Modeling the role of myosin 1c in neuronal growth cone turning

We addressed the mechanical basis for how embryonic chick dorsal root ganglion growth cones turn on a uniform substrate of laminin-1. Turning is significantly correlated with lamellipodial area but not with filopodial length. We assessed the lamellipodial contribution to turning by asymmetric micro-CALI of myosin isoforms that causes localized lamellipodial expansion (myosin 1c) or filopodial retraction (myosin V). Episodes of asymmetric micro-CALI of myosin 1c (or myosin 1c and V together) caused significant turning of the growth cone. In contrast, repeated micro-CALI of myosin V or irradiation without added antibody did not turn growth cones. These findings argue that lamellipodia and not filopodia are necessary for growth cone turning. To model the role of myosin 1c on growth cone turning, we fitted the measured trajectories from asymmetric micro-CALI of myosin 1c-treated and untreated growth cones to the persistent random walk model. The first parameter in this equation, root-mean-square speed, is indistinguishable between the two data sets whereas the second parameter, the persistence of motion, is significantly increased (2.5-fold) as a result of asymmetric inactivation of myosin 1c by micro-CALI. This analysis demonstrates that growth cone turning results from an increase in the persistence of directional motion rather than a change in speed. Taken together, our results suggest that myosin 1c is a molecular correlate for directional persistence underlying growth cone motility.     

The search and prime hypothesis for growth cone turning

In the fields of axonal and dendritic guidance, there is now a significant accumulation of knowledge of how extracellular signaling molecules activate their cognate growth cone receptors. Relatively little is known about the subsequent activation of intracellular signaling pathways and actin reorganization, and very little is known about how microtubules (MTs) reorganize during growth cone turning. I hypothesize that dynamic MTs are required in order to catalyze the polarized actin assembly necessary for growth cone turning, that MTs and actin filaments promote each other's assembly through positive feedback, that MT stability is enhanced further through the formation of membrane-associated MT attachment sites, and that these MT stabilization events subsequently accelerate axonal/dendritic shaft formation.     

The role of cytoskeleton in organizing growth cones: a microfilament- associated growth cone component depends upon microtubules for its localization

We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament- associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to- distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.     

Agrin regulates growth cone turning of Xenopus spinal motoneurons

The pivotal role of agrin in inducing postsynaptic specializations at neuromuscular junctions has been well characterized. Increasing evidence suggests that agrin is also involved in neuronal development. In this study, we found that agrin inhibited neurite extension and, more importantly, a gradient of agrin induced repulsive growth-cone turning in cultured Xenopus spinal neurons. Incubation with a neutralizing antibody to agrin or expression of the extracellular domain of muscle-specific kinase, a component of the agrin receptor complex, abolished these effects of agrin. Agrin-induced repulsive growth-cone turning requires the activity of PI3-kinase and Ca2+ signaling. In addition, the expression of dominant-negative Rac1 inhibited neurite extension and blocked agrin-mediated growth-cone turning. Taken together, our findings suggest that agrin regulates neurite extension and provide evidence for an unanticipated role of agrin in growth-cone steering in developing neurons.     

Autonomous right-screw rotation of growth cone filopodia drives neurite turning

The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.     

Pursuing a ‘turning point’ in growth cone research

Growth cones are highly motile structures found at the leading edge of developing and regenerating nerve processes. Their role in axonal pathfinding has been well established and many guidance cues that influence growth cone behavior have now been identified. Many studies are now providing insights into the transduction and integration of signals in the growth cone, though a full understanding of growth cone behavior still eludes us. This review focuses on recent studies adding to the growing body of literature on growth cone behavior, focusing particularly on the level of autonomy the growth cone possesses and the role of local protein synthesis.     

Involvement of microtubules in the regulation of neuronal growth cone morphologic remodeling

The guidance of nerve fibers depends on the constant protrusion, movement, and retraction (i.e., remodeling) of growth cone lamellae and filopodia. We used drugs that interfere with the dynamics of microtubules to investigate the role of microtubules in the remodeling of larval amphibian spinal cord neuronal growth cones. Vinblastine (8–100 nM), taxol (10 nM), and nocodazole (330 nM) altered microtubule distributions in growth cones and decreased the percentage of lamellar perimeter undergoing remodeling, while not affecting the rates of lamellar protrusion and retraction. Also, 8–20 nM vinblastine caused temporary losses of the continuity of the originally fan-shaped lamella, resulting in two or more lamellae at the growth cone. At higher concentrations of microtubule drugs, the originally fan-shaped lamella broke up into separate smaller lamellae followed by the centrifugal displacement from the base of the growth cone and eventual collapse of the resultant lamellae. Low doses of cytochalasin B prevented the centrifugal displacement of lamellae in response to microtubule drugs. During microtubule drug-mediated loss of growth cone lamellae, some filopodia were observed to elongate to greater than normal lengths. Similarly, exposure to 20 nM vinblastine resulted in an increase in filopodial length but not filopodial number. As evidenced by DiOC₆(3) staining, 8–20 nM vinblastine altered the distribution of membranous organelles within growth cones, suggesting that the effects of microtubule drugs on growth cones may be mediated in part by alterations in organelle localization. Our data show that microtubules are involved in the maintenance and regulation of lamellar and filopodial structures at the neuronal growth cone. These findings have implications for the mechanisms by which growth cones are guided during development and regeneration. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 121–140, 1998     

The role of microtubule dynamics in growth cone motility and axonal growth

The growth cone contains dynamic and relatively stable microtubule populations, whose function in motility and axonal growth is uncharacterized. We have used vinblastine at low doses to inhibit microtubule dynamics without appreciable depolymerization to probe the role of these dynamics in growth cone behavior. At doses of vinblastine that interfere only with dynamics, the forward and persistent movement of the growth cone is inhibited and the growth cone wanders without appreciable forward translocation; it quickly resumes forward growth after the vinblastine is washed out. Direct visualization of fluorescently tagged microtubules in these neurons shows that in the absence of dynamic microtubules, the remaining mass of polymer does not invade the peripheral lamella and does not undergo the usual cycle of bundling and splaying and the growth cone stops forward movement. These experiments argue for a role for dynamic microtubules in allowing microtubule rearrangements in the growth cone. These rearrangements seem to be necessary for microtubule bundling, the subsequent coalescence of the cortex around the bundle to form new axon, and forward translocation of the growth cone.     

Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein

Cytoplasmic second messengers, Ca2+ and cAMP, regulate nerve growth cone turning responses induced by many guidance cues, but the causal relationship between these signaling pathways has been unclear. We here report that, for growth cone turning induced by a gradient of myelin-associated glycoprotein (MAG), cAMP acts by modulating MAG-induced Ca2+ signaling. Growth cone repulsion induced by MAG was accompanied by localized Ca2+ signals on the side of the growth cone facing the MAG source, due to Ca2+ release from intracellular stores. Elevating cAMP signaling activity or membrane depolarization enhanced MAG-induced Ca2+ signals and converted growth cone repulsion to attraction. Directly imposing high- or low-amplitude Ca2+ signals with an extracellular gradient of Ca2+ ionophore was sufficient to trigger either attractive or repulsive turning, respectively. Thus, distinct Ca2+ signaling, which can be modulated by cAMP, mediates the bidirectional turning responses induced by MAG.     

Retinoic acid induces neurite outgrowth and growth cone turning in invertebrate neurons

Identification of molecules involved in neurite outgrowth during development and/or regeneration is a major goal in the field of neuroscience. Retinoic acid (RA) is a biologically important metabolite of vitamin A that acts as a trophic factor and has been implicated in neurite outgrowth and regeneration in many vertebrate species. Although abundant in the CNS of many vertebrates, the precise role of RA in neural regeneration has yet to be determined. Moreover, very little information is available regarding the role of RA in invertebrate nervous systems. Here, we demonstrate for the first time that RA induces neurite outgrowth from invertebrate neurons. Using individually identified neurons isolated from the CNS of Lymnaea stagnalis, we demonstrated that a significantly greater proportion of cells produced neurite outgrowth in RA. RA also extended the duration of time that cells remained electrically excitable in vitro, and we showed that exogenously applied RA acted as a chemoattractive factor and induced growth cone turning toward the source of RA. This is the first demonstration that RA can induce turning of an individual growth cone. These data strongly suggest that the actions of RA on neurite outgrowth and cell survival are highly conserved across species.     

The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.     

Focal loss of actin bundles causes microtubule redistribution and growth cone turning

It is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.     

Stochastic dynamics of the nerve growth cone and its microtubules during neurite outgrowth

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345-363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of approximately 4 min, meaning that growth cone advances tended to be followed by growth cone retractions approximately 4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cone's microtubule array. Cross-correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of approximately 0-2 min, while microtubules tended to precede growth cone dynamics on a approximately 0-20-s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean-squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance. (c) 1996 John Wiley & Sons, Inc.     

Effects of substrate geometry on growth cone behavior and axon branching

At the leading edge of a growing axon, the growth cone determines the path the axon takes and also plays a role in the formation of branches, decisions that are regulated by a complex array of chemical signals. Here, we used microfabrication technology to determine whether differences in substrate geometry, independent of changes in substrate chemistry, can modulate growth cone motility and branching, by patterning a polylysine grid of narrow (2 or 5 microm wide) intersecting lines. The shape of the intersections varied from circular nodes 15 microm in diameter to simple crossed lines (nodeless intersections). Time-lapse recordings of cultured hippocampal neurons showed that simple variations in substrate geometry changed growth cone shape, and altered the rate of growth and the probability of branching. When crossing onto a node intersection the growth cone paused, often for hours, and microtubules appeared to defasciculate. Once beyond the node, filopodia and lamellipodia persisted at that site, sometimes forming a collateral branch. At nodeless intersections, the growth cone passed through with minimal hesitation, often becoming divided into separate areas of motility that led to the growth of separate branches. When several lines intersected at a common point, growth cones sometimes split into several subdivisions, resulting in the emergence of as many as five branches. Such experiments revealed an intrinsic preference for branches to form at angles less than 90 degrees . These data show that simple changes in the geometry of a chemically homogeneous substrate are detected by the growth cone and can regulate axonal growth and the formation of branches.     

Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone

Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F-actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F-actin from the leading lamella. The loss of F-actin was nearly complete within 2-3 min of CB application and was largely reversed within 5-6 min of CB removal. The loss and recovery of F-actin were found to occur with a very distinctive spatial organization. Within 20-30 s of CB application, F-actin networks receded from the entire peripheral margin of the lamella forming a band devoid of F-actin. This band widened as F-actin receded at rates of 3-6 microns/min. Upon removal of CB, F-actin began to reappear within 20-30 s. The initial reappearance of F-actin took two forms: a coarse isotropic matrix of F-actin bundles throughout the lamella, and a denser matrix along the peripheral margin. The denser peripheral matrix then expanded in width, extending centrally to replace the coarse matrix at rates again between 3-6 microns/min. These results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min. CB treatment was also observed to alter the distribution of microtubules, assayed by antitubulin antibody staining. Normally, microtubules are restricted to the neurite shaft and a central growth cone domain. Within approximately 5 min after CB application, however, microtubules began extending into the lamellar region, often reaching the peripheral margin. Upon removal of CB, the microtubules were restored to their former central localization. The timing of these microtubule redistributions is consistent with their being secondary to effects of CB on lamellar F-actin.     

IRES‐mediated translation of cofilin regulates axonal growth cone extension and turning

In neuronal development, dynamic rearrangement of actin promotes axonal growth cone extension, and spatiotemporal translation of local mRNAs in response to guidance cues directs axonal growth cone steering, where cofilin plays a critical role. While regulation of cofilin activity is well studied, regulatory mechanism for cofilin mRNA translation in neurons is unknown. In eukaryotic cells, proteins can be synthesized by cap‐dependent or cap‐independent mechanism via internal ribosome entry site (IRES)‐mediated translation. IRES‐mediated translation has been reported in various pathophysiological conditions, but its role in normal physiological environment is poorly understood. Here, we report that 5′UTR of cofilin mRNA contains an IRES element, and cofilin is predominantly translated by IRES‐mediated mechanism in neurons. Furthermore, we show that IRES‐mediated translation of cofilin is required for both axon extension and axonal growth cone steering. Our results provide new insights into the function of IRES‐mediated translation in neuronal development.     

The role of microtubules in guard cell function

Guard cells are able to sense a multitude of environmental signals and appropriately adjust the stomatal pore to regulate gas exchange in and out of the leaf. The role of the microtubule cytoskeleton during these stomatal movements has been debated. To help resolve this debate, in vivo stomatal aperture assays with different microtubule inhibitors were performed. We observed that guard cells expressing the microtubule-binding green fluorescent fusion protein (green fluorescent protein::microtubule binding domain) fail to open for all major environmental triggers of stomatal opening. Furthermore, guard cells treated with the anti-microtubule drugs, propyzamide, oryzalin, and trifluralin also failed to open under the same environmental conditions. The inhibitory conditions caused by green fluorescent protein::microtubule binding domain and these anti-microtubule drugs could be reversed using the proton pump activator, fusicoccin. Therefore, we conclude that microtubules are involved in an upstream event prior to the ionic fluxes leading to stomatal opening. In a mechanistic manner, evidence is presented to implicate a microtubule-associated protein in this putative microtubule-based signal transduction event.     

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.     

The role of microtubules in processive kinesin movement

Kinesins are microtubule-based motors that are important for various intracellular transport processes. To understand the mechanism of kinesin movement, X-ray crystallography has been used to study the atomic structures of kinesin. However, as crystal structures of kinesin alone accumulate, it is becoming clear that kinesin structures should also be investigated with the microtubule to understand the contribution of the microtubule track to the nucleotide-induced conformational changes of kinesin. Recently, several high-resolution structures of kinesin with microtubules were obtained using cryo-electron microscopy. Comparison with X-ray crystallographic structures revealed the importance of the microtubule in determining the conformation of kinesin. Together with recent biophysical data, we describe different structural models of processive kinesin movement and provide a framework for future experiments.     

Myristoylated, alanine-rich C-kinase substrate phosphorylation regulates growth cone adhesion and pathfinding

Repellents evoke growth cone turning by eliciting asymmetric, localized loss of actin cytoskeleton together with changes in substratum attachment. We have demonstrated that semaphorin-3A (Sema3A)-induced growth cone detachment and collapse require eicosanoid-mediated activation of protein kinase C epsilon (PKC epsilon) and that the major PKC epsilon target is the myristoylated, alanine-rich C-kinase substrate (MARCKS). Here, we show that PKC activation is necessary for growth cone turning and that MARCKS, while at the membrane, colocalizes with alpha3-integrin in a peripheral adhesive zone of the growth cone. Phosphorylation of MARCKS causes its translocation from the membrane to the cytosol. Silencing MARCKS expression dramatically reduces growth cone spread, whereas overexpression of wild-type MARCKS inhibits growth cone collapse triggered by PKC activation. Expression of phosphorylation-deficient, mutant MARCKS greatly expands growth cone adhesion, and this is characterized by extensive colocalization of MARCKS and alpha3-integrin, resistance to eicosanoid-triggered detachment and collapse, and reversal of Sema3A-induced repulsion into attraction. We conclude that MARCKS is involved in regulating growth cone adhesion as follows: its nonphosphorylated form stabilizes integrin-mediated adhesions, and its phosphorylation-triggered release from adhesions causes localized growth cone detachment critical for turning and collapse.