F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation.

Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits.

Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.

Cell Motility and Cytokinesis: From Mysteries to Molecular Mechanisms in Five Decades.

This is the story of someone who has been fortunate to work in a field of research where essentially nothing was known at the outset but that blossomed with the discovery of profound insights about two basic biological processes: cell motility and cytokinesis. The field started with no molecules, just a few people, and primitive methods. Over time, technological advances in biophysics, biochemistry, and microscopy allowed the combined efforts of scientists in hundreds of laboratories to explain mysterious processes with molecular mechanisms that can be embodied in mathematical equations and simulated by computers. The success of this field is a tribute to the power of the reductionist strategy for understanding biology.

Regulation of branched versus linear Arp2/3-generated actin filaments.

Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.

Inactivating negative regulators of cortical branched actin enhances persistence of single cell migration.

The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

Neutrophil motility is regulated by both cell intrinsic and endothelial cell ARPC1B.

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.

Actin-capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans.

Actin dynamics play an important role in tissue morphogenesis, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report an in vivo role of the actin-capping protein CAP-1 in the Caenorhabditis elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, and its depletion or overexpression led to severe structural defects in the syncytial germline and oocytes. A 60% reduction in the level of CAP-1 caused a twofold increase in F-actin and non-muscle myosin II activity, and laser incision experiments revealed an increase in rachis contractility. Cytosim simulations pointed to increased myosin as the main driver of increased contractility following loss of actin-capping protein. Double depletion of CAP-1 and myosin or Rho kinase demonstrated that the rachis architecture defects associated with CAP-1 depletion require contractility of the rachis actomyosin corset. Thus, we uncovered a physiological role for actin-capping protein in regulating actomyosin contractility to maintain reproductive tissue architecture.

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and ß subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Polymerization force-regulated actin filament-Arp2/3 complex interaction dominates self-adaptive cell migrations.

Cells migrate by adapting their leading-edge behaviors to heterogeneous extracellular microenvironments (ECMs) during cancer invasions and immune responses. Yet it remains poorly understood how such complicated dynamic behaviors emerge from millisecond-scale assembling activities of protein molecules, which are hard to probe experimentally. To address this gap, we establish a spatiotemporal "resistance-adaptive propulsion" theory based on the interactions between Arp2/3 complexes and polymerizing actin filaments and a multiscale dynamic modeling system spanning from molecular proteins to the cell. We quantitatively find that cells can accurately self-adapt propulsive forces to overcome heterogeneous ECMs via a resistance-triggered positive feedback mechanism, dominated by polymerization-induced actin filament bending and the bending-regulated actin-Arp2/3 binding. However, for high resistance regions, resistance triggers a negative feedback, hindering branched filament assembly, which adapts cellular morphologies to circumnavigate the obstacles. Strikingly, the synergy of the two opposite feedbacks not only empowers the cell with both powerful and flexible migratory capabilities to deal with complex ECMs but also enables efficient utilization of intracellular proteins by the cell. In addition, we identify that the nature of cell migration velocity depending on ECM history stems from the inherent temporal hysteresis of cytoskeleton remodeling. We also show that directional cell migration is dictated by the competition between the local stiffness of ECMs and the local polymerizing rate of actin network caused by chemotactic cues. Our results reveal that it is the polymerization force-regulated actin filament-Arp2/3 complex binding interaction that dominates self-adaptive cell migrations in complex ECMs, and we provide a predictive theory and a spatiotemporal multiscale modeling system at the protein level.

The WAVE complex drives the morphogenesis of the photoreceptor outer segment cilium.

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened "disc" membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae.

Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

N-WASP-Arp2/3 signaling controls multiple steps of dendrite maturation in Purkinje cells in vivo.

During neural development, the actin filament network must be precisely regulated to form elaborate neurite structures. N-WASP tightly controls actin polymerization dynamics by activating an actin nucleator Arp2/3. However, the importance of N-WASP-Arp2/3 signaling in the assembly of neurite architecture in vivo has not been clarified. Here, we demonstrate that N-WASP-Arp2/3 signaling plays a crucial role in the maturation of cerebellar Purkinje cell (PC) dendrites in vivo in mice. N-WASP was expressed and activated in developing PCs. Inhibition of Arp2/3 and N-WASP from the beginning of dendrite formation severely disrupted the establishment of a single stem dendrite, which is a characteristic basic structure of PC dendrites. Inhibition of Arp2/3 after stem dendrite formation resulted in hypoplasia of the PC dendritic tree. Cdc42, an upstream activator of N-WASP, is required for N-WASP-Arp2/3 signaling-mediated PC dendrite maturation. In addition, overactivation of N-WASP is also detrimental to dendrite formation in PCs. These findings reveal that proper activation of N-WASP-Arp2/3 signaling is crucial for multiple steps of PC dendrite maturation in vivo.

Arp2/3 mediated dynamic lamellipodia of the hPSC colony edges promote liposome-based DNA delivery.

Cationic liposome-mediated delivery of drugs, DNA, or RNA plays a pivotal role in small molecule therapy, gene editing, and immunization. However, our current knowledge regarding the cellular structures that facilitate this process remains limited. Here, we used human pluripotent stem cells (hPSCs), which form compact colonies consisting of dynamically active cells at the periphery and epithelial-like cells at the core. We discovered that cells at the colony edges selectively got transfected by cationic liposomes through actin-related protein 2/3 (Arp2/3) dependent dynamic lamellipodia, which is augmented by myosin II inhibition. Conversely, cells at the core establish tight junctions at their apical surfaces, impeding liposomal access to the basal lamellipodia and thereby inhibiting transfection. In contrast, liposomes incorporating mannosylated lipids are internalized throughout the entire colony via receptor-mediated endocytosis. These findings contribute a novel mechanistic insight into enhancing therapeutic delivery via liposomes, particularly in cell types characterized by dynamic lamellipodia, such as immune cells or those comprising the epithelial layer.

Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy.

Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, and multiple classes of nucleation promoting factors (NPFs) stimulate its nucleation activity. When activated by WASP family NPFs, the complex must bind to the side of a preexisting (mother) filament of actin to complete the nucleation process, ensuring that WASP-mediated activation creates branched rather than linear actin filaments. How actin filaments contribute to activation is currently not understood, largely due to the lack of high-resolution structures of activated Arp2/3 complex bound to the side of a filament. Here, we present the 3.9-Å cryo-electron microscopy structure of the Arp2/3 complex at a branch junction. The structure reveals contacts between Arp2/3 complex and the side of the mother actin filament that likely stimulate subunit flattening, a conformational change that allows the actin-related protein subunits in the complex (Arp2 and Arp3) to mimic filamentous actin subunits. In contrast, limited contact between the bottom half of the complex and the mother filament suggests that clamp twisting, a second major conformational change observed in the active state, is not stimulated by actin filaments, potentially explaining why actin filaments are required but insufficient to trigger nucleation during WASP-mediated activation. Along with biochemical and live-cell imaging data and molecular dynamics simulations, the structure reveals features critical for the interaction of Arp2/3 complex with actin filaments and regulated assembly of branched actin filament networks in cells.

Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high-resolution cryo-EM structureof the branch junction.

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 cryo-microscopy. 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.

Single-molecule analysis of actin filament debranching by cofilin and GMF.

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.

RNAi screen in the Drosophila wing of genes encoding proteins related to cytoskeleton organization and cell division.

Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. In this manuscript we describe the phenotypes in the adult fly wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. We also compile a molecular classification of these proteins into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, we analyze in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and define the consequences of interfering with cell division for wing growth and patterning.

Molecular dynamics simulations support a multistep pathway for activation of branched actin filament nucleation by Arp2/3 complex.

Actin-related protein 2/3 complex (Arp2/3 complex) catalyzes the nucleation of branched actin filaments that push against membranes in processes like cellular motility and endocytosis. During activation by WASP proteins, the complex must bind WASP and engage the side of a pre-existing (mother) filament before a branched filament is nucleated. Recent high-resolution structures of activated Arp2/3 complex revealed two major sets of activating conformational changes. How these activating conformational changes are triggered by interactions of Arp2/3 complex with actin filaments and WASP remains unclear. Here we use a recent high-resolution structure of Arp2/3 complex at a branch junction to design all-atom molecular dynamics simulations that elucidate the pathway between the active and inactive states. We ran a total of ∼4.6 microseconds of both unbiased and steered all-atom molecular dynamics simulations starting from three different binding states, including Arp2/3 complex within a branch junction, bound only to a mother filament, and alone in solution. These simulations indicate that the contacts with the mother filament are mostly insensitive to the massive rigid body motion that moves Arp2 and Arp3 into a short pitch helical (filament-like) arrangement, suggesting actin filaments alone do not stimulate the short pitch conformational change. In contrast, contacts with the mother filament stabilize subunit flattening in Arp3, an intrasubunit change that converts Arp3 from a conformation that mimics an actin monomer to one that mimics a filamentous actin subunit. Our results support a multistep activation pathway that has important implications for understanding how WASP-mediated activation allows Arp2/3 complex to assemble force-producing actin networks.