Lamellipodial Network Research Articles

Liquid-like condensates mediate competition between actin branching and bundling.

Cellular remodeling of actin networks underlies cell motility during key morphological events, from embryogenesis to metastasis. In these transformations, there is an inherent competition between actin branching and bundling, because steric clashes among branches create a mechanical barrier to bundling. Recently, liquid-like condensates consisting purely of proteins involved in either branching or bundling of the cytoskeleton have been found to catalyze their respective functions. Yet in the cell, proteins that drive branching and bundling are present simultaneously. In this complex environment, which factors determine whether a condensate drives filaments to branch or become bundled? To answer this question, we added the branched actin nucleator, Arp2/3, to condensates composed of VASP, an actin bundling protein. At low actin to VASP ratios, branching activity, mediated by Arp2/3, robustly inhibited VASP-mediated bundling of filaments, in agreement with agent-based simulations. In contrast, as the actin to VASP ratio increased, addition of Arp2/3 led to formation of aster-shaped structures, in which bundled filaments emerged from a branched actin core, analogous to filopodia emerging from a branched lamellipodial network. These results demonstrate that multi-component, liquid-like condensates can modulate the inherent competition between bundled and branched actin morphologies, leading to organized, higher-order structures, similar to those found in motile cells.

N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters

Antigen-induced B-cell receptor (BCR) signaling is critical for initiating and regulating B-cell activation. The actin cytoskeleton plays essential roles in BCR signaling. Upon encountering cell-surface antigens, actin-driven B-cell spreading amplifies signaling, while B-cell contraction following spreading leads to signal attenuation. However, the mechanism by which actin dynamics switch BCR signaling from amplification to attenuation is unknown. Here, we show that Arp2/3-mediated branched actin polymerization is required for mouse splenic B-cell contraction. Contracting B-cells generate centripetally moving actin foci from lamellipodial F-actin networks in the plasma membrane region contacting antigen-presenting surfaces. Actin polymerization driven by N-WASP, but not WASP, initiates these actin foci and facilitates non-muscle myosin II recruitment to the contact zone, creating actomyosin ring-like structures. B-cell contraction increases BCR molecular density in individual clusters, leading to decreased BCR phosphorylation. Increased BCR molecular density reduced levels of the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. These results suggest that N-WASP-activated Arp2/3, coordinating with myosin, generates centripetally moving foci and contractile actomyosin ring-like structures from lamellipodial networks, enabling contraction. B-cell contraction attenuates BCR signaling by pushing out both stimulatory kinases and inhibitory phosphatases from BCR clusters, providing novel insights into actin-facilitated signal attenuation.

N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters.

Antigen-induced B-cell receptor (BCR) signaling is critical for initiating and regulating B-cell activation. The actin cytoskeleton plays essential roles in BCR signaling. Upon encountering cell-surface antigens, actin-driven B-cell spreading amplifies signaling, while B-cell contraction following spreading leads to signal attenuation. However, the mechanism by which actin dynamics switch BCR signaling from amplification to attenuation is unknown. Here, we show that Arp2/3-mediated branched actin polymerization is required for mouse splenic B-cell contraction. Contracting B-cells generate centripetally moving actin foci from lamellipodial F-actin networks in the plasma membrane region contacting antigen-presenting surfaces. Actin polymerization driven by N-WASP, but not WASP, initiates these actin foci and facilitates non-muscle myosin II recruitment to the contact zone, creating actomyosin ring-like structures. B-cell contraction increases BCR molecular density in individual clusters, leading to decreased BCR phosphorylation. Increased BCR molecular density reduced levels of the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. These results suggest that N-WASP-activated Arp2/3, coordinating with myosin, generates centripetally moving foci and contractile actomyosin ring-like structures from lamellipodial networks, enabling contraction. B-cell contraction attenuates BCR signaling by pushing out both stimulatory kinases and inhibitory phosphatases from BCR clusters, providing novel insights into actin-facilitated signal attenuation.

PD-1 inhibits T cell actin remodeling at the immunological synapse independently of its signaling motifs.

Engagement of the receptor programmed cell death molecule 1 (PD-1) by its ligands PD-L1 and PD-L2 inhibits T cell-mediated immune responses. Blocking such signaling provides the clinical effects of PD-1-targeted immunotherapy. Here, we investigated the mechanisms underlying PD-1-mediated inhibition. Because dynamic actin remodeling is crucial for T cell functions, we characterized the effects of PD-1 engagement on actin remodeling at the immunological synapse, the interface between a T cell and an antigen-presenting cell (APC) or target cell. We used microscopy to analyze the formation of immunological synapses between PD-1+ Jurkat cells or primary human CD8+ cytotoxic T cells and APCs that presented T cell-activating antibodies and were either positive or negative for PD-L1. PD-1 binding to PD-L1 inhibited T cell spreading induced by antibody-mediated activation, which was characterized by the absence of the F-actin-dense distal lamellipodial network at the immunological synapse and the Arp2/3 complex, which mediates branched actin formation. PD-1-induced inhibition of actin remodeling also prevented the characteristic deformation of T cells that contact APCs and the release of cytotoxic granules. We showed that the effects of PD-1 on actin remodeling did not require its tyrosine-based signaling motifs, which are thought to mediate the co-inhibitory effects of PD-1. Our study highlights a previously unappreciated mechanism of PD-1-mediated suppression of T cell activity, which depends on the regulation of actin cytoskeleton dynamics in a signaling motif-independent manner.

New insights into the molecular mechanisms of axon guidance receptor regulation and signaling.

As the nervous system develops, newly differentiated neurons need to extend their axons toward their synaptic targets to form functional neural circuits. During this highly dynamic process of axon pathfinding, guidance receptors expressed at the tips of motile axons interact with soluble guidance cues or membrane tethered molecules present in the environment to be either attracted toward or repelled away from the source of these cues. As competing cues are often present at the same location and during the same developmental period, guidance receptors need to be both spatially and temporally regulated in order for the navigating axons to make appropriate guidance decisions. This regulation is exerted by a diverse array of molecular mechanisms that have come into focus over the past several decades and these mechanisms ensure that the correct complement of surface receptors is present on the growth cone, a fan-shaped expansion at the tip of the axon. This dynamic, highly motile structure is defined by a lamellipodial network lining the periphery of the growth cone interspersed with finger-like filopodial projections that serve to explore the surrounding environment. Once axon guidance receptors are deployed at the right place and time at the growth cone surface, they respond to their respective ligands by initiating a complex set of signaling events that serve to rearrange the growth cone membrane and the actin and microtubule cytoskeleton to affect axon growth and guidance. In this review, we highlight recent advances that shed light on the rich complexity of mechanisms that regulate axon guidance receptor distribution, activation and downstream signaling.

Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity

The cell's dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

Actin Turnover in Lamellipodial Fragments

Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively invitro. However, a comprehensive picture of actin turnover invivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of the fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and extraction experiments to show that about two-thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, whereas the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the invitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a non-polymerizable "reserve" pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust yet rapidly adaptable and amenable to regulation.

Buckle up: Membrane tension drives lamellipodial network compression and adhesion deposition.

Whether to spread on a surface or to crawl, cells must apply traction forces to the underlying substrate via adhesion complexes. In this issue, Pontes et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611117) shed new light on how the interplay among membrane tension, the lamellipodial actin network, and adhesions coordinate the dynamics of spreading fibroblasts.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion ofleukocytes.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Functional Hierarchy of Redundant Actin Assembly Factors Revealed by Fine-Grained Registration of Intrinsic Image Fluctuations

Highly redundant pathways often contain components whose functions are difficult to decipher from the responses induced by genetic or molecular perturbations. Here, we present a statistical approach that samples and registers events observed in images of intrinsic fluctuations in unperturbed cells to establish the functional hierarchy of events in systems with redundant pathways. We apply this approach to study the recruitment of actin assembly factors involved in the protrusion of the cell membrane. We find that the formin mDia1, along with nascent adhesion components, is recruited to the leading edge of the cell before protrusion onset, initiating linear growth of the lamellipodial network. Recruitment of Arp2/3, VASP, cofilin, and the formin mDia2 then promotes sustained exponential growth of the network. Experiments changing membrane tension suggest that Arp2/3 recruitment is mechano-responsive. These results indicate that cells adjust the overlapping contributions of multiple factors to actin filament assembly during protrusion on a ten-second timescale and in response to mechanical cues.

Bidirectional coupling between integrin-mediated signaling and actomyosin mechanics explains matrix-dependent intermittency of leading-edge motility

Animal cell migration is a complex process characterized by the coupling of adhesion, cytoskeletal, and signaling dynamics. Here we model local protrusion of the cell edge as a function of the load-bearing properties of integrin-based adhesions, actin polymerization fostered by adhesion-mediated signaling, and mechanosensitive activation of RhoA that promotes myosin II-generated stress on the lamellipodial F-actin network. Analysis of stochastic model simulations illustrates how these pleiotropic functions of nascent adhesions may be integrated to govern temporal persistence and frequency of protrusions. The simulations give mechanistic insight into the documented effects of extracellular matrix density and myosin abundance, and they show characteristic, nonnormal distributions of protrusion duration times that are similar to those extracted from live-cell imaging experiments. Analysis of the model further predicts relationships between measurable quantities that reflect the partitioning of stress between tension on F-actin-bound adhesions, which act as a molecular clutch, and dissipation by retrograde F-actin flow.

Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.

An Elastic Actomyosin Network in Motile Fish Keratocytes

Crawling motility of eukaryotic cells is driven by net assembly of an actin network at the leading edge and net disassembly at the cell rear, orchestrated by a combination of biochemical and mechanical processes. To understand the distribution and function of mechanical forces in motile cells, it is necessary to determine the mechanical properties and connectivities of the structural components of the cell.We have investigated the mechanical properties of the actin network using detergent-extracted cytoskeletons from fish epithelial keratocytes. When stretched by a microneedle, the extracted lamellipodial network stretched significantly, up to several-fold its original dimensions. At moderate strains, the network was found to be highly elastic, at least over the timescales of 0.2 s to 30 s. These observations suggest that there is strong mechanical coupling within the cytoskeleton over distance scales comparable to the whole cell.Previously we have shown that activation of myosin II in detergent-extracted cytoskeletons by addition of ATP results in contraction and disassembly of the rear network. We have found that ATP analogs (AMP-PNP and ADP-fluoroberyllate) also cause partial disintegration and inward sliding of the rear network, toward the cell body, but without observable contraction within the network. The accompanied deformation of the cell body suggests that the cell body is stretched laterally in the live cell, anchored by the flanking actomyosin networks, and that release of actomyosin bonds (triggered by binding of ATP analogs to myosin) is sufficient for the network to rupture, allowing the cell body to relax. This raises the possibility that myosin may be playing a structural role, maintaining the integrity of the rear network under tension, while simultaneously driving its contraction. The cell body may serve as a reservoir for elastic energy, stress being distributed across the rear actomyosin network.

The cytoskeletal mechanisms of cell–cell junction formation in endothelial cells

The actin cytoskeleton and associated proteins play a vital role in cell-cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell-cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin-rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell-cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context.

How does the antagonism between capping and anti-capping proteins affect actin networkdynamics?

Actin-based cell motility is essential to many biological processes. We built a simplified,three-dimensional computational model and subsequently performed stochastic simulationsto study the growth dynamics of lamellipodia-like branched networks. In this work, we shedlight on the antagonism between capping and anti-capping proteins in regulatingactin dynamics in the filamentous network. We discuss detailed mechanisms bywhich capping and anti-capping proteins affect the protrusion speed of the actinnetwork and the rate of nucleation of filaments. We computed a phase diagramshowing the regimes of motility enhancement and inhibition by these proteins.Our work shows that the effects of capping and anti-capping proteins are mainlytransmitted by modulation of the filamentous network density and local availability ofmonomeric actin. We discovered that the combination of the capping/anti-cappingregulatory network with nucleation-promoting proteins introduces robustnessand redundancy in cell motility machinery, allowing the cell to easily achievemaximal protrusion speeds under a broader set of conditions. Finally, we discussdistributions of filament lengths under various conditions and speculate on theirpotential implication for the emergence of filopodia from the lamellipodial network.

Stochastic Simulation Study of the Role of Capping and Anti-Capping Proteins in Regulating Actin Network Growth

Actin-based cell motility is essential to many biological processes. We build simplified computational model and perform stochastic simulation to study the growth of lamellipodia-like branched filamentous network. Here we investigate how capping proteins and anti-capping proteins affect the speed of protrusion and the nucleation of filaments in the model system. Phase diagram showing the regimes of motility enhancement and motility inhibition is presented. Furthermore, we discuss filament length distribution and its implication in the formation of filopodia from lamellipodial network.

Control of Actin Filament Treadmilling in Cell Motility

Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have furthered our understanding of the molecular mechanisms by which regulated assembly dynamics of actin filaments drive motile processes. Attention is focused on lamellipodium protrusion, powered by the turnover of a branched filament array. ATP hydrolysis on actin is the key reaction that allows filament treadmilling. It regulates barbed-end dynamics and length fluctuations at steady state and specifies the functional interaction of actin with essential regulatory proteins such as profilin and ADF/cofilin. ATP hydrolysis on actin and Arp2/3 acts as a timer, regulating the assembly and disassembly of the branched array to generate tropomyosin-mediated heterogeneity in the structure and dynamics of the lamellipodial network. The detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly. Novel biophysical methods involving single-molecule measurements should foster progress in these crucial issues.

Roles of Arp2/3 complex and mDia2 in actin‐based protrusions

Actin polymerization is a key mechanism of force generation in cells. Actin filament nucleators, formins and the Arp2/3 complex, are believed to have non‐overlapping functions in inducing actin filament bundles and dendritic networks, respectively. We investigated roles of the Arp2/3 complex and an mDia2 formin in lamellipodia and filopodia, cellular protrusions containing dendritic and bundled actin filaments, respectively. Contrary to the above paradigm, we have found that mDia2 is an important player in lamellipodia, while Arp2/3 complex significantly contributes to generation of filopodia. The contribution of mDia2 to lamellipodial protrusion was related to generation of long filaments in the lamellipodial network. These long filaments displayed a high tendency to converge into bundles during elongation, thereby producing filopodia. Depletion of the Arp2/3 complex by siRNA in neuronal cells, as expected, inhibited lamellipodia, but remarkably, also filopodia. Correlative structural and kinetic analyses suggested that Arp2/3‐nucleated filaments become incorporated into filopodial bundles in the course of elongation, and that this process contributes significantly to filopodia initiation. Together, our data show that each of two distinct actin nucleators, Arp2/3 complex and mDia2, jointly participate in generation of both lamellipodia and filopodia. Supported by NIH grant GM 070898 to TMS.

Novel Roles of Formin mDia2 in Lamellipodia and Filopodia Formation in Motile Cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.

Spatial and Temporal Relationships between Actin-Filament Nucleation, Capping, and Disassembly

The leading actin network in motile cells is composed of two compartments, the lamellipod and the lamellum. Construction of the lamellipod requires a set of conserved proteins that form a biochemical cycle. The timing of this cycle and the roles of its components in determining actin network architecture in vivo, however, are not well understood. We performed fluorescent speckle microscopy on spreading Drosophila S2 cells by using labeled derivatives of actin, the Arp2/3 complex, capping protein, and tropomyosin. We find that capping protein and the Arp2/3 complex both incorporate at the cell edge but that capping protein dissociates after covering less than half the width of the lamellipod, whereas the Arp2/3 complex dissociates after crossing two thirds of the lamellipod. The lamellipodial actin network itself persists long after the loss of the Arp2/3 complex. Depletion of capping protein by RNAi results in the displacement of the Arp2/3 complex and disappearance of the lamellipod. In contrast, depletion of cofilin, slingshot, twinfilin, and tropomyosin, all factors that control the stability of actin filaments, dramatically expanded the lamellipod at the expense of the lamellum. The Arp2/3 complex is incorporated into the lamellipodial network at the cell edge but debranches well before the lamellipodial network itself is disassembled. Capping protein is required for the formation of a lamellipodial network but dissociates from the network precisely when filament disassembly is first detected. Cofilin, twinfilin, and tropomyosin appear to play no role in lamellipodial network assembly but function to limit its size.