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Polymerization of actin filaments against membranes produces force for numerous cellular processes, such as migration, morphogenesis, endocytosis, phagocytosis and organelle dynamics. Consequently, aberrant actin cytoskeleton dynamics are linked to various diseases, including cancer, as well as immunological and neurological disorders. Understanding how actin filaments generate forces in cells, how force production is regulated by the interplay between actin-binding proteins and how the actin-regulatory machinery responds to mechanical load are at the heart of many cellular, developmental and pathological processes. During the past few years, our understanding of the mechanisms controlling actin filament assembly and disassembly has evolved substantially. It has also become evident that the activities of key actin-binding proteins are not regulated solely by biochemical signalling pathways, as mechanical regulation is critical for these proteins. Indeed, the architecture and dynamics of the actin cytoskeleton are directly tuned by mechanical load. Here we discuss the general mechanisms by which key actin regulators, often in synergy with each other, control actin filament assembly, disassembly, and monomer recycling. By using an updated view of actin dynamics as a framework, we discuss how the mechanics and geometry of actin networks control actin-binding proteins, and how this translates into force production in endocytosis and mesenchymal cell migration.Actin cytoskeleton underlies key cellular processes, such as membrane dynamics and cell migration. Despite years of research, how cells regulate actin filament assembly and disassembly to establish dynamic actin structures that fulfil these functions remains an exciting area of study.

Key Points Cells alter their migratory phenotypes and velocity in response to the physical properties of their extracellular environment. Confinement, adhesion, stiffness and topology of the extracellular environment are key physical variables influencing cell migration. Univariate profiles and phase diagrams enable an understanding of how physical variables influence cell migration. Numerical simulations enable systematic exploration of the phase space to highlight regions for experimental exploration. The physical properties of the extracellular environment — in terms of confinement, rigidity, surface topology and adhesion-ligand density — can have profound effects on the migration strategy and migration velocity of cells in different in vivo contexts. The way in which a cell migrates is influenced by the physical properties of its surroundings, in particular the properties of the extracellular matrix. How the physical aspects of the cell's environment affect cell migration poses a considerable challenge when trying to understand migration in complex tissue environments and hinders the extrapolation of in vitro analyses to in vivo situations. A comprehensive understanding of these problems requires an integrated biochemical and biophysical approach. In this Review, we outline the findings that have emerged from approaches that span these disciplines, with a focus on actin-based cell migration in environments with different stiffness, dimensionality and geometry.

Living cells actively migrate in their environment to perform key biological functions—from unicellular organisms looking for food to single cells such as fibroblasts, leukocytes or cancer cells that can shape, patrol or invade tissues. Cell migration results from complex intracellular processes that enable cell self-propulsion, and has been shown to also integrate various chemical or physical extracellular signals. While it is established that cells can modify their environment by depositing biochemical signals or mechanically remodelling the extracellular matrix, the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here, we show that cells can retrieve their path: by confining motile cells on 1D and 2D micropatterned surfaces, we demonstrate that they leave long-lived physicochemical footprints along their way, which determine their future path. On this basis, we argue that cell trajectories belong to the general class of self-interacting random walks, and show that self-interactions can rule large scale exploration by inducing long-lived ageing, subdiffusion and anomalous first-passage statistics. Altogether, our joint experimental and theoretical approach points to a generic coupling between motile cells and their environment, which endows cells with a spatial memory of their path and can dramatically change their space exploration. Cells can modify their environment by depositing biochemical signals or mechanically remodelling the extracellular matrix; the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here authors show that motile cells leave long-lived physicochemical footprints along their way, which determine their future path.

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly. The assembly of branched actin networks depends on the heterodimeric capping protein CP/CapZ. Combining cryoEM, in vitro reconstitution and cell biological assays, the authors show that CP not only prevents actin filament elongation but also selectively masks actin filament ends to promote nucleation.

Key Points Lamellipodial protrusion depends on the force generated by actin polymerization. Actin polymerization is the sum of the activities of nucleators — for example, the actin-related protein 2/3 (ARP2/3) complex — and elongators — formins and ENA/VASP proteins. Small GTPases, such as RAC and CDC42, control both actin nucleators and actin elongators; RAC activates the WASP family verprolin-homologous protein (WAVE) complex upstream of the ARP2/3 complex independently of the activation of the formin FMNL2 by CDC42, but RAC may coordinate ARP2/3 with ENA/VASP proteins by inducing a complex between WAVE and lamellipodin. The speed of cell migration depends on the turnover of actin branched junctions and on the elongation of actin networks. An intrinsic instability of lamellipodia is due to ARP2/3 inhibitory proteins, such as Arpin, which is also activated downstream of RAC. The persistence of lamellipodia is the major controller of cell directionality. Directional persistence (that is, the characteristic time during which a cell sustains its migration in the same direction) is the combinatory result of several intertwined positive- and negative-feedback loops that sustain or stop actin polymerization at the leading edge. Lamellipodial protrusion is powered by actin polymerization that is mediated through the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. These processes are regulated by positive and negative feedback loops centred around the GTPase RAC, and the balance between them determines lamellipodial and directional persistence during cell migration. Membrane protrusions at the leading edge of cells, known as lamellipodia, drive cell migration in many normal and pathological situations. Lamellipodial protrusion is powered by actin polymerization, which is mediated by the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. Recently, advances have been made in our understanding of positive and negative ARP2/3 regulators (such as the SCAR/WAVE (SCAR/WASP family verprolin-homologous protein) complex and Arpin, respectively) and of proteins that control actin branch stability (such as glial maturation factor (GMF)) or actin filament elongation (such as ENA/VASP proteins) in lamellipodium dynamics and cell migration. This Review highlights how the balance between actin filament branching and elongation, and between the positive and negative feedback loops that regulate these activities, determines lamellipodial persistence. Importantly, directional persistence, which results from lamellipodial persistence, emerges as a critical factor in steering cell migration.

