Changsong Yang

αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

αE-catenin has cell–cell contact–dependent and –independent functions in regulating actin and membrane dynamics.

Filopodia initiation: Focus on the Arp2/3 complex and formins

Filopodia are long, slender, actin-rich cellular protrusions, which recently have become a focus of cell biology research because of their proposed roles as sensory and exploratory organelles that allow for “intelligent” cell behavior. Actin nucleation, elongation and bundling are believed to be essential for filopodia formation and functions. However, the identity of actin filament nucleators responsible for the initiation of filopodia remains controversial. Two alternative models, the convergent elongation and tip nucleation, emphasize two different actin filament nucleators, the Arp2/3 complex or formins, respectively, as key players during filopodia initiation. Although these two models in principle are not mutually exclusive, it is important to understand which of them is actually employed by cells. In this review, we discuss the existing evidence regarding the relative roles of the Arp2/3 complex and formins in filopodia initiation.

Coordination of Membrane and Actin Cytoskeleton Dynamics during Filopodia Protrusion

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.

Mechanism of Actin Filament Bundling by Fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

Functions of nonmuscle myosin II in assembly of the cellular contractile system.

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.

Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis.

Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

We investigated the poorly understood mechanism of plasma membrane targeting of formin mDia2 and found that its N terminus plays important roles in this process by binding acidic phospholipids through its N-terminal basic domain and by binding small GTPase Rif through direct interaction with the GTPase binding region and the diaphanous inhibitory domain.

Visualizing branched actin filaments in lamellipodia by electron tomography

To the editor: Urban et al.1 recently challenged the dendritic nucleation model of lamellipodia protrusion based on their inability to detect branched actin filaments in lamellipodia by electron tomography. We have carefully analysed the primary data that was provided as Supplementary Information by Urban et al., and report that there are numerous branches that have been overlooked by the authors. Lamellipodia are thin veil-like protrusions of the cell edge that have important roles in cell migration. Their advance is driven by the polymerization of actin filaments. The exact mechanisms of lamellipodia protrusion are still debated, owing in part to controversy regarding the structural organization of the actin filaments in lamellipodia. Initial structural data obtained by Small et al.2 using negative-staining electron microscopy suggested that lamellipodia contained a network of long diagonally oriented actin filaments. Based on this structure, the authors proposed a treadmilling model of lamellipodia protrusion stating that actin filaments continuously elongate at their distal barbed (plus) ends, thus pushing the membrane, and continuously depolymerize from the proximal pointed (minus) ends, thus maintaining actin turnover. A large body of structural, biochemical, kinetic and functional data accumulated over the subsequent three decades has led to a revised model of lamellipodia protrusion, termed the dendritic nucleation model3. A key additional point of this model is that the actin filaments in lamellipodia are constantly nucleated by the Arp2/3 complex as branches on pre-existing filaments. However, despite the compelling evidence, Small and

Cdc42-induced actin filaments are protected from capping protein

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.

Exo70 Stimulates the Arp2/3 Complex for Lamellipodia Formation and Directional Cell Migration

Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration. Using in vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.