Growth Cone Size Research Articles

Polyglutamine binding protein 1 regulates neurite outgrowth through recruiting N-WASP

Neurite outgrowth is a critical step in neural development, leading to the generation of neurite branches that allow individual neurons to make contacts with multiple neurons within the target region. Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein with a key role in neural development. Our recent mass spectrometric analysis showed that PQBP1 associates with neural Wiskott-Aldrich syndrome protein (N-WASP), an important actin polymerization-promoting factor involved in neurite outgrowth. Here, we report that the WW domain of PQBP1 directly interacts with the proline-rich domain of N-WASP. The disruption of this interaction leads to impaired neurite outgrowth and growth cone size. Furthermore, we demonstrate that PQBP1/N-WASP interaction is critical for the recruitment of N-WASP to the growth cone, but does not affect N-WASP protein levels or N-WASP-induced actin polymerization. Our results indicated that PQBP1 regulates neurite outgrowth by recruiting N-WASP to the growth cone, thus representing an alternative molecular mechanism via which PQBP1-mediates neurite outgrowth.

CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.

Nifedipine Ameliorates Cellular Differentiation Defects of Smn-Deficient Motor Neurons and Enhances Neuromuscular Transmission in SMA Mice.

In spinal muscular atrophy (SMA), mutations in or loss of the Survival Motor Neuron 1 (SMN1) gene reduce full-length SMN protein levels, which leads to the degeneration of a percentage of motor neurons. In mouse models of SMA, the development and maintenance of spinal motor neurons and the neuromuscular junction (NMJ) function are altered. Since nifedipine is known to be neuroprotective and increases neurotransmission in nerve terminals, we investigated its effects on cultured spinal cord motor neurons and motor nerve terminals of control and SMA mice. We found that application of nifedipine increased the frequency of spontaneous Ca2+ transients, growth cone size, cluster-like formations of Cav2.2 channels, and it normalized axon extension in SMA neurons in culture. At the NMJ, nifedipine significantly increased evoked and spontaneous release at low-frequency stimulation in both genotypes. High-strength stimulation revealed that nifedipine increased the size of the readily releasable pool (RRP) of vesicles in control but not SMA mice. These findings provide experimental evidence about the ability of nifedipine to prevent the appearance of developmental defects in SMA embryonic motor neurons in culture and reveal to which extent nifedipine could still increase neurotransmission at the NMJ in SMA mice under different functional demands.

Microcephaly gene Cenpj regulates axonal growth in cortical neurons through microtubule destabilization.

Neocortex development comprises of a complex series of time- and space-specific processes to generate the typical interconnected six-layered architecture of adult mammals. Axon growth is required for the proper establishment of cortical circuits. Malformations in axonal growth and pathfinding might lead to severe neuropathologies, such as corpus callosum dysgenesis. Cenpj, a microcephaly gene, encodes a scaffold protein that regulates centrosome biogenesis and microtubule stabilization. During corticogenesis, Cenpj regulates progenitor division and neuronal migration. Since microtubule stabilization is crucial for axon extension, we investigated the role of Cenpj in axon growth during cortical development in a mouse model. Through loss- and gain-of-function assays ex vivo and in utero, we quantified callosal axonal length, branching, and growth cone size compared to controls. We observed that silencing Cenpj results in an increased axonal length. Ex vivo, we assessed the number of branches, the area of growth cones and the stability of microtubules. In silenced Cenpj axons, there were more branches, larger growth cone area, and more stable microtubules. Rescue experiments confirmed that neurons present axonal length comparable to controls. Here we propose that Cenpj regulates axon growth by destabilizing microtubules during cortical development. Finally, our findings suggest that Cenpj might be a novel target for axonal regeneration.

SHANK2 Mutations Result in Dysregulation of the ERK1/2 Pathway in Human Induced Pluripotent Stem Cells-Derived Neurons and Shank2(-/-) Mice.

SHANK2 (ProSAP1) is a postsynaptic scaffolding protein of excitatory synapses in the central nervous system and implicated in the development of autism spectrum disorders (ASD). Patients with mutations in SHANK2 show autism-like behaviors, developmental delay, and intellectual disability. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying a heterozygous deletion of SHANK2 and from the unaffected parents. In patient hiPSCs and derived neurons SHANK2 mRNA and protein expression was reduced. During neuronal maturation, a reduction in growth cone size and a transient increase in neuronal soma size were observed. Neuronal proliferation was increased, and apoptosis was decreased in young and mature neurons. Additionally, mature patient hiPSC-derived neurons showed dysregulated excitatory signaling and a decrease of a broad range of signaling molecules of the ERK-MAP kinase pathway. These findings could be confirmed in brain samples from Shank2(−/−) mice, which also showed decreased mGluR5 and phospho-ERK1/2 expression. Our study broadens the current knowledge of SHANK2-related ASD. We highlight the importance of excitatory-inhibitory balance and mGluR5 dysregulation with disturbed downstream ERK1/2 signaling in ASD, which provides possible future therapeutic strategies for SHANK2-related ASD.

Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size.

The cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where it promoted neuritogenesis in immature human neurons. Super-resolution microscopy revealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by autism spectrum disorder (ASD)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.

Functional Redundancy of Cyclase-Associated Proteins CAP1 and CAP2 in Differentiating Neurons

Cyclase-associated proteins (CAPs) are evolutionary-conserved actin-binding proteins with crucial functions in regulating actin dynamics, the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). Mammals possess two family members (CAP1 and CAP2) with different expression patterns. Unlike most other tissues, both CAPs are expressed in the brain and present in hippocampal neurons. We recently reported crucial roles for CAP1 in growth cone function, neuron differentiation, and neuron connectivity in the mouse brain. Instead, CAP2 controls dendritic spine morphology and synaptic plasticity, and its dysregulation contributes to Alzheimer’s disease pathology. These findings are in line with a model in which CAP1 controls important aspects during neuron differentiation, while CAP2 is relevant in differentiated neurons. We here report CAP2 expression during neuron differentiation and its enrichment in growth cones. We therefore hypothesized that CAP2 is relevant not only in excitatory synapses, but also in differentiating neurons. However, CAP2 inactivation neither impaired growth cone morphology and motility nor neuron differentiation. Moreover, CAP2 mutant mice did not display any obvious changes in brain anatomy. Hence, differently from CAP1, CAP2 was dispensable for neuron differentiation and brain development. Interestingly, overexpression of CAP2 rescued not only growth cone size in CAP1-deficient neurons, but also their morphology and differentiation. Our data provide evidence for functional redundancy of CAP1 and CAP2 in differentiating neurons, and they suggest compensatory mechanisms in single mutant neurons.

Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons-New Insights into the Role of the C-Terminal Region of Tpm3.1.

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

HDAC6 and Miro1: Another interaction causing trouble in neurons.

Myelin-associated glycoprotein and chondroitin sulfate proteoglycans in the extracellular matrix can prevent regeneration of injured axons. In this issue, Kalinski et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201702187) report that inhibition of HDAC6 prevents the deacetylation of Miro1, increases mitochondrial axonal transport, and restores the size of axonal growth cones.

Cholesterol Depletion Regulates Axonal Growth and Enhances Central and Peripheral Nerve Regeneration.

Axonal growth during normal development and axonal regeneration rely on the action of many receptor signaling systems and complexes, most of them located in specialized raft membrane microdomains with a precise lipid composition. Cholesterol is a component of membrane rafts and the integrity of these structures depends on the concentrations present of this compound. Here we explored the effect of cholesterol depletion in both developing neurons and regenerating axons. First, we show that cholesterol depletion in vitro in developing neurons from the central and peripheral nervous systems increases the size of growth cones, the density of filopodium-like structures and the number of neurite branching points. Next, we demonstrate that cholesterol depletion enhances axonal regeneration after axotomy in vitro both in a microfluidic system using dissociated hippocampal neurons and in a slice-coculture organotypic model of axotomy and regeneration. Finally, using axotomy experiments in the sciatic nerve, we also show that cholesterol depletion favors axonal regeneration in vivo. Importantly, the enhanced regeneration observed in peripheral axons also correlated with earlier electrophysiological responses, thereby indicating functional recovery following the regeneration. Taken together, our results suggest that cholesterol depletion per se is able to promote axonal growth in developing axons and to increase axonal regeneration in vitro and in vivo both in the central and peripheral nervous systems.

Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization.

Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.

Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture.

As one major component of extracellular matrix (ECM) in the central nervous system, chondroitin sulfate proteoglycans (CSPGs) have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN) culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM) which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Nogo-B is the major form of Nogo at the floor plate and likely mediates crossing of commissural axons in the mouse spinal cord.

Using Nogo antibodies with defined binding specificity, Nogo-B, but not Nogo-A, was localized on radial glia in the floor plate of mouse embryos. The presence of Nogo-B was confirmed in Nogo-A knockout mice. In explant cultures of embryonic day (E) 11 and E12 spinal cord, blocking of NgR function with antagonist peptide NEP1-40 reduced the crossing of newly arrived commissural axons, resulting in an accumulation of growth cones in the floor plate. Analysis of growth cone morphology demonstrated an increase in size of growth cones in the floor plate after peptide treatment, which was not detected in axons growing toward the midline. In knockout embryos, midline crossing was not affected by absence of Nogo-A. In co-culture experiments using collagen gel, floor plate showed a strong inhibitory effect on the extension of post-commissural neurites from the spinal cord. This effect was abolished by NEP1-40, and was observed neither in pre-commissural neurites, nor in post-commissural neurites grown with floor plate derived from Nogo-A knockout embryo. Furthermore, western blot analysis of conditioned medium from floor plates showed a truncated form of Nogo with molecular weight of 37 kDa, which could mediate the diffusible effect to axon growth. We conclude that Nogo-B is expressed in the floor plate of mouse embryo, which probably mediates axon crossing in the spinal cord by repelling axons out of the midline when they start upregulate NgR. Nogo acts on axon growth not only through a contact-mediated mechanism, but also through a diffusible mechanism.

Increase in Growth Cone Size Correlates with Decrease in Neurite Growth Rate.

Several important discoveries in growth cone cell biology were made possible by the use of growth cones derived from cultured Aplysia bag cell neurons, including the characterization of the organization and dynamics of the cytoskeleton. The majority of these Aplysia studies focused on large growth cones induced by poly-L-lysine substrates at early stages in cell culture. Under these conditions, the growth cones are in a steady state with very little net advancement. Here, we offer a comprehensive cellular analysis of the motile behavior of Aplysia growth cones in culture beyond this pausing state. We found that average growth cone size decreased with cell culture time whereas average growth rate increased. This inverse correlation of growth rate and growth cone size was due to the occurrence of large growth cones with a peripheral domain larger than 100 μm2. The large pausing growth cones had central domains that were less consistently aligned with the direction of growth and could be converted into smaller, faster-growing growth cones by addition of a three-dimensional collagen gel. We conclude that the significant lateral expansion of lamellipodia and filopodia as observed during these culture conditions has a negative effect on neurite growth.

Cytoskeleton involvement in lithium-induced SH-SY5Y neuritogenesis and the role of glycogen synthase kinase 3β.

Lithium modulates signals impacting on the cytoskeleton, a dynamic system contributing to neural plasticity at multiple levels. In this study, SH-SY5Y human neuronal cells were cultured in the absence (C) or in presence (Li) of a 0.5 mM Li2CO3 (i.e. 1 mM lithium ion) for 25-50 weeks. We investigated the effect of this treatment on (1) morphological changes of cells observed using Hemalun eosin staining assay, (2) cytoskeletal changes by indirect immunofluorescence (IIF) staining of microtubules (α-tubulin) and heavy neurofilaments subunits (NF-H) and by measuring the expression rate changes of genes coding for receptor for activated C kinase (RACK1), casein kinase2 (CK2) and thymosine beta-10 using cDNA arrays technology, (3) cell adhesion properties by IIF staining of β-catenin protein. Besides, we have tried to understand the molecular mechanism of lithium action that triggers changes in cytoskeleton and neurites outgrowth. Thus, we examined the effect of this treatment on glycogen synthase kinase 3 (GSK3) expression and activity using western blotting of GSK3 and phosphorylated β-catenin, a downstream GSK3 target protein. Our results showed that lithium treatment reduces axon length, increases axonal spreading, enhances neurites growth and neurites branching with an increase of growth cone size. Moreover, genes coding for CK2 and thymosine beta-10 were significantly up-regulated, however, that coding for RACK1 was down-regulated. The most interesting result in this work is that mechanism underlying lithium action was not related to the inhibition of GSK3 activity. In fact, neither expression rate nor activity of this protein was changed.

Phosphatidylinositol 4-phosphate 5-kinase β regulates growth cone morphology and Semaphorin 3A-triggered growth cone collapse in mouse dorsal root ganglion neurons

Growth cone motility and morphology, which are critical for axon guidance, are controlled through intracellular events such as actin cytoskeletal reorganization and vesicular trafficking. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] has been implicated in regulation of these cellular processes in a diverse range of cell types. The main kinases involved in the production of PI(4,5)P2 are the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family, which consist of three isozymes, α, β and γ. Here, we demonstrate the involvement of PIP5Kβ in growth cone dynamics. Overexpression of a lipid kinase-deficient mutant of PIP5Kβ (PIP5Kβ-KD) in mouse dorsal root ganglion (DRG) neurons stimulated axon elongation and increased growth cone size, whereas wild-type PIP5Kβ tended to show opposite effects. Furthermore, PIP5Kβ-KD inhibited growth cone collapse of DRG neurons induced by semaphorin 3A (Sema3A). These results provide evidence that PIP5Kβ negatively regulates axon elongation and growth cone size and is involved in the cellular signaling pathway for Sema3A-triggered repulsion in DRG neurons.

VEGF-Induced Growth Cone Enhancement Is Diminished by Inhibiting Tyrosine-Residue 1214 of VEGFR-2

Axonal outgrowth is of paramount significance for establishing the intricate neuronal network both during embryogenesis and nerve regeneration. Vascular endothelial growth factor (VEGF), which is known for its essential role in vascular sprouting and its involvement in cancer, has recently been found to exert a trophic activity on neurons leading to an increased axonal outgrowth. Although two receptors, VEGFR-2 and neuropilin-1, were identified on neurons, the signaling pathways associated with them are not well understood. The aim of this study was to analyze the influence of VEGF on the growth cone morphology and motility of dorsal root ganglia (DRG) neurons. Moreover, we aimed for a deeper understanding of VEGFR-2 on growth cones that potentially mediates the stimulating and attractive effects. We cultivated chicken DRG in medium containing mouse VEGF and analyzed growth cone size. The data presented here show a positive effect of VEGF on growth cone size. Furthermore, we interrupted the activity of VEGFR-2 by either blocking the tyrosine residue 1214 (tyr1214) or by inhibiting the receptor phosphorylation with axitinib, a novel small molecule, which has recently entered phase III trials for cancer treatment. Disruption of the VEGFR-2 leads to a significantly diminished growth cone size. Based on these findings, we propose a positive effect of VEGF on peripheral nervous system growth cone size and show for the first time quantitative data to underline this hypothesis. Additionally, we propose that VEGFR-2 and especially the tyr1214-dependent pathway of VEGFR-2 are of importance in VEGF signaling in the growth cone of DRG neurons.

