Concentration-dependent stimulation and inhibition of growth cone behavior and neurite elongation by protein kinase inhibitors KT5926 and K-252a

We examined the concentration- and time-dependent effects of two related protein kinase inhibitors, KT5926 and K-252a, on neurite formation and nerve growth cone migration of chick embryo sensory neurons. The effects of these drugs on neurite formation over an 18-h period were dissimilar. KT5926 stimulated neurite formation at concentrations between 100 and 500 nM and inhibited neurite formation at 5 μM. K-252a had no stimulatory effects on neurite formation, and it inhibited neurite formation at concentrations above 50 nM. This difference may occur because K-252a inhibits activation of the nerve growth factor receptor trk A, while KT5926 does not inhibit trk A. Both drugs, however, had similar immediate effects on growth cone migration. Growth cone migration and lamellipodial spreading were rapidly stimulated by 500 nM concentrations of KT5926 and K-252a. At 2 μM levels of either drug, growth cone spreading was still stimulated, but growth cone migration was inhibited by both drugs. These results show that changes in protein phosphorylation/dephosphorylation can rapidly regulate the cellular machinery that is responsible for driving growth cone migration and neurite elongation. The different effects of 2 μM concentrations of either KT5926 or K-252a on growth cone spreading versus migration suggests that the actin-dependent protrusive motility of the growth cone leading margin is regulated differently by changes in protein phosphorylation and dephosphorylation than the cytoskeletal mechanism that drives neurite elongation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 161–171, 1997     

C-kinase manipulations disrupt activity-driven retinotopic sharpening in regenerating goldfish retinotectal projection

Regenerating optic axons initially branch over a wide area in tectum to form a crude retinotopic map. The map is sharpened, and retinotopically appropriate synapses are stabilized via NMDA receptors that detect, via summation of EPSPs, the coincident activity of neighboring ganglion cells that make synapses onto common tectal cells. Sharpening shares a number of properties with long-term potentiation (LTP) in hippocampus. This study tested whether protein kinase C (PKC) activation is necessary for sharpening as it is for LTP. Intracular (IO) or intracranial (IC) injections of kinase inhibitors or activators were made every other day from 19 to 37 days postcrush (sensitive period), and the projections formed were later recorded. Retinotopic sharpening was prevented by IC injection of the following agents: (1) general kinase inhibitors sphingosine and H7 (100-200 μM in fluid above brain), (2) active but not inactive