Formation of filopodia-like bundles in vitro from a dendritic network

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.     

Pyramidal neuron morphogenesis requires a septin network that stabilizes filopodia and suppresses lamellipodia during neurite initiation

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EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.     

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

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Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell-cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell-cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin-actin cooperation that supports productive contact formation.     

F-actin binding region of SPIN90 C-terminus is essential for actin polymerization and lamellipodia formation

We recently reported that SPIN90 is able to bind with several proteins involved in regulating actin cytoskeleton networks, including dynamin, WASP, β PIX, and Nck. Based on these findings, we investigated how SPIN90 regulates the actin cytoskeleton and promotes actin assembly. This study demonstrated that aluminium fluoride-induced localization of SPIN90 to lamellipodia requires amino acids 582-722 at the SPIN90 C-terminus, which is also essential for F-actin binding and Arp2/3 complex mediated polymerization of actin into branched actin filaments. Furthermore, after deletion of the F-actin binding region (582-722 SPIN90) failed to localize at the membrane edge and was unable to promote lamellipodia formation, suggesting that the F-actin binding region in the SPIN90 C-terminus is essential for the formation of branched actin networks and regulation of the actin cytoskeleton at the leading edge of cells.     

Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.     

Disruption of the cytoskeleton during Semaphorin 3A induced growth cone collapse correlates with differences in actin organization and associated binding proteins

Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin‐rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here, we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp‐2 and cortactin decreases relative to total protein; whereas in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f‐actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

New insights into the formation and the function of lamellipodia and ruffles in mesenchymal cell migration

ABSTRACT

Lamellipodia and ruffles are veil-shaped cell protrusions composed of a highly branched actin filament meshwork assembled by the Arp2/3 complex. These structures not only hallmark the leading edge of cells adopting the adhesion-based mesenchymal mode of migration but are also thought to drive cell movement.

Although regarded as textbook knowledge, the mechanism of formation of lamellipodia and ruffles has been revisited in the last years leveraging new technologies. Furthermore, recent observations have also challenged our current view of the function of lamellipodia and ruffles in mesenchymal cell migration.

Here, I review this literature and compare it with older studies to highlight the controversies and the outstanding open issues in the field. Moreover, I outline simple and plausible explanations to reconcile conflicting results and conclusions. Finally, I integrate the mechanisms regulating actin-based protrusion in a unifying model that accounts for random and ballistic mesenchymal cell migration.     

Actin binding proteins

In order to metastasize away from the primary tumor site and migrate into adjacent tissues, cancer cells will stimulate cellular motility through the regulation of their cytoskeletal structures. Through the coordinated polymerization of actin filaments, these cells will control the geometry of distinct structures, namely lamella, lamellipodia and filopodia, as well as the more recently characterized invadopodia. Because actin binding proteins play fundamental functions in regulating the dynamics of actin polymerization, they have been at the forefront of cancer research. This review focuses on a subset of actin binding proteins involved in the regulation of these cellular structures and protrusions, and presents some general principles summarizing how these proteins may remodel the structure of actin. The main body of this review aims to provide new insights into how the expression of these actin binding proteins is regulated during carcinogenesis and highlights new mechanisms that may be initiated by the metastatic cells to induce aberrant expression of such proteins.     

Tropomyosins are present in lamellipodia of motile cells

This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised.     

Synapse formation is regulated by the signaling adaptor GIT1

Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.     

The mammalian Verprolin, WIRE induces filopodia independent of N-WASP through IRSp53

The mammalian verprolin family of proteins, WIP (WASP Interacting Protein), CR16 (Corticoid Regulated) and WIRE (WIp-RElated) regulate the actin cytoskeleton through WASP/N-WASP (Wiskott Aldrich Syndrome Protein and Neural-WASP). In order to characterize the WASP/N-WASP-independent function of WIRE, we screened and identified IRSp53 (Insulin Receptor Substrate) as a WIRE interacting protein. Expression of IRSp53 with WIRE in N-WASP^−/− mouse fibroblast cells induced filopodia while co-expression of IRSp53 with WIP did not. The induction of filopodia is dependent on WIRE-IRSp53 interaction as mutation in the SH3 domain of IRSp53 abolished WIRE-IRSp53 interaction as well as the ability to induce filopodia. Similarly, the Verprolin (V)-domain of WIRE is critical for IRSp53-WIRE interaction and for filopodia formation. The interaction between WIRE and IRSp53 is regulated by Cdc42 as mutations which abolish Cdc42-IRSp53 interaction lead to loss of IRSp53-WIRE interaction as shown by pull down assay. The plasma membrane localization of IRSp53 is dependent on Cdc42 and WIRE. Expression of Cdc42^G12V (active mutant) with WIRE-IRSp53 caused significant increase in the number of filopodia per cell. Thus our results show that Cdc42 regulates the activity of IRSp53 by regulating the IRSp53-WIRE interaction as well as localization of the complex to plasma membrane to generate filopodia.     

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.     

Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.     

Get to grips: steering local actin dynamics with IQGAPs

IQGAPs are actin-binding proteins that scaffold numerous interaction partners, transmitting extracellular signals that influence mitogenic, morphological and migratory cell behaviour. However, the precise mechanisms by which IQGAP proteins influence actin dynamics and actin filament structures have been elusive. Now that IQGAP1 has emerged as a potential key regulator of actin-cytoskeletal dynamics by recruiting both the actin related protein (Arp)2/3 complex and/or formin-dependent actin polymerizing machineries, we propose that IQGAP1 might coordinate the function of mechanistically different actin nucleators for cooperative localized actin filament production in various cellular processes.