Kinesin-12, a Mitotic Microtubule-Associated Motor Protein, Impacts Axonal Growth, Navigation, and Branching

Kinesin-12 (also called Kif15) is a mitotic motor protein that continues to be expressed in developing neurons. Depletion of kinesin-12 causes axons to grow faster, more than doubles the frequency of microtubule transport in both directions in the axon, prevents growth cones from turning properly, and enhances the invasion of microtubules into filopodia. These results are remarkably similar to those obtained in previous studies in which neurons were depleted of kinesin-5 (also called Eg5 or Kif11), another mitotic motor protein that continues to be expressed in developing neurons. However, there are also notable differences in the phenotypes obtained with depleting each of these motors. Depleting kinesin-12 decreases axonal branching and growth cone size, whereas inhibiting kinesin-5 increases these parameters. In addition, depleting kinesin-12 diminishes the appearance of growth-cone-like waves along the length of the axon, an effect not observed with depletion of kinesin-5. Finally, depletion of kinesin-12 abolishes the "waggling" behavior of microtubules that occurs as they assemble along actin bundles within filopodia, whereas inhibition of kinesin-5 does not. Interestingly, and perhaps relevant to these differences in phenotype, in biochemical studies, kinesin-12 coimmunoprecipitates with actin but kinesin-5 does not. Collectively, these findings support a scenario whereby kinesin-12 shares functions with kinesin-5 related to microtubule-microtubule interactions, but kinesin-12 has other functions not shared by kinesin-5 that are related to the ability of kinesin-12 to interact with actin.

PTEN depletion rescues axonal growth defect and improves survival in SMN-deficient motor neurons

Phosphatase and tensin homolog (PTEN), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, is widely involved in the regulation of protein synthesis. Here we show that the PTEN protein is enriched in cell bodies and axon terminals of purified motor neurons. We explored the role of the PTEN pathway by manipulating PTEN expression in healthy and diseased motor neurons. PTEN depletion led to an increase in growth cone size, promotion of axonal elongation and increased survival of these cells. These changes were associated with alterations of downstream signaling pathways for local protein synthesis as revealed by an increase in pAKT and p70S6. Most notably, this treatment also restores beta-actin protein levels in axonal growth cones of SMN-deficient motor neurons. Furthermore, we report here that a single injection of adeno-associated virus serotype 6 (AAV6) expressing siPTEN into hind limb muscles at postnatal day 1 in SMNDelta7 mice leads to a significant PTEN depletion and robust improvement in motor neuron survival. Taken together, these data indicate that PTEN-mediated regulation of protein synthesis in motor neurons could represent a target for therapy in spinal muscular atrophy.

The Arp2/3 complex, UNC-115/abLIM, and UNC-34/Enabled regulate axon guidance and growth cone filopodia formation in Caenorhabditis elegans

BackgroundWhile many molecules involved in axon guidance have been identified, the cellular and molecular mechanisms by which these molecules regulate growth cone morphology during axon outgrowth remain to be elucidated. The actin cytoskeleton of the growth cone underlies the formation of lamellipodia and filopodia that control growth cone outgrowth and guidance. The role of the Arp2/3 complex in growth cone filopodia formation has been controversial, and other mechanisms of growth cone filopodia formation remain to be described.ResultsHere we show that mutations in genes encoding the Arp2/3 complex (arx genes) caused defects in axon guidance. Analysis of developing growth cones in vivo showed that arx mutants displayed defects in filopodia and reduced growth cone size. Time-lapse analysis of growth cones in living animals indicated that arx mutants affected the rate of growth cone filopodia formation but not filopodia stability or length. Two other actin modulatory proteins, UNC-115/abLIM and UNC-34/Enabled, that had been shown previously to affect axon guidance had overlapping roles with Arp2/3 in axon guidance and also affected the rate of filopodia initiation but not stability or length.ConclusionOur results indicate that the Arp2/3 complex is required cell-autonomously for axon guidance and growth cone filopodia initiation. Furthermore, they show that two other actin-binding proteins, UNC-115/abLIM and UNC-34/Enabled, also control growth cone filopodia formation, possibly in parallel to Arp2/3. These studies indicate that, in vivo, multiple actin modulatory pathways including the Arp2/3 complex contribute to growth cone filopodia formation during growth cone outgrowth.