In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.     

Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Contractile proteins in phagocytosis: an example of cell surface-to-cytoplasm communication.

Phagocytosis is a prime example of a cellular event in which cell surface perturbation activates the assembly of a filamentous gel beneath the plasma membrane. This gel may be responsible for movement of the membrane around ingestible particles. The molecular mechanism of these events is being approached by the purification of actin, myosin and associated proteins from phagocytic cells and by the study of a human disease, neutrophil actin dysfunction. Novel contractile proteins discovered in mammalian phagocytes include a cofactor that regulates actin:myosin interaction and an actin-binding protein that promotes assembly and gelation of actin. There is evidence that phagocytosis alters the state of the actin-binding protein, and that this alteration may be an early event in the assembly of the actin gel. Cytochalasin B, which inhibits phagocytosis, acts by interfering with the interaction between actin-binding protein and actin. Actin polymerized poorly in the neutrophils of a human infant, and the affected neutrophils were deficient in phagocytosis. Actin assembly is important in phagocytosis and is amenable to biochemical analysis.     

The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba

The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.     

Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton

Podocytes of the renal glomerulus are unique cells with a complex cellular organization consisting of a cell body, major processes and foot processes. Podocyte foot processes form a characteristic interdigitating pattern with foot processes of neighboring podocytes, leaving in between the filtration slits that are bridged by the glomerular slit diaphragm. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells or pericytes. Mutations affecting several podocyte proteins lead to rearrangement of the actin cytoskeleton, disruption of the filtration barrier and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context.     

The contractile basis of ameboid movement. VI. The solation-contraction coupling hypothesis

The contracted pellets derived from a high-speed supernate of Dictyostelium discoideum (S3) were investigated to determine the functional activity associated with this specific subset of the cellular motile apparatus. A partially purified model system of gelation and contraction (S6) was prepared from the contracted pellets, and the presence of calcium- and pH-sensitive gelation and contraction in this model demonstrated that a functional cytoskeletal-contratile complex remained at least partially associated with the actin and myosin during contraction. Semi-quantitative assays of gelation and solation in the myosin-free preparation S6 included measurements of turbidity, relative viscosity, and strain birefringence. The extent of gelation was optimal at pH 6.8 and a free calcium ion concentration of approximately 3.0 x 10(-8) M. Solation was favored when the free calcium ion concentration was greater than 7.6 x 10(-7) M or when the pH was increased or decreased from pH 6.8. Gelation was reversibly inhibited by increasing the free calcium ion concentration to approxomately 4.6 x 10(-6) M at pH 6.8. The solation-gelation process of this model has been interpreted to involve the reversible cross-linking of actin filaments. The addition of purified D. discoideum myosin to S6 served to reconstitute calcium- and pH-regulated contraction. The results from this study indicate that contraction is coupled functionally to the local breakdown (solation) of the gel. Therefore, solation has been identified as a structural requirement for extensive shortening during contraction. We have called this concept the solation-contraction coupling hypothesis. Fractionation of a preparation derived from the contracted pellets yielded a fraction consisting of actin and a 95,000-dalton polypeptide that exhibited calcium-sensitive gelation at 28 degrees C and a fraction composed of actin and 30,000- and 18,000-dalton polypeptides that demonstrated calcium-sensitive genlation at 0 degrees C.     

The role of solation-contraction coupling in regulating stress fiber dynamics in nonmuscle cells

Serum-deprived Swiss 3T3 fibroblasts constitutively form stress fibers at their edges. These fibers move centripetally towards the perinuclear region where they disassemble. Serum stimulation causes shortening of fibers in a manner suggesting active actin-myosin-based contraction (Giuliano, K.A. and D.L. Taylor. 1990. Cell Motil. and Cytoskeleton. 16:14-21). To elucidated the role of actin-based gel structure in these movements, we examined the effects of disrupting actin organization with cytochalasin. Serum-deprived fibroblasts were microinjected with rhodamine analogs of actin or myosin II and fiber dynamics were monitored with a multimode light microscope workstation using video-enhanced contrast and fluorescence modes. When cells were perfused with greater than or equal to 3 microM cytochalasin B or 0.5 microM cytochalasin D, formation and transport of stress fibers were reversibly inhibited, and rapid and immediate shortening of existing fibers was induced. Quantification of actin and myosin II fluorescence associated with individual shortening fibers demonstrated that fluorescence per length of fiber increased for both components, suggesting sliding filament contraction. However, there was also a net loss of both actin and myosin II from fibers as they shortened, indicating a self-destructive process. Loss of material from fibers coupled with increased overlap of actin and myosin II remaining in the fibers suggested that contraction could be induced not only by increasing the force exerted by contractile motors, but also by decreasing gel structure through partial solation. Finally, cytochalasin accelerated contraction of actin-myosin-based gels reconstituted from purified proteins in the absence of myosin-based regulation, further supporting solation-contraction coupling as a possible mechanism for modulating cytoplasmic contractility (Taylor, D.L. and M. Fechheimer. 1982. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 299:185-197).     

A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium

We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.     

Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge.     

The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses.

