Reversible stress softening of actin networks

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.     

Dependence of the mechanical properties of actin/alpha-actinin gels on deformation rate

The cortical cytoplasm, including the cleavage furrow, is largely composed of a network of actin filaments that is rigid even as it is extensively deformed during cytokinesis. Here we address the question of how actin-filament networks such as those in the cortex can be simultaneously rigid (solid-like) and fluid-like. Conventional explanations are that actin filaments rearrange by some combination of depolymerization and repolymerization; fragmentation and annealing of filaments; and inactivation and reestablishment of crosslinks between filaments. We describe the mechanical properties of a model system consisting of actin filaments and Acanthamoeba alpha-actinin, one of several actin crosslinking proteins found in amoeba and other cells. The results suggest another molecular mechanism that may account for the paradoxical mechanical properties of the cortex. When deformed rapidly, these mixtures are 40 times more rigid than actin filaments without alpha-actinin, but when deformed slowly these mixtures were indistinguishable from filaments alone. These time-dependent mechanical properties can be explained by multiple, rapidly rearranging alpha-actinin crosslinks between the actin filaments, a mechanism proposed by Frey-Wyssling to account for the behaviour of cytoplasm long before the discovery of cytoplasmic actin or alpha-actinin. If other actin-filament crosslinking proteins behave like Acanthamoeba alpha-actinin, this mechanism may explain how the cortex recoils elastically from small rapid insults but deforms extensively when minute forces are applied over long periods of time.     

Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate

The actin-binding protein gelsolin requires micromolar concentrations of calcium ions to sever actin filaments, to potentiate its binding to the end of the filament and to promote the polymerization of monomeric actin into filaments. Because transient increases in both intracellular [Ca2+] and actin polymerization accompany the cellular response to certain stimuli, it has been suggested that gelsolin regulates the reversible assembly of actin filaments that accompanies such cellular activations. But other evidence suggests that these activities do not need increased cytoplasmic [Ca2+] and that once actin-gelsolin complexes form in the presence of Ca2+ in vitro, removal of free Ca2+ causes dissociation of only one of two bound actin monomers from gelsolin and the resultant binary complexes cannot sever actin filaments. The finding that cellular gelsolin-actin complexes can be dissociated suggests that a Ca2+-independent regulation of gelsolin also occurs. Here we show that, like the dissociation of profilin-actin complexes, phosphatidylinositol 4,5-bisphosphate, which undergoes rapid turnover during cell stimulation, strongly inhibits the actin filament-severing properties of gelsolin, inhibits less strongly the nucleating ability of this protein and restores the potential for filament-severing activity to gelsolin-actin complexes.     

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.     

A gelsolin-like Ca2+-dependent actin-binding domain in villin

Villin is an actin-binding protein of relative molecular mass (Mr) 95,000 found in the core bundle of microfilaments in brush border microvilli from intestine. In physiological calcium concentrations (less than 1 microM), villin crosslinks actin filaments into bundles. However, in free calcium concentrations (greater than 1 microM), villin severs actin filaments into short pieces. To understand how villin can sever and bundle actin filaments, we are studying the molecular basis of villin-actin binding interactions by identifying important actin-binding domains in villin. Here, we report the purification and preliminary characterization of a 44,000-Mr fragment of villin which contains a calcium-dependent actin-severing activity. In addition, the partial amino-acid sequence from the amino terminus of this fragment reveals homology with a 16-residue region near the amino terminus of gelsolin, an actin-severing protein found in many cells and sera. The sequence homology suggests a common structural basis for the calcium-regulated actin-severing properties shared by villin and gelsolin.     

Effect of ATP on actin filament stiffness

Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function.     

Requirement of phosphatidylinositol 4,5-bisphosphate for alpha-actinin function.

Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.     

Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.     

Cytochalasin D does not produce net depolymerization of actin filaments in HEp-2 cells

The altered morphology, disappearance or 'disruption' of actin filaments (microfilaments) in cells treated with cytochalasin has sometimes been attributed to depolymerization of filamentous actin (F-actin) to its globular subunit (G-actin), but attempts to confirm that mechanism have been inconclusive. Treatment of purified actin filaments with cytochalasin B (CB) decreased their viscosity, consistent with depolymerization, which was not, however, revealed by electron microscopy, although the filaments appeared abnormal. CB also increased the ATP-ase activity of F-actin, suggesting that it had been destabilized, while actin filaments in the acrosomal process were not depolymerized. CB or cytochalasin D (CD) can dissolve actin gels (reviewed in ref. 7, see also refs 8 and 9) without depolymerizing their filaments. The 'disrupted' actin structures in CD-treated cells bound heavy meromysin, indicating that at least some of the cellular actin was filamentous. Using a rapid assay for G- and F-actin in cell extracts, based on the inhibition of DNase I, we have found that neither short-nor long-term exposure of HEp-2 cells to CD produce net depolymerization of actin filaments.     

Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I

In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. We have now cloned and sequenced the gene encoding ABP1p. ABP1p is a novel protein with a 50 amino-acid C-terminal domain that is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. We also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton.     

Drosophila Spire is an actin nucleation factor

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.     

Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.     

Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain

Gelsolin is representative of a class of actin-modulating proteins found in lower eukaryotes to mammals, which sever actin filaments. Gelsolin found in the cytoplasm of cells is functionally similar to a mammalian plasma protein of similar size, originally called ADF or brevin. Human plasma and rabbit macrophage gelsolins differ by the presence of a 25-amino-acid residue extension on plasma gelsolin which appears to account for the difference in relative molecular mass (Mr) between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (PAGE), 93,000 (93K) and 90K, respectively. Here we report the isolation of full-length human plasma gelsolin complementary DNA clones from a HepG2 library. The inferred amino-acid sequence reveals the presence of a signal peptide, a long tandem repeat that matches the actin-binding domains of gelsolin, a tetrapeptide present in actin and extended regions of identical sequence with rabbit macrophage gelsolin. Southern blot analysis indicates that a single gene in the haploid genome encodes both protein forms.     

Identification of an actin-binding protein from Dictyostelium as elongation factor 1a

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.     

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.     

Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.     

Expression of a cotton profilin gene GhPFN1 is associated with fiber cell elongation

Profilin is an important actin-binding protein involved in regulating the organization of actin filaments. A cotton profilin gene (GhPFN1) that shares 71% identity to profilin1 of Arabidopsis in its amino acid sequence was isolated. Semi-quantitative RT-PCR showed that GhPFN1 was expressed preferentially in the developing cotton fibers and reached the highest level at the fast elongation stage. The function of GhPFN1 in vivo was analyzed using the S. pombe system, and results suggested that GhPFN1 plays a role in fiber cell elongation.     

Sliding movement of single actin filaments on one-headed myosin filaments

The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread, superprecipitation and chemical modification of muscle fibres. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.     

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a β-actin-GFP fusion, and evidence was found that actin is replaced, from the top down, in 2-3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12-24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells. In contrast, turnover in chick stereocilia in vivo is much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a (15)N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing β-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of β- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.     

Requirement of yeast fimbrin for actin organization and morphogenesis in vivo

The SAC6 gene was found by suppression of a yeast actin mutation. Its protein product, Sac6p (previously referred to as ABP67), was independently isolated by actin-filament affinity chromatography and colocalizes with actin in vivo. Thus Sac6p binds to actin in vitro, and functionally associates with actin structures involved in the development and maintenance of cell polarity in vivo. We report here that Sac6p is an actin-filament bundling protein 43% identical in amino-acid sequence to the vertebrate bundling protein fimbrin. This yeast fimbrin homologue contains two putative actin-binding regions homologous to domains of dystrophin, beta-spectrin, filamin, actin-gelation protein and alpha-actinin. Mutants lacking Sac6p do not form normal actin structures and are defective in morphogenesis. These findings demonstrate an in vivo role for the well-documented biochemical interaction between fimbrin and actin.