Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles. I. PREFACE ON ACTIN ORGANIZATION AND CELL MECHANICSAnimal cells (i.e., those without a cell wall) have the ability to change their shape to adapt to their environment, move through narrow spaces, divide, or allow exo-and endocytosis. The machinery of these shape changes relies on the assembly of proteins, in particular actin, a globular protein that polymerizes into filaments of different types of organization: branched and crosslinked networks, parallel bundles, and anti-parallel contractile structures (FIGURE 1A). These different architectures can be envisioned as a series of interconnected active springs and dashpots (green and red symbols in FIGURE 1B, respectively) that act as mechanical elements to drive cell shape changes and motility. The purpose of this review is to correlate recent progress in our understanding of the interplay between biochemical elements and mechanical properties. Instead of describing biochemical and mechanical properties separately, our main goal here is to address piece by piece the integrated feedback loop between biochemistry and mechanics.At the front of the cell, branched and crosslinked networks in a quasi two-dimensional sheet make up the lamellipodium, and are the major engine of cell movement since they push the cell membrane by polymerizing against it (FIGURE 1A, iii). Aligned bundles underlie filopodia that are the fingerlike structures at the front of the cell, important for directional response of the cell (FIGURE 1A, iv). A thin layer of actin, called the cell cortex, coats the plasma membrane at the back and sides of the cell, important for cell shape maintenance and changes (FIGURE 1A, i). The rest of the cell contains a three-dimensional network of crosslinked filaments interspersed with contractile bundles, including stress fibers that connect the cell cytoskeleton to the extracellular matrix via focal adhesion sites (FIGURE 1A, ii). Contraction in the cell is produced by the molecular motor protein myosin. Myosin assembles into anti...

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture is maintained in a dynamic steady-state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization myosins selectively contracted and disassembled anti-parallel actin structures while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This “orientation selection” mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

Summary Cell motility driven by actin filament assembly demands the spatial and temporal coordination of numerous regulatory Actin Binding Proteins (ABPs) [1], many of which bind with affinities and kinetics that depend on the chemical state (ATP, ADP-Pi or ADP) of actin filament subunits. ADF/cofilin, one of three ABPs that precisely choreograph actin assembly and organization into “comet-tails” that drive motility in reconstituted in vitro systems [2], binds and stochastically severs “aged” ADP actin filament segments of de novo growing actin filaments [3]. Severing increases the density of filament ends from which subunits can add and dissociate, thereby increasing overall actin filament assembly dynamics. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments as well as ADF/cofilin severing limits the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled ADF/cofilin that retains actin filament binding and severing activities. Since ADF/cofilin binding depends strongly on the actin-bound nucleotide direct visualization of fluorescent ADF/cofilin binding serves as a marker of the actin filament nucleotide state and permits assessment of the “ATP/ADP-Pi cap” length of individual actin filaments during assembly and elongation. Bound ADF/cofilin allosterically accelerates Pi release from unoccupied filament subunits, which shortens the filament ATP/ADP-Pi cap length by nearly an order of magnitude. Rapid elongation far exceeds ADF/cofilin-acceleration of Pi release under in vivo conditions; thereby filament barbed end capping is required for efficient ADF/cofilin binding and severing. Real time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and ADF/cofilin decorated filament segments, thereby controlling the overall filament length depending on the ADF/cofilin activity and filament binding density.

, and colleagues. Actin fi laments power multiple cellular processes such as motility, morphogen-esis, and polarity, but the mechanisms controlling their dynamics is poorly understood. Michelot et al. investigated the dynamics of actin building, bundling, and breakage in vitro by covering polystyrene beads with an actin fi lament-promoting protein called formin and then adding fl uorescently labeled actin monomers. The formin and actin monomers were enough to induce continuous actin poly-merization at the surface of the bead. When an actin-severing factor called cofi lin was added to the mix, fi laments began to switch rapidly between elongation and shortening. The shortening always occurred from the fi lament's "older" end (the one further from the bead). According to Blanchoin, this end preference results from the gradual conversion of each actin monomer's ATP to ADP after its incorporation into the fi lament, as only the ADP form is a suitable cofi lin substrate. As fi laments grew out from the bead, neighboring fi laments often "zipped" together to form thicker multifilament cables. These cables were considerably more resistant to cofi lin severing than were individual fi laments, probably because cofi lin cuts one fi lament at a time and thus would only nick the cable rather than chop right through. The in vitro system used by the authors involved a minimal set of proteins and yet show that actin fi laments can generate their own stability simply by grouping together. The team now plans to add other actin associated factors, one by one, to observe how increasing complexity affects actin dynamics.

Cells use actin assembly to generate forces for membrane protrusions during movement [1] or, in the case of pathogens, to propel themselves in the host cells, in crude extracts [2], 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 [4-6]. 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.

Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators 1-3 in vivo [4][5][6][7] . Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices [8][9][10][11][12][13][14][15][16][17][18] . However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied 14,19 . Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures.In cells, actin nucleation occurs at various locations at the plasma membrane, and bundles of parallel actin filaments are initiated at focal adhesion sites 1 or result from the rearrangement of the dynamic branched actin network of the lamellipodium 6 . Here, we modulate the positioning of nucleation sites at scales corresponding to cellular dimensions. As a first step and to precisely regulate the position of actin nucleation sites in vitro, we used a recently developed ultraviolet-based micropatterning approach 20 to create a template for the localization of the nucleation promoting factor pWA (Fig. 1a). pWA comprises the C-terminal domains from the WASP/Scar proteins, a ubiquitous family of proteins that initiate actin polymerization on a pre-existing actin filament in the presence of the Arp2/3 complex and an actin monomer [21][22][23] . A small volume of solution made of a minimal set of purified proteins ensuring actin polymerization 8,10 was placed between the pWA-coated micropatterned slide and a glass support. Functionalized micropatterns specifically initiate actin filament nucleation on their surface and promote two-dimensional growth of actin filaments (Fig. 1b). Real-time visualization of actin-filament nucleation and growth highlighted the autocatalytic process of network formation (see Supplementary Fig. S1). These networks consist of filaments growing from the pWA-coated regions, with their fast-growing, barbed end oriented outwards ( Fig. 1c and Supplementary Fig. S2 and Videos S1-S3). In agreement with actin-filament growth on glass rods 15 , as the nucleation waves propagate, dense and interconnected

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.

No abstract