Coronin 2B regulates dendrite outgrowth by modulating actin dynamics.

Cytoskeletal remodeling is indispensable for the development and maintenance of neuronal structures and functions. However, the molecular machinery that controls the balance between actin polymerization and depolymerization during these processes is incompletely understood. Here, we report that coronin 2B, a conserved actin-binding protein, is concentrated at the tips of developing dendrites and that knockdown of coronin 2B inhibits the growth of dendrites. Importantly, coronin 2B interacts with actin and reduces the F-actin/G-actin ratio. Furthermore, the coiled-coil domain of coronin 2B is required for its oligomerization, thus confining coronin 2B to neurite tips. Our findings collectively suggest that coronin 2B is important for promoting dendrite outgrowth by limiting the speed of actin polymerization at growth cones.

Semaphorin-3A Promotes Degradation of Fragile X Mental Retardation Protein in Growth Cones via the Ubiquitin-Proteasome Pathway.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates local translation in dendrites and spines for synaptic plasticity. In axons, FMRP is implicated in axonal extension and axon guidance. We previously demonstrated the involvement of FMRP in growth cone collapse via a translation-dependent response to Semaphorin-3A (Sema3A), a repulsive axon guidance factor. In the case of attractive axon guidance factors, RNA-binding proteins such as zipcode binding protein 1 (ZBP1) accumulate towards the stimulated side of growth cones for local translation. However, it remains unclear how Sema3A effects FMRP localization in growth cones. Here, we show that levels of FMRP in growth cones of hippocampal neurons decreased after Sema3A stimulation. This decrease in FMRP was suppressed by the ubiquitin-activating enzyme E1 enzyme inhibitor PYR-41 and proteasome inhibitor MG132, suggesting that the ubiquitin-proteasome pathway is involved in Sema3A-induced FMRP degradation in growth cones. Moreover, the E1 enzyme or proteasome inhibitor suppressed Sema3A-induced increases in microtubule-associated protein 1B (MAP1B) in growth cones, suggesting that the ubiquitin-proteasome pathway promotes local translation of MAP1B, whose translation is mediated by FMRP. These inhibitors also blocked the Sema3A-induced growth cone collapse. Collectively, our results suggest that Sema3A promotes degradation of FMRP in growth cones through the ubiquitin-proteasome pathway, leading to growth cone collapse via local translation of MAP1B. These findings reveal a new mechanism of axon guidance regulation: degradation of the translational suppressor FMRP via the ubiquitin-proteasome pathway.

Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis.

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia.

Shank3 is localized in axons and presynaptic specializations of developing hippocampal neurons and involved in the modulation of NMDA receptor levels at axon terminals.

Autism-related Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses. A few studies, however, have already indicated that within a neuron, the presence of Shank family members is not limited to the postsynaptic density. By separating axons from dendrites of developing hippocampal neurons in microfluidic chambers, we show that RNA of all three Shank family members is present within axons. Immunostaining confirms these findings as all three Shanks are indeed found within separated axons and further co-localize with well-known proteins of the presynaptic specialization in axon terminals. Therefore, Shank proteins might not only serve as postsynaptic scaffold proteins, but also play a crucial role during axonal outgrowth and presynaptic development and function. This is supported by our findings that shRNA-mediated knockdown of Shank3 results in up-regulation of the NMDA receptor subunit GluN1 in axon terminals. Taken together, our findings will have major implications for the future analysis of neuronal Shank biology in both health and disease. Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses strongly related to several neuropsychiatric disorders. However, a few studies have already implicated a functional role of the Shanks beyond the postsynaptic density (PSD). We here show that all three Shanks are localized in both axons and pre-synaptic specializiations of developing hippocampal neurons in culture. We further provide evidence that Shank3 is involved in the modulation of NMDA receptor levels at axon terminals. Taken together, our study will open up novel avenues for the future analysis of neuronal Shank biology in both health and disease.

Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8).

The role of actin turnover in retrograde actin network flow in neuronal growth cones.

The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.

Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions.

Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, because classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.

Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones.

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant. Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette. Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

Localized alteration of microtubule polymerization in response to guidance cues.

Inhibition of microtubule dynamic instability prevents growth cone turning in response to guidance cues, yet specific changes in microtubule polymerization as growth cones encounter boundaries have not been investigated. In this study, we examined the rate and direction of microtubule polymerization in response to soluble nerve growth factor (NGF) and immobilized chondroitin sulfate proteoglycans (CSPGs) by expressing enhanced GFP-EB3 in rat pheochromocytoma (PC12) cells. GFP-EB3 comets were monitored in live cells using time-lapse epifluorescent microscopy. With an automated tracking system, the rate of microtubule polymerization was calculated as the frame-to-frame displacement of EB3 comets. Our results demonstrate that the rate of microtubule polymerization is increased following NGF treatment, whereas contact with CSPGs decreases microtubule polymerization rates. This reduction in microtubule polymerization rates was specifically localized to neurites in direct contact with CSPGs and not at noncontacting neurites. Additionally, we found an increase in the percentage of microtubules polymerizing in the retrograde direction in neurites at CSPG boundaries, with a concomitant decrease in the rate of retrograde microtubule polymerization. These results implicate localized changes in microtubule dynamics as an important component of the growth cone response to guidance cues.

Proteomics analysis of the temporal changes in axonal proteins during maturation.

After the initial primary projection, axons undergo various structural and functional changes to establish mature neural circuits. The changes in protein expression associated with this maturation were investigated in lateral olfactory tract axons using two-dimensional gel electrophoresis. The most prominent group upregulated during the period consisted of calcium-dependent membrane-binding proteins including VILIP1, neurocalcin delta, copine 6, and annexin A6 from three structurally different families. During maturation of primary cultured neurons, annexin A6 gradually became concentrated on the axon initial segment, and its overexpression significantly enhanced axon branching. On the other hand, overexpression of VILIP1 and neurocalcin delta reduced axon outgrowth and branching. The second group upregulated during axon maturation comprised tubulin- and microtubule-binding proteins including CRMP2, guanine deaminase, MAP1B, and fibronectin type3 SPRY domain-containing protein. Because the maturation of lateral olfactory axons involves massive extension of secondary collateral branches, the augmentation of these proteins during these stages may underlie the drastic restructuring of the axon cytoskeleton.

Stochastic actin polymerization and steady retrograde flow determine growth cone advancement.

Neuronal growth is an extremely complex yet reliable process that is directed by a dynamic lamellipodial structure at the tip of every growing neurite, called the growth cone. Lamellipodial edge fluctuations are controlled by the interplay between actin polymerization pushing the edge forward and molecular motor driven retrograde actin flow retracting the actin network. The leading edge switches randomly between extension and retraction processes. We identify switching of "on/off" states in actin polymerization as the main determinant of lamellipodial advancement. Our analysis of motility statistics allows for a prediction of growth direction. This was used in simulations explaining the amazing signal detection capabilities of neuronal growth by the experimentally found biased stochastic processes. Our measurements show that the intensity of stochastic fluctuations depend on changes in the underlying active intracellular processes and we find a power law eta = a*x(alpha) with exponent alpha = 2.63 +/- 0.12 between noise intensity eta and growth cone activity x, defined as the sum of protrusion and retraction velocity. Differences in the lamellipodial dynamics between primary neurons and a neuronal cell line further suggests that active processes tune the observed stochastic fluctuations. This hints at a possible role of noise intensity in determining signal detection sensitivity.

Drebrin E is involved in the regulation of axonal growth through actin-myosin interactions.

Drebrin is a well-known side-binding protein of F-actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F-actins and microtubules overlapped. Over-expression of drebrin E caused drebrin E and F-actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F-actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over-expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin-myosin interactions in the transitional zone of axonal growth cones.

The CRMP family of proteins and their role in Sema3A signaling.

The CRMP proteins were originally identified as mediators of Sema3A signaling and neuronal differentiation. Much has been learned about the mechanism by which CRMPs regulate cellular responses to Sema3A. In this review, the evidence for CRMP as a component of the Sema3A signaling cascade and the modulation of CRMP by plexin and phosphorylation are considered. In addition, current knowledge of the function of CRMP in a variety of cellular processes, including regulation of the cytoskeleton and endocytosis, is discussed in relationship to the mechanisms of axonal growth cone Sema3A response. The secreted protein Sema3A (collapsin-1) was the first identified vertebrate semaphorin. Sema3A acts primarily as a repulsive axon guidance cue, and can cause a dramatic collapse of the growth cone lamellipodium. This process results from the redistribution of the F-actin cytoskeleton and endocytosis of the growth cone cell membrane. Neuropilin-1 (NP1) and members of the class A plexins (PlexA) form a Sema3A receptor complex, with NP1 serving as a high-affinity ligand binding partner, and PlexA transducing the signal into the cell via its large intracellular domain. Although the effect of Sema3A on growth cones was first described nearly 15 years ago, the intracellular signaling pathways that lead to the cellular effects have only recently begun to be understood. Monomeric G-proteins, various kinases, the redox protein, MICAL, and protein turnover have all been implicated in PlexA transduction. In addition, the collapsin-response-mediator protein (CRMP) family of cytosolic phosphoproteins plays a crucial role in Sema3A/NP1/PlexA signal transduction. Current knowledge regarding CRMP functions are reviewed here.

Signaling of secreted semaphorins in growth cone steering.

Despite a tremendous amount of progress in the identification and characterization of many new players as components of class 3 secreted semaphorin signaling in growth cone steering (Fig. 1), our understanding of the molecular mechanisms is far from complete. More questions remain to be answered: how are differential cytoskeletal changes within a growth cone achieved in response to semaphorins? What are the target(s) of cyclic nucleotide modulation? How does a growth cone make a reliable decision in response to a shallow gradient? And finally, how does a growth cone maintain its sensitivity to a decreasing concentration ofsemaphorins when it is growing away from the source? With a high degree of interest in the field with the development of novel technologies in analyzing growth cone steering, we expect to see a much more complete picture of semaphoring signaling in the near future.

Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve growth cones analyzed by single quantum dot imaging.

During development of the nervous system, the tip of a growing axon, the growth cone (GC), must respond accurately to stimuli that direct its growth. This axonal navigation depends on extracellular concentration gradients of numerous guidance cues, including GABA. GCs can detect even weak directional signals, yet the mechanisms underlying this sensitivity remain unclear. Past studies in other eukaryotic chemotactic systems have pointed to the role of the spatial reorganization of the transduction pathway in their sensitive response. Here we have developed a single-molecule assay to observe individual GABA(A) receptors (GABA(A)Rs) in the plasma membrane of nerve GCs subjected to directional stimuli. We report that in the presence of an external GABA gradient GABA(A)Rs redistribute asymmetrically across the GC toward the gradient source. Single-particle tracking of GABA(A)Rs shows that the redistribution results from transient interactions between the receptors and the microtubules. Moreover, the relocalization is accompanied by an enhancement in the asymmetry of intracellular calcium concentration. Altogether, our results reveal a microtubule-dependent polarized reorganization of chemoreceptors at the cell surface and suggest that this polarization serves as an amplification step in GABA gradient sensing by nerve GCs.

Cytoplasmic dynein and LIS1 are required for microtubule advance during growth cone remodeling and fast axonal outgrowth.

Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent protein-tubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.

Single quantum dot tracking of membrane receptors.

The dynamics of membrane proteins in living cells has become a major issue to understand important biological questions such as chemotaxis, synaptic regulation, or signal transduction. The advent of semi-conductor quantum dots (QDs) has opened new perspectives for the study of membrane properties because these new nanomaterials enable measurements at the single molecule level with high signal-to-noise ratio. Probes used until now indeed encounter significant limitations: organic fluorophores and fluorescent proteins rapidly photobleach, whereas gold particles and latex beads, although more stable, are bulky and usually stain only one protein per experiment. In comparison, QDs are bright and photostable fluorescent probes with a size on the order of 10 nm can be used with standard immunochemical methods. We present the experimental protocols and methods of analysis which we used to investigate the dynamics of individual GABAA receptors in the axonal growth cone of spinal neurons in culture. Single QD tracking is nevertheless a general method, suitable to study many transmembrane proteins.

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

Membrane-bound CSPG mediates growth cone outgrowth and substrate specificity by Schwann cell contact with the DRG neuron cell body and not via growth cone contact.

The central nervous system and peripheral nervous system (CNS/PNS) contain factors that inhibit axon regeneration, including myelin-associated glycoprotein (MAG), the Nogo protein, and chondroitin sulfate proteoglycan (CSPG). They also contain factors that promote axon regeneration, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Axon regeneration into and within the CNS fails because the balance of factor favors inhibiting regeneration, while in the PNS, the balance of factor favors promoting regeneration. The balance of influences in the CNS can be shifted toward promoting axon regeneration by eliminating the regeneration-inhibiting factors, overwhelming them with regeneration-promoting factors, or making axon growth cones non-receptive to regeneration-inhibiting factors. The present in vitro experiments, using adult rat dorsal root ganglion (DRG) neurons, were designed to determine whether the regeneration-inhibiting influences of Schwann cell CSPG are mediated via Schwann cell membrane contact with the DRG neuron cell body or their growth cones. The average longest neurite of neurons in cell body contact with Schwann cells was 7.4-fold shorter than those of neurons without Schwann cell-neuron cell body contact (naked neurons), and the neurites showed substrate specificity, growing only on the Schwann cell membranes and not extending onto the laminin substrate. The neurites of naked neurons showed no substrate specificity and extended over the laminin substrate, as well as onto and off the Schwann cells. After digesting the Schwann cell CSPG with the enzyme C-ABC, neurons in cell body contact with Schwann cells extended neurites the same length as those of naked neurons, and their neurites showed no substrate selectivity. Further, the neurites of naked neurons were not longer than those of naked neurons not exposed to C-ABC. These data indicate that the extent of neurite outgrowth from adult rat DRG neurons and substrate specificity of their growth cone is mediated via contact between the Schwann cell membrane-bound CSPG and the DRG neuron cell body and not with their growth cones. Further, there was no apparent influence of diffusible or substrate-bound CSPG on neurite outgrowth. These results show that eliminating the CSPG of Schwann cells in contact with the cell body of DRG neurons eliminates the sensitivity of their growth cones to the CSPG-induced outgrowth inhibition. This may in turn allow the axons of these neurons to regenerate through the dorsal roots and into the spinal cord.