Smooth muscle exhibits biophysical characteristics and physiological behaviors that are not readily explained by present paradigms of cytoskeletal and cross-bridge mechanics. There is increasing evidence that contractile activation of the smooth muscle cell involves an array of cytoskeletal processes that extend beyond cross-bridge cycling and the sliding of thick and thin filaments. We review here the evidence suggesting that the biophysical and mechanical properties of the smooth muscle cell reflect the integrated interactions of an array of highly dynamic cytoskeletal processes that both react to and transform the dynamics of cross-bridge interactions over the course of the contraction cycle. The activation of the smooth muscle cell is proposed to trigger dynamic remodeling of the actin filament lattice within cellular microdomains in response to local mechanical and pharmacological events, enabling the cell to adapt to its external environment. As the contraction progresses, the cytoskeletal lattice stabilizes, solidifies, and forms a rigid structure well suited for transmission of tension generated by the interaction of myosin and actin. The integrated molecular transitions that occur within the contractile cycle are interpreted in the context of microscale agitation mechanisms and resulting remodeling events within the intracellular microenvironment. Such an interpretation suggests that the cytoskeleton may behave as a glassy substance whose mechanical function is governed by an effective temperature.     

Actin-based centripetal flow: phosphatase inhibition by calyculin-A alters flow pattern, actin organization, and actomyosin distribution

Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.     

Invasion inhibition by a MEK inhibitor correlates with the actin-based cytoskeleton in lung cancer A549 cells

Metastasis remains the primary cause of lung cancer. The molecules involved in metastasis may be candidates for new targets in the therapy of lung cancer. The MEK/ERK signaling pathway has been highlighted in a number of studies on invasiveness and metastasis. In this paper, we show that the MEK inhibitor U0126 induces flattened morphology, remodels the actin-based cytoskeleton, and potently inhibits chemotaxis and Matrigel invasion in the human lung cancer A549 cell line. Furthermore, downregulation of ERK by small interfering RNA significantly inhibits the invasion of A549 cells and induces stress fiber formation. Taken together, our findings provide the first evidence that the inhibition of invasion of lung cancer A549 cells by inhibiting MEK/ERK signaling activity is associated with remodeling of the actin cytoskeleton, suggesting a novel link between MEK/ERK signaling-mediated cell invasion and the actin-based cytoskeleton.     

A novel biosensor for mercuric ions based on motor proteins

We explored the potential of contractile proteins, actin and myosin, as biosensors of solutions containing mercuric ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 microm long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.     

Reconstitution of contractile actomyosin bundles

Contractile actomyosin bundles are critical for numerous aspects of muscle and nonmuscle cell physiology. Due to the varying composition and structure of actomyosin bundles in vivo, the minimal requirements for their contraction remain unclear. Here, we demonstrate that actin filaments and filaments of smooth muscle myosin motors can self-assemble into bundles with contractile elements that efficiently transmit actomyosin forces to cellular length scales. The contractile and force-generating potential of these minimal actomyosin bundles is sharply sensitive to the myosin density. Above a critical myosin density, these bundles are contractile and generate large tensile forces. Below this threshold, insufficient cross-linking of F-actin by myosin thick filaments prevents efficient force transmission and can result in rapid bundle disintegration. For contractile bundles, the rate of contraction decreases as forces build and stalls under loads of ∼0.5 nN. The dependence of contraction speed and stall force on bundle length is consistent with bundle contraction occurring by several contractile elements connected in series. Thus, contraction in reconstituted actomyosin bundles captures essential biophysical characteristics of myofibrils while lacking numerous molecular constituents and structural signatures of sarcomeres. These results provide insight into nonsarcomeric mechanisms of actomyosin contraction found in smooth muscle and nonmuscle cells.     

A multi-modular tensegrity model of an actin stress fiber

Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on the extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model can also explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors and represent a new handle on multi-scale modeling of living materials.     

Calcium Regulation of an Actin Spring

Calcium is essential for many biological processes involved in cellular motility. However, the pathway by which calcium influences motility, in processes such as muscle contraction and neuronal growth, is often indirect and complex. We establish a simple and direct mechanochemical link that shows how calcium quantitatively regulates the dynamics of a primitive motile system, the actin-based acrosomal bundle of horseshoe crab sperm. The extension of this bundle requires the continuous presence of external calcium. Furthermore, the extension rate increases with calcium concentration, but at a given concentration, we find that the volumetric rate of extension is constant. Our experiments and theory suggest that calcium sequentially binds to calmodulin molecules decorating the actin filaments. This binding leads to a collective wave of untwisting of the actin filaments that drives bundle extension.     

The Contractile Proteins and Calcium Mediated Gelation and Contraction of Pollen Tube Extracts

The crude extracts of pollen tubes, like other nonmuscle ceils, showed gelation at Iow Ga2+ concentrations and ATP-dependent contraction at higher Ga2+ concentrations. The contracted cytoplasmic clots contained a lot of filaments which were mainly composed of actin, myosin, 105 kD, 67 kD, 48 kD, 38 kD, 34 kD and 28 kD proteins. It is likely that Ca2+ are able to mediate tranformation of acfin from a less ordered state to a more oriented filaments, which interact with actin-binding proteins to form the filamentous network, thus to induce the gel formation of cytoplasm, to regulate the interaction of actin and myosin which transform the chemical energy of ATP into mechanical work of contractile movement of cytoplasm.     

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.