highly conserved 12-15 kDa eukaryotic proteins that bind actin, phosphoinositides, poly-l-proline (PLP) and proteins with proline-rich motifs. Some proteins with proline-rich motifs form complexes that have been implicated in the dynamics of the actin cytoskeleton and processes such as vesicular trafficking. A major unanswered question in the field is how profilin achieves the required specificity to bind such an array of proteins. It is now becoming clear that profilin isoforms are subject to differential regulation and that they may play distinct roles within the cell. Considerable evidence suggests that these isoforms have different functional roles in the sorting of diverse proteins with proline-rich motifs. All profilins contain highly conserved aromatic residues involved in PLP binding which are presumably implicated in the interaction with proline-rich motif proteins. We have previously shown that profilin is phosphorylated on tyrosine residues. Here, we show that profilin can bind directly to Phaseolus vulgaris phosphoinositide 3-kinase (PI3K) type III. We demonstrate that a new region around Y72 of profilin, as well as the N- and C-terminal PLP-binding domain, recognizes and binds PLP and PI3K. In vitro binding assays indicate that PI3K type III forms a complex with profilin in a manner that depends on the tyrosine phosphorylation status within the proline-rich-binding domain in profilin. Profilin-PI3K type III interaction suggests that profilin may be involved in membrane trafficking and in linking the endocytic pathway with actin reorganization dynamics.     

Deformations in actin comets from rocketing beads

The mechanical and dynamical properties of the actin network are essential for many cellular processes like motility or division, and there is a growing body of evidence that they are also important for adhesion and trafficking. The leading edge of migrating cells is pushed out by the polymerization of actin networks, a process orchestrated by cross-linkers and other actin-binding proteins. In vitro physical characterizations show that these same proteins control the elastic properties of actin gels. Here we use a biomimetic system of Listeria monocytogenes, beads coated with an activator of actin polymerization, to assess the role of various actin-binding proteins in propulsion. We find that the properties of actin-based movement are clearly affected by the presence of cross-linkers. By monitoring the evolution of marked parts of the comet, we provide direct experimental evidence that the actin gel continuously undergoes deformations during the growth of the comet. Depending on the protein composition in the motility medium, deformations arise from either gel elasticity or monomer diffusion through the actin comet. Our findings demonstrate that actin-based movement is governed by the mechanical properties of the actin network, which are fine-tuned by proteins involved in actin dynamics and assembly.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Isolation of low molecular weight actin-binding proteins from porcine brain

Three new actin-binding proteins having molecular weights of 26,000, 21,000, and 19,000 were isolated from porcine brain by DNase I affinity column chromatography. These proteins were released from the DNase I column by elution with a solution of high ionic strength. They were further purified by column chromatographies using hydroxyapatite, phosphocellulose, and Sephadex G-75. All of these actin-binding proteins behaved as monomeric particles in the gel filtration chromatography. After elution of the three actin-binding proteins, actin and profilin were recovered from the DNase I column with 2 M urea solution. The eluted was further purified by a cycle of polymerization and depolymerization and finally by gel filtration. Little difference in polymerizability was detected between the purified brain actin and muscle actin. After sedimentation of the polymerized brain actin, profilin was purified by DEAE-cellulose and gel filtration column chromatographies. In the assay of the action of these actin-binding proteins, the 26K protein was found to cause a large decrease in the rate of actin polymerization, while showing little effect on the extent of polymerization. The 21K protein decreased the steady-state viscosity of actin solution in a concentration-dependent manner irrespective of whether it was added before or after actin polymerization. It reacted with actin at a 1:1 molar ratio.     

Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells

Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell “softness” is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.     

Acto-myosin cytoskeleton dependent viscosity and shear-thinning behavior of the amoeba cytoplasm

The mechanical behavior of the human parasite Entamoeba histolytica plays a major role in the invasive process of host tissues and vessels. In this study, we set up an intracellular rheological technique derived from magnetic tweezers to measure the viscoelastic properties within living amoebae. The experimental setup combines two magnetic fields at 90° from each other and is adapted to an inverted microscope, which allows monitoring of the rotation of pairs of magnetic phagosomes. We observe either the response of the phagosome pair to an instantaneous 45° rotation of the magnetic field or the response to a permanent uniform rotation of the field at a given frequency. By the first method, we concluded that the phagosome pairs experience a soft viscoelastic medium, represented by the same mechanical model previously described for the cytoplasm of Dictyostelium discoideum [Feneberg et al. in Eur Biophys J 30(4):284–294 2001]. By the second method, the permanent rotation of a pair allowed us to apply a constant shear rate and to calculate the apparent viscosity of the cytoplasm. As found for entangled polymers, the viscosity decreases with the shear rate applied (shear-thinning behavior) and exhibits a power-law-type thinning, with a corresponding exponent of 0.65. Treatment of amoeba with drugs that affect the actin polymer content demonstrated that the shear-thinning behavior of the cytoplasm depends on the presence of an intact actin cytoskeleton. These data present a physiologic relevance for Entamoeba histolytica virulence. The shear-thinning behavior could facilitate cytoplasm streamings during cell movement and cell deformation, under important shear experienced by the amoeba during the invasion of human tissues. In this study, we also investigated the role of the actin-based motor myosin II and concluded that myosin II stiffens the F-actin gel in living parasites likely by its cross-linking activity.     

Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non- cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum- deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.