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Spatial control of cortical actin nucleation is indispensable for proper establishment and plasticity of cell morphology. Cobl is a novel WH2 domain‐based actin nucleator. The cellular coordination of Cobl's nucleation activity, however, has remained elusive. Here, we reveal that Cobl's cellular functions are dependent on syndapin. Cobl/syndapin complexes form in vivo , as demonstrated by colocalization, coimmunoprecipitation and subcellular recruitment studies. In vitro reconstitutions and subcellular fractionations demonstrate that, via its lipid‐binding Fer/CIP4 Homology (FCH)‐Bin/Amphiphysin/Rvs (F‐BAR) domain, syndapin recruits Cobl to membranes. Consistently, syndapin I RNAi impairs cortical localization of Cobl. Further functional studies in neurons show that Cobl and syndapin I work together in dendritic arbor development. Importantly, both proteins are crucial for dendritogenesis. Cobl‐mediated functions in neuromorphogenesis critically rely on syndapin I and interestingly also on Arp3. Endogenous Cobl, syndapin I and the Arp2/3 complex activator and syndapin‐binding partner N‐WASP were present in one complex, as demonstrated by coimmunoprecipitations. Together, these data provide detailed insights into the molecular basis for Cobl‐mediated functions and reveal that different actin nucleators are functionally intertwined by syndapin I during neuromorphogenesis. Here, an interaction between the actin nucleator Cobl and the F‐BAR domain protein Syndapin is shown to recruit Cobl to the membrane, where it regulates the actin cytoskeleton during neuronal dendritogenesis.

We reconstructed the structure of actin filament branch junctions formed by fission yeast Arp2/3 complex at 3.5 Å resolution from images collected by electron cryomicroscopy. During specimen preparation, all of the actin subunits and Arp3 hydrolyzed their bound adenosine triphosphate (ATP) and dissociated the γ-phosphate, but Arp2 retained the γ-phosphate. Binding tightly to the side of the mother filament and nucleating the daughter filament growing as a branch requires Arp2/3 complex to undergo a dramatic conformational change where two blocks of structure rotate relative to each other about 25° to align Arp2 and Arp3 as the first two subunits in the branch. During branch formation, Arp2/3 complex acquires more than 8,000 Å2 of new buried surface, accounting for the stability of the branch. Inactive Arp2/3 complex binds only transiently to the side of an actin filament, because its conformation allows only a subset of the interactions found in the branch junction.

Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed.

Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.

Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to < 1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name “young/strong” and “old/weak.” The “young/strong” state 1 has adenosine 5′- diphosphate (ADP)−Pi bound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the “old/weak” state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP−Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP−Pi−Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling “old” actin filament branches in cells.

Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch–junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin–actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament–Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes, including phagocytosis, endocytosis, and pseudopod protrusion. When confronted with opposing forces, the growth rate of these networks slows and their density increases, but the stoichiometry of key components does not change. The molecular mechanisms governing this force response are not well understood, so we used single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Although load forces are observed to increase the density of growing filaments, we find that they actually decrease the rate of filament nucleation due to inhibitory interactions between actin filament ends and nucleation promoting factors. The force-induced increase in network density turns out to result from an exponential drop in the rate constant that governs filament capping. The force dependence of filament capping matches that of filament elongation and can be explained by expanding Brownian Ratchet theory to cover both processes. We tested a key prediction of this expanded theory by measuring the force-dependent activity of engineered capping protein variants and found that increasing the size of the capping protein increases its sensitivity to applied forces. In summary, we find that Brownian Ratchets underlie not only the ability of growing actin filaments to generate force but also the ability of branched actin networks to adapt their architecture to changing loads.

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. Our cells contain a network of filaments made up of a protein called actin. Just like the skeleton that supports our body, the actin ‘cytoskeleton’ gives a cell its shape and strength. Actin filaments are also critical for many other processes including enabling cells to move and divide. The assembly of actin filaments must be properly controlled so that they are formed at the right time and place within the cell. A complex of proteins known as the WAVE Regulatory Complex (WRC) promotes the assembly of actin filaments. The complex contains a region called the VCA, which is able to bind to and activate another protein to make the new actin filaments. The WRC regulates filament assembly by controlling the availability of the VCA in a way that is similar to opening and closing a safe box. When new actin filaments are not needed, the safe box is closed and the VCA is not available. However, when cells need to make new actin filaments, the WRC is opened to release the VCA region so that it is able to bind to the filament-producing protein. Previous studies have shown that a protein called Rac1 acts as a key to open the WRC and trigger actin filament assembly. But it remains unclear how this works. A major obstacle to studying this process is that Rac1 and the WRC only weakly interact with each other, which makes it difficult to capture the interaction under experimental conditions. To overcome this obstacle, Chen et al. tethered a Rac1 molecule to the WRC in order to make the interaction more stable. A technique called cryo-electron microscopy was used to study the three-dimensional shape of this Rac1-WRC complex. Unexpectedly, Rac1 was attached to a different part of the WRC than the site predicted by previous studies. Further experiments showed that Rac1 needs to bind to both of these sites at the same time in order to open the WRC and release VCA, similar to using two keys to open one safe box for increased security. Some cancers, neurological disorders and other diseases can be caused by defects in WRC and Rac1 activity. Therefore, these findings could lead to new ways to treat these conditions in human patients.

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.

Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes.