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Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells. Hakala et al. report that twinfilin dissociates capping proteins from the actin filament barbed ends to promote actin turnover at leading-edge lamellipodia.

The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes. The cofilin family proteins are actin disassembly factors but the disassembly mechanism is poorly understood. Here authors show that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold and reveal how CAP destabilizes the interface between terminal actin subunits.

Experiments using purified proteins reveal how the network of filaments that underlie cell movement becomes denser when pushing against a stronger mechanical force.Experiments using purified proteins reveal how the network of filaments that underlie cell movement becomes denser when pushing against a stronger mechanical force.

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division. Cytokinetic abscission relies on the local constriction after cytoskeleton disassembly, but it is not known how the actin filaments are disassembled. Here, the authors show that the redox enzyme MICAL1 is recruited by Rab35 and induces oxidation-mediated depolymerization of actin, which is required to recruit ESCRT-III and complete abscission.

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90–Arp2/3 nucleated filaments and formation of a ternary SPIN90–Arp2/3–mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90–Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.Cao et al. show that SPIN90 enhances formation of Arp2/3-mediated unbranched filaments and promotes a formin-based cortex by recruiting mDia1 or forming a ternary SPIN90–Arp2/3–mDia1 complex.

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.

Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. Formins promote actin filament polymerization and capping protein blocks polymerization; both proteins are thought to exclude each other from barbed ends. Here the authors show that both proteins can simultaneously bind barbed ends in a ternary complex while enhancing each other's dissociation from the barbed end.

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high‐density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53‐dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair. The mechanisms underlying filopodia initiation and localization at the plasma membrane are only incompletely understood. This study presents a new model in which active Cdc42 promotes IRSp53‐dependent VASP localization at discrete foci, where it drives actin polymerization and initiates filopodia formation.

The regulated assembly of actin filaments is essential in nearly all cell types. Studying actin assembly dynamics can pose many technical challenges. A number of these challenges can be overcome by using microfluidics to observe and manipulate single actin filaments under an optical microscope. In particular, microfluidics can be tremendously useful for applying different mechanical stresses to actin filaments and determining how the physical context of the filaments affects their regulation by biochemical factors. In this review, we summarize the main features of microfluidics for the study of actin assembly dynamics, and we highlight some recent developments that have emerged from the combination of microfluidics and other techniques. We use two case studies to illustrate our points: the rapid assembly of actin filaments by formins and the disassembly of filaments by actin depolymerizing factor (ADF)/cofilin. Both of these protein families play important roles in cells. They regulate actin assembly through complex molecular mechanisms that are sensitive to the filaments’ mechanical context, with multiple activities that need to be quantified separately. Microfluidics-based experiments have been extremely useful for gaining insight into the regulatory actions of these two protein families.

SignificanceActin filaments assemble into ordered networks able to exert forces and shape cells. In response, filaments are exposed to mechanical stress which can potentially modulate their interactions with regulatory proteins. We developed in vitro tools to manipulate single filaments and study the impact of mechanics on the activity of actin depolymerizing factor (ADF)/cofilin, the central player in actin disassembly. While tension has almost no effect, curvature enhances severing by ADF/cofilin. We also discovered a mechanism that boosts the severing of anchored filaments: When binding to these filaments, ADF/cofilin locally increases their natural helicity, generating a torque that accelerates filament fragmentation up to 100-fold. As a consequence, interconnected filament networks are severed far more efficiently than independent filaments. Proteins of the actin depolymerizing factor (ADF)/cofilin family are the central regulators of actin filament disassembly. A key function of ADF/cofilin is to sever actin filaments. However, how it does so in a physiological context, where filaments are interconnected and under mechanical stress, remains unclear. Here, we monitor and quantify the action of ADF/cofilin in different mechanical situations by using single-molecule, single-filament, and filament network techniques, coupled to microfluidics. We find that local curvature favors severing, while tension surprisingly has no effect on cofilin binding and weakly enhances severing. Remarkably, we observe that filament segments that are held between two anchoring points, thereby constraining their twist, experience a mechanical torque upon cofilin binding. We find that this ADF/cofilin-induced torque does not hinder ADF/cofilin binding, but dramatically enhances severing. A simple model, which faithfully recapitulates our experimental observations, indicates that the ADF/cofilin-induced torque increases the severing rate constant 100-fold. A consequence of this mechanism, which we verify experimentally, is that cross-linked filament networks are severed by cofilin far more efficiently than nonconnected filaments. We propose that this mechanochemical mechanism is critical to boost ADF/cofilin’s ability to sever highly connected filament networks in cells.