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α 2a - and β 1 -adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME). This study describes a fast, clathrin-independent endocytic pathway mediated by endophilin, dynamin and actin; the pathway is activated by ligand binding to a variety of cargo receptors, and endophilin-mediated endocytosis occurs primarily at the leading edges of cells where lamellipodin and the lipid PtdIns(3,4)P 2 ensure endophilin targeting. Endocytosis and cell signalling Cells internalize nutrients and turnover membrane components through the process of endocytosis, which in most cases involves the protein clathrin. Endophilin has been thought to be a component of clathrin-mediated endocytosis, but two studies published in this issue of Nature show that this protein mediates a fast-acting, clathrin-independent form of endocytosis which involves formation of tubular vesicles. Emmanuel Boucrot et al . report that this pathway is triggered by binding of ligands to cargo receptors, and requires the proteins dynamin and actin. Endophilin-mediated endocytosis also seems to have distinct cellular homes, occurring at the leading edges of cells where the lipid PtdIns(3,4)P 2 ensures endophilin engagement. This form of endocytosis is shown to mediate the uptake of several physiological and disease-relevant receptors including G-protein-coupled receptors and receptor tyrosine kinases. In the second paper, Henri-François Renard et al . provide evidence that bacterial toxins take advantage of the same pathway to enter cells, and also find that endophilin-A2 acts together with dynamin and actin.

Key Points Capping protein (CP) is a major regulator of actin assembly dynamics via the capping of actin filament barbed ends. The capping activity of CP can be regulated by a number of different proteins and phospholipids in various ways, some direct and others indirect. The capping protein interacting (CPI) motif is a 30-amino acid region necessary and sufficient to bind and inhibit CP. This motif is found in a set of unrelated proteins, many of which are involved in membrane interactions. CARMIL (capping protein, ARP2/3 and myosin I linker) family proteins contain a CPI motif, and they also contain a separate CARMIL-specific interacting (CSI) motif. In CARMIL, the CPI motif is necessary for distinct cellular functions, such as macropinocytosis. The CPI and CSI motifs are unstructured in the unbound state, but they adopt a specific structure when they bind to CP, applying themselves to the surface of CP. The CPI and CSI motifs decrease the actin capping activity of CP via an allosteric mechanism. The complex of a CPI motif-containing protein with CP retains a low level of capping activity, which raises the possibility that CPI motif-containing proteins may target CP to certain cellular locations, in addition to, or as an alternative to, simply decreasing the capping activity. Vertebrates have three distinct conserved CARMIL genes, which seem to have distinct functions in cells. Of note, CARMIL2 localizes with vimentin filaments, representing a potential novel link between the actin and intermediate filament cytoskeleton systems. The actin capping activity of capping protein (CP) is indirectly regulated by competing with other factors for filament binding, or directly by factors that bind CP and sterically inhibit its interactions with filaments. Other proteins interact with CP through their 'capping protein interaction' (CPI) motif and modulate its activity via allosteric effects. Capping protein (CP) binds the fast growing barbed end of the actin filament and regulates actin assembly by blocking the addition and loss of actin subunits. Recent studies provide new insights into how CP and barbed-end capping are regulated. Filament elongation factors, such as formins and ENA/VASP (enabled/vasodilator-stimulated phosphoprotein), indirectly regulate CP by competing with CP for binding to the barbed end, whereas other molecules, including V-1 and phospholipids, directly bind to CP and sterically block its interaction with the filament. In addition, a diverse and unrelated group of proteins interact with CP through a conserved 'capping protein interaction' (CPI) motif. These proteins, including CARMIL (capping protein, ARP2/3 and myosin I linker), CD2AP (CD2-associated protein) and the WASH (WASP and SCAR homologue) complex subunit FAM21, recruit CP to specific subcellular locations and modulate its actin-capping activity via allosteric effects.

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped. In vivo, cells migrate across a diverse landscape of extracellular matrix containing gaps which present a challenge for cells to protrude across. Here, the authors show that T-Plastin strengthens protrusive actin networks to promote protrusion, extracellular matrix gap-bridging, and cell migration.

In animal cells, actin is dynamically distributed between multiple coexisting arrays. Carlier and Shekhar propose that a global treadmilling process — whereby the various actin networks grow and shrink depending on the local activity of actin regulators — establishes a steady-state concentration of actin monomers that supports this homeostatic actin turnover. Various cellular processes (including cell motility) are driven by the regulated, polarized assembly of actin filaments into distinct force-producing arrays of defined size and architecture. Branched, linear, contractile and cytosolic arrays coexist in vivo , and cells intricately control the number, length and assembly rate of filaments in these arrays. Recent in vitro and in vivo studies have revealed novel molecular mechanisms that regulate the number of filament barbed and pointed ends and their respective assembly and disassembly rates, thus defining classes of dynamically different filaments, which coexist in the same cell. We propose that a global treadmilling process, in which a steady-state amount of polymerizable actin monomers is established by the dynamics of each network, is responsible for defining the size and turnover of coexisting actin networks. Furthermore, signal-induced changes in the partitioning of actin to distinct arrays (mediated by RHO GTPases) result in the establishment of various steady-state concentrations of polymerizable monomers, thereby globally influencing the growth rate of actin filaments.

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro . In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching. Actin polymerization in lamellipodia of cells is regulated by the Arp2/3 complex and FMNL family formins. Here the authors show that both FMNL2 and FMNL3 contribute to lamellipodium protrusion and structure, and abolishing FMNL2/3 reduces protrusion force generation and migration, without affecting Arp2/3 incorporation.