Recent studies indicate the actin and microtubule cytoskeletons are a final common target of many signaling cascades that influence the developing neuron. Regulation of polymer dynamics and transport are crucial for the proper growth cone motility. This review addresses how actin filaments, microtubules, and their associated proteins play crucial roles in growth cone motility, axon outgrowth, and guidance. We present a working model for cytoskeletal regulation of directed axon outgrowth. An important goal for the future will be to understand the coordinated response of the cytoskeleton to signaling cascades induced by guidance receptor activation.

Ena/VASP proteins are a conserved family of actin regulatory proteins made up of EVH1, EVH2 domains, and a proline-rich central region. They have been implicated in actin-based processes such as fibroblast migration, axon guidance, and T cell polarization and are important for the actin-based motility of the intracellular pathogen Listeria monocytogenes. Mechanistically, these proteins associate with barbed ends of actin filaments and antagonize filament capping by capping protein (CapZ). In addition, they reduce the density of Arp2/3-dependent actin filament branches and bind Profilin at sites of actin polymerization. Vertebrate Ena/VASP proteins are substrates for PKA/PKG serine/threonine kinases. Phosphorylation by these kinases appears to modulate Ena/VASP function within cells, although the mechanism underlying this regulation remains to be determined.

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.

Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence timelapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth. Key words: microtubule; actin filament; growth cone; collateral axon branching; cortical development; fluorescence timelapse imagingAxons are guided in new directions by reorientation of their growth cones as well as extension of collateral branches (O'Leary et al., 1990). We have shown previously (Szebenyi et al., 1998) that cortical axon branching occurs in vitro through changes in growth cone morphologies and behaviors. Growth cones at the tips of rapidly extending cortical axons are typically small and highly motile. However, in preparation for branching, growth cones pause for many hours, greatly enlarge, and maintain motility without forward advance. Subsequently, a new growth cone develops from the tip of the large paused growth cone and forms the new leading axon. Remnants of the large paused growth cone remain behind on the axon shaft as filopodial and lamellar expansions that subsequently give rise to interstitial axon collaterals. In living cortical slices (Halloran and Kalil, 1994), similar growth cone pausing behaviors were observed in the corpus callosum in regions where collateral axon branches develop and extend toward cortical targets, suggesting that growth cone pausing is closely related to branching mechanisms in vivo.Changes in the direction of axon outgrowth depend on reorganization of the microtubule and actin cytoskeleton (Lin and

Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. We found that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation was dependent upon Ena/VASP function and directly correlated with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function was required for filopodial formation from the growth cone in response to global PKA activation. We propose that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues.

Dendritic spines are the primary sites of contact with presynaptic axons on excitatory hippocampal and cortical neurons. During development and plasticity spines undergo marked changes in structure that directly affect the functional communication between neurons. Elucidating the cytoskeletal events that induce these structural changes is fundamental to understanding synaptic biology. Actin plays a central role in the spine cytoskeleton, however the role of microtubules in spine function has been studied little. Although microtubules have a prominent role in transporting material throughout the dendrite that is destined for spines, they are not thought to directly influence spine structure or function. Using total internal reflectance fluorescent microscopy we discovered that microtubules rapidly invade dendritic protrusions of mature CNS neurons (up to 63 d in vitro), occasionally being associated with marked changes in spine morphology in the form of transient spine head protrusions (tSHPs). Two microtubules can occupy a spine simultaneously and microtubule targeting can occur from both the proximal and distal dendrite. A small percentage of spines are targeted at a time and all targeting events are transient, averaging only a few minutes. Nevertheless, over time many spines on a dendrite are targeted by microtubules. Importantly, we show that increasing neuronal activity enhances both the number of spines invaded by microtubules and the duration of these invasions. This study provides new insight into the dynamics of the neuronal cytoskeleton in mature CNS neurons and suggests that microtubules play an important, direct role in spine morphology and function.

The remarkable ability of a single axon to extend multiple branches and form terminal arbors allows vertebrate neurons to integrate information from divergent regions of the nervous system. Axons select appropriate pathways during development, but it is the branches that extend interstitially from the axon shaft and arborize at specific targets that are responsible for virtually all of the synaptic connectivity in the vertebrate CNS. How do axons form branches at specific target regions? Recent studies have identified molecular cues that activate intracellular signalling pathways in axons and mediate dynamic reorganization of the cytoskeleton to promote the formation of axon branches.