Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin (original) (raw)
Related papers
Control of Actin Filament Treadmilling in Cell Motility
Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have furthered our understanding of the molecular mechanisms by which regulated assembly dynamics of actin filaments drive motile processes. Attention is focused on lamellipodium protrusion, powered by the turnover of a branched filament array. ATP hydrolysis on actin is the key reaction that allows filament treadmilling. It regulates barbed-end dynamics and length fluctuations at steady state and specifies the functional interaction of actin with essential regulatory proteins such as profilin and ADF/cofilin. ATP hydrolysis on actin and Arp2/3 acts as a timer, regulating the assembly and disassembly of the branched array to generate tropomyosin-mediated heterogeneity in the structure and dynamics of the lamellipodial network. The detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly. Novel biophysical methods involving single-molecule measurements should foster progress in these crucial issues. 449 Annu. Rev. Biophys. 2010.39:449-470. Downloaded from arjournals.annualreviews.org by 89.133.73.22 on 05/25/10. For personal use only.
Cytoskeleton, 2011
Membrane protrusion at the leading edge of migrating cells is driven by the polymerization of actin. Recent studies using advanced imaging techniques raised a lively controversy about the morphology of these filaments; however, common ground between the two sides now appears to have been found. Here we discuss how the controversy has led to a deeper consideration of the architecture of actin networks underlying cell migration, and has helped define new challenges that lie ahead.
A “Primer”-Based Mechanism Underlies Branched Actin Filament Network Formation and Motility
Current Biology, 2010
Cells use actin assembly to generate forces for membrane protrusions during movement or, in the case of pathogens, to propel themselves in the host cells, in crude extracts , or in mixtures of actin and other purified proteins [3]. Significant progress has been made in understanding the mechanism of actin-based motility at a macroscopic level by using biomimetic systems in vitro . Here, we combined such a system with evanescent wave microscopy to visualize Arp2/3-mediated actin network formation at single-actin-filament resolution. We found that actin filaments that we call ''primers'' determine the origin of the autocatalytic and propagative formation of the actin network. In the presence of capping protein, multiple ''primers'' generate independent networks that merge around the object to form an outer ''shell'' made of entangled and capped filaments. Simultaneously, newly created filaments on the surface of the particle initiate mechanical stress, which develops until symmetry breaking. Our results and extensive modeling support that the stress, which releases into propulsive forces [7], is controlled not by any specific orientation of actin filaments toward the nucleation sites but only by new monomers added near the load surface.
Arp2/3 Branched Actin Network Mediates Filopodia-Like Bundles Formation In Vitro
PLoS ONE, 2008
During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.
Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins
Journal of Biological Chemistry, 2005
Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG and gelsolin:actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly and restrict the access of barbed end capping proteins.
Proceedings of the National Academy of Sciences of the United States of America , 2017
Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin–binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligo-merization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules syner-gize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays. actin | Ena/VASP | TIRF | cluster | formin
2022
Actin filament polymerization can be branched or linear, which depends on the associated regulatory proteins. Competition for Actin monomers occurs between proteins that induce branched or linear actin polymerization. Cell specialization requires the regulation of actin filaments to allow the formation of cell-type specific structures, like cuticular hairs in Drosophila, formed by linear actin filaments. Here, we report the functional analysis of CG34401/pelado, a gene encoding a SWIM domain containing protein, conserved throughout the animal kingdom. Mutant pelado epithelial cells display actin hair elongation defects. This phenotype is reversed by increasing Actin monomer levels or by either pushing linear actin polymerization or reducing branched actin polymerization. The same behavior occurs in hemocytes, where Pelado is essential to induce filopodia, a linear actin-based structure. We further show that this function of Pelado, ZSWIM8 in mammals, is conserved in human cells, whe...
Actin’s propensity for dynamic filament patterning
FEBS Letters, 2002
Actin, through its various forms of assembly, provides the basic framework for cell motility, cell shape and intracellular organization in all eukaryotic cells. Many other cellular processes, for example endocytosis and cytokinesis, are also associated with dynamic changes of the actin cytoskeleton. Important prerequisites for actin's functional diversity are its intrinsic ability to rapidly assemble and disassemble ¢laments and its spatially and temporally well-controlled supramolecular organization. A large number of proteins that interact with actin, collectively referred to as actin-binding proteins (ABPs), carefully orchestrate di¡erent scenarios. Since its isolation in
Emergence of Large-Scale Cell Morphology and Movement from Local Actin Filament Growth Dynamics
PLoS Biology, 2007
Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actinremodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged ''canoe'' shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features-cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence-that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes-which had VASP highly enriched at their leading edges and migrated fast with straight trajectories-to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior phenotypes as large-scale consequences of kinetic contributions of VASP to actin filament growth and protection from capping at the leading edge. This work shows that the local effects of actinremodeling proteins on cytoskeletal dynamics and organization can manifest as global modifications of the shape and behavior of migrating cells and that mathematical modeling can elucidate these large-scale cell behaviors from knowledge of detailed multiscale protein interactions. Citation: Lacayo CI, Pincus Z, VanDuijn MM, Wilson CA, Fletcher DA, et al. (2007) Emergence of large-scale cell morphology and movement from local actin filament growth dynamics. PLoS Biol 5(9): e233.