Cell migration initiates by extension of the actin cytoskeleton at the leading edge. Computational analysis of fluorescent speckle microscopy movies of migrating epithelial cells revealed this process is mediated by two spatially colocalized but kinematically, kinetically, molecularly, and functionally distinct actin networks. A lamellipodium network assembled at the leading edge but completely disassembled within 1 to 3 micrometers. It was weakly coupled to the rest of the cytoskeleton and promoted the random protrusion and retraction of the leading edge. Productive cell advance was a function of the second colocalized network, the lamella, where actomyosin contraction was integrated with substrate adhesion.

Cells exhibit a biphasic migration-velocity response to increasing adhesion strength, with fast migration occurring at intermediate extracellular matrix (ECM) concentration and slow migration occurring at low and high ECM concentration. A simple mechanical model has been proposed to explain this observation, in which too little adhesion does not provide sufficient traction whereas too much adhesion renders cells immobile. Here we characterize a phenotype for rapid cell migration, which in contrast to the previous model reveals a complex interdependence of subcellular systems that mediates optimal cell migration in response to increasing adhesion strength. The organization and activity of actin, myosin II, and focal adhesions (FAs) are spatially and temporally highly variable and do not exhibit a simple correlation with optimal motility rates. Furthermore, we can recapitulate rapid migration at a nonoptimal ECM concentration by manipulating myosin II activity. Thus, the interplay between actomyosin and FA dynamics results in a specific balance between adhesion and contraction, which induces maximal migration velocity.

Axon outgrowth and guidance to the proper target requires the coordination of filamentous (F)-actin and microtubules (MTs), the dynamic cytoskeletal polymers that promote shape change and locomotion. Over the past two decades, our knowledge of the many guidance cues, receptors, and downstream signaling cascades involved in neuronal outgrowth and guidance has increased dramatically. Less is known, however, about how those cascades of information converge and direct appropriate remodeling and interaction of cytoskeletal polymers, the ultimate effectors of movement and guidance. During development, much of the communication that occurs between environmental guidance cues and the cytoskeleton takes place at the growing tip of the axon, the neuronal growth cone. Several articles on this topic focus on the "input" to the growth cone, the myriad of receptor types, and their corresponding cognate ligands. Others investigate the signaling cascades initiated by receptors and propagated by second messenger pathways (i.e., kinases, phosphatases, GTPases). Ultimately, this plethora of information converges on proteins that associate directly with the actin and microtubule cytoskeletons. The role of these cytoskeletal-associated proteins, as well as the cytoskeleton itself in axon outgrowth and guidance, is the subject of this article.A s evidenced by other articles on this topic, our understanding of the cues, receptors, and signaling events underlying axon outgrowth and guidance has grown dramatically in recent years. Here, we focus on recent research involving cytoskeletal dynamics downstream of guidance receptor signaling that has begun to unravel the mechanisms underlying guided growth cone movement during development. We begin by covering how growth cone morphology changes during outgrowth and guidance. Since cytoskeletal dynamics underlie changes in growth cone morphology, we discuss where different cytoskeletal polymers reside in the growth cone and what forms of dynamic reorganization they undergo. We then present a description of selected actin-and microtubule-associated proteins that have been identified in developing neurons and discuss how they may function in axon outgrowth and guidance. Finally, we present a working model of how growth cones integrate multiple signaling

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Localized plasma membrane expansion during axon branching mediated by Netrin-1 occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

Filopodia: The Fingers That Do the WalkingThis information is current as of 1 May 2012.The following resources related to this article are available online at http://stke.sciencemag.org. Filopodia are actin-based structures composed of parallel bundles of actin filaments and various actinassociated proteins, and they play important roles in cell-cell signaling, guidance toward chemoattractants, and adhesion to the extracellular matrix. Two mechanisms for the formation of filopodia have been suggested, each using different sets of actin-regulating proteins, creating some controversy in the field. New molecules, some of unknown functions, have also been implicated in filopodium formation, suggesting that other possible mechanisms of filopodium formation exist. We discuss established and novel proteins that mediate the formation and dynamics of filopodia, different mechanisms of filopodium formation, and the various functions that distinct filopodia perform. Article Tools

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that Ϸ90% of the polymer disassembles at the lamellipodiumlamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction. C ell migration involves the assembly of a meshwork of filamentous actin (F-actin) at the cell edge facing the direction of movement (1-3), which is balanced by depolymerization in other places (4). In many cells, this continuous turnover of the meshwork is accompanied by retrograde flow away from the leading edge (5). It is likely that turnover and flow are coregulatory mechanisms of cell protrusion and hence the trajectory of motion. However, how the molecular effectors of actin dynamics are orchestrated to produce directed movement at the cellular level is poorly understood.It is fairly obvious to hypothesize that turnover and flow should be interdependent parameters of F-actin dynamics. First, theoretical (6) and experimental studies (7) have indicated that actin polymerization can generate the forces required for protrusion of the cell plasma membrane. Conversely, these forces may be responsible for the pushing of the meshwork toward the cell center, implying a relationship between retrograde flow speed and the rate of polymerization at the leading edge. Second, the maps of retrograde flow we (8, 9) and others (10) have published exhibit noncoherent F-actin movement with zones of convergence. Using ratio imaging of labeled F-actin and a fluorescent volume marker suggested that convergence zones are not accompanied by a significant increase of F-actin density (11). Thus, depolymerization may be coupled to meshwork contraction.To test these hypotheses, we have advanced our analysis...

Neurons establish their unique morphology by elaborating multiple neurites that subsequently form axons and dendrites. Neurite initiation entails significant surface area expansion, necessitating addition to the plasma membrane. We report that regulated membrane delivery coordinated with the actin cytoskeleton is crucial for neuritogenesis, and identify two independent pathways that use distinct exocytic and cytoskeletal machinery to drive neuritogenesis. One pathway employs Ena/VASP-regulated actin dynamics coordinated with VAMP2-mediated exocytosis, and involves a novel role for Ena/VASP in exocytosis. A second mechanism occurs in the presence of laminin through integrin-dependent activation of FAK and src, and utilizes coordinated activity of the Arp2/3 complex and VAMP7-mediated exocytosis. We conclude that neuritogenesis can be driven by two distinct pathways that differentially coordinate cytoskeletal dynamics and exocytosis. These regulated changes and coordination of cytoskeletal and exocytic machinery may be utilized in other physiological contexts involving cell motility and morphogenesis.