Actin-filament stochastic dynamics mediated by ADF/cofilin

BACKGROUND: The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, we combined evanescent-wave microscopy with a biomimetic system and followed the behavior of single actin filaments in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. RESULTS: In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. CONCLUSIONS: Our proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells.     

Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin

Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics.     

The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE₂ release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE₂ release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE₂ release. In fact, PGE₂ release was increased threefold in the polymerization inhibited cells and this PGE₂ release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE₂ release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE₂ release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton. mechanotransduction; cell mechanics     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo. phosphatidylinositol 4,5-bisphosphate; cytoskeleton; membrane traffic; Arp2/3; actin comets     

利用激光AFM和TEM探索肌动蛋白体外自装配聚合结构

细胞内肌动蛋白(actin)通过与actin结合蛋白(actin binding proteins,ABPs)相互作用,形成以F-actin为基础多种ABPs参与装配的高度有序的超分子聚合结构,行使各种重要生理功能。在体外聚合条件下,不存在F-actin稳定剂时纯化的actin主要通过自装配形成大尺度的聚集堆积结构;这种表观无序的结构体系由于被认为不具备细胞功能活性而受到忽视。利用激光原子力显微镜(atomic force microscope,AFM)和透射电子显微镜(transmission electron microscope,TEM)技术,对actin体外通过自装配过程形成的大尺度聚集结构进行了细致的观察和分析。研究发现,actin在体外通过自装配过程除了形成无序的蛋白堆积物之外,还能够聚合形成复杂的离散结构,包括树状分支的纤维丛、无规卷曲的纤维簇以及具有不同直径的长纤维等;这些大尺度纤维复合物明显不同于在ABPs或过量F-actin稳定剂参与下形成的由单根微丝和微丝束构成的聚合结构。表明无ABPs或F-actin稳定剂存在的情况下,体外聚合的F-actin在一定条件下可进一步聚集缠绕形成复杂的纤维结构或无序的蛋白堆积物。事实上,actin自装配过程反映了其固有的聚合热力学特性,深入探索将有助于理解ABPs在体内actin超分子聚合结构体系装配中的调控作用及其分子机制。     

ActA and human zyxin harbour Arp2/3-independent actin-polymerization activity

The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.     

Mechanisms of actin disassembly

The actin cytoskeleton is constantly assembling and disassembling. Cells harness the energy of these turnover dynamics to drive cell motility and organize cytoplasm. Although much is known about how cells control actin polymerization, we do not understand how actin filaments depolymerize inside cells. I briefly describe how the combination of imaging actin filament dynamics in cells and using in vitro biochemistry progressively altered our views of actin depolymerization. I describe why I do not think that the prevailing model of actin filament turnover—cofilin-mediated actin filament severing—can account for actin filament disassembly detected in cells. Finally, I speculate that cells might be able to tune the mechanism of actin depolymerization to meet physiological demands and selectively control the stabilities of different actin arrays.     

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.     

Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts

BACKGROUND: Actin filaments polymerize in vivo primarily from their fast-growing barbed ends. In cells and extracts, GTPgammaS and Rho-family GTPases, including Cdc42, stimulate barbed-end actin polymerization; however, the mechanism responsible for the initiation of polymerization is unknown. There are three formal possibilities for how free barbed ends may be generated in response to cellular signals: uncapping of existing filaments; severing of existing filaments; or de novo nucleation. The Arp2/3 complex localizes to regions of dynamic actin polymerization, including the leading edges of motile cells and motile actin patches in yeast, and in vitro it nucleates the formation of actin filaments with free barbed ends. Here, we investigated actin polymerization in soluble extracts of Acanthamoeba. RESULTS: Addition of actin filaments with free barbed ends to Acanthamoeba extracts is sufficient to induce polymerization of endogenous actin. Addition of activated Cdc42 or activation of Rho-family GTPases in these extracts by the non-hydrolyzable GTP analog GTPgammaS stimulated barbed-end polymerization, whereas immunodepletion of Arp2 or sequestration of Arp2 using solution-binding antibodies blocked Rho-family GTPase-induced actin polymerization. CONCLUSIONS: For this system, we conclude that the accessibility of free barbed ends regulates actin polymerization, that Rho-family GTPases stimulate polymerization catalytically by de novo nucleation of free barbed ends and that the primary nucleation factor in this pathway is the Arp2/3 complex.     

Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin

Pathogen-induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmonella by non-phagocytic intestinal epithelial cells. Two Salmonella actin-binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F-actin bundling, we demonstrate that SipA stimulates substantially SipC-mediated nucleation of actin polymerization. SipA additionally enhances SipC-mediated F-actin bundling, and SipC-SipA collaboration generates stable networks of F-actin bundles. The data show that bacterial SipC and SipA cooperate to direct efficient modulation of actin dynamics, independently of host cell proteins. The ability of SipA to enhance SipC-induced reorganization of the actin cytoskeleton in vivo was confirmed using semi-permeabilized cultured mammalian cells.     

Actin network growth under load

Many processes in eukaryotic cells, including the crawling motion of the whole cell, rely on the growth of branched actin networks from surfaces. In addition to their well-known role in generating propulsive forces, actin networks can also sustain substantial pulling loads thanks to their persistent attachment to the surface from which they grow. The simultaneous network elongation and surface attachment inevitably generate a force that opposes network growth. Here, we study the local dynamics of a growing actin network, accounting for simultaneous network elongation and surface attachment, and show that there exist several dynamical regimes that depend on both network elasticity and the kinetic parameters of actin polymerization. We characterize this in terms of a phase diagram and provide a connection between mesoscopic theories and the microscopic dynamics of an actin network at a surface. Our framework predicts the onset of instabilities that lead to the local detachment of the network and translate to oscillatory behavior and waves, as observed in many cellular phenomena and in vitro systems involving actin network growth, such as the saltatory dynamics of actin-propelled oil drops.     

Selective assay of monomeric and filamentous actin in cell extracts,using inhibition of deoxyribonuclease I

A simple and selective assay for monomeric and filamentous actin is presented, based on the inhibition of DNAase I by actin. In mixtures of monomeric and filamentous actin, only the monomeric form is measured as DNAase inhibitor. The total amount of actin in a sample can be determined after depolymerization of F actin with guanidine hydrochloride. The assay is rapid enough to detect changes in the polymerization state of actin in vitro over time intervals as short as 3 min. Data characterizing unpolymerized and filamentous actin pools in extracts of human platelets, lymphocytes and HeLa cells are presented.     

Gelsolin-actin interaction and actin polymerization in human neutrophils

The fraction of polymerized actin in human blood neutrophils increases after exposure to formyl-methionyl-leucyl-phenylalanine (fmlp), is maximal 10 s after peptide addition, and decreases after 300 s. Most of the gelsolin (85 +/- 11%) in resting ficoll-hypaque (FH)-purified neutrophils is in an EGTA resistant, 1:1 gelsolin-actin complex, and, within 5 s after 10(-7) M fmlp activation, the amount of gelsolin complexed with actin decreases to 42 +/- 12%. Reversal of gelsolin binding to actin occurs concurrently with an increase in F-actin content, and the appearance of barbed-end nucleating activity. The rate of dissociation of EGTA resistant, 1:1 gelsolin-actin complexes is more rapid in cells exposed to 10(-7) M fmlp than in cells exposed to 10(-9) M fmlp, and the extent of dissociation 10 s after activation depends upon the fmlp concentration. Furthermore, 300 s after fmlp activation when F-actin content is decreasing, gelsolin reassociates with actin as evidenced by an increase in the amount of EGTA resistant, 1:1 gelsolin-actin complex. Since fmlp induces barbed end actin polymerization in neutrophils and since in vitro the gelsolin-actin complex caps the barbed ends of actin filaments and blocks their growth, the data suggests that in FH neutrophils fmlp-induced actin polymerization could be initiated by the reversal of gelsolin binding to actin and the uncapping of actin filaments or nuclei. The data shows that formation and dissociation of gelsolin-actin complexes, together with the effects of other actin regulatory proteins, are important steps in the regulation of actin polymerization in neutrophils. Finally, finding increased amounts of gelsolin-actin complex in basal FH cells and dissociation of the complex in fmlp-activated cells suggests a mechanism by which fmlp can cause actin polymerization without an acute increase in cytosolic Ca++.     

N′-[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide is a dynamin GTPase inhibitor that suppresses cancer cell migration and invasion by inhibiting actin polymerization

Dynasore, a specific dynamin GTPase inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments. In search for novel dynamin inhibitors that suppress actin dynamics more efficiently, dynasore analogues were screened. N′-[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide (DBHA) markedly reduced in vitro actin polymerization, and dose-dependently inhibited phosphatidylserine-stimulated dynamin GTPase activity. DBHA significantly suppressed both the recruitment of dynamin 2 to the leading edge in U2OS cells and ruffle formation in H1299 cells. Furthermore, DBHA suppressed both the migration and invasion of H1299 cells by approximately 70%. Furthermore, intratumoral DBHA delivery significantly repressed tumor growth. DBHA was much less cytotoxic than dynasore. These results strongly suggest that DBHA inhibits dynamin-dependent actin polymerization by altering the interactions between dynamin and lipid membranes. DBHA and its derivative may be potential candidates for potent anti-cancer drugs.     

Fluxes of Water through Aquaporin 9 Weaken Membrane-Cytoskeleton Anchorage and Promote Formation of Membrane Protrusions

All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia, and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs) has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i) AQP9 induced and accumulated in filopodia, (ii) AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii) minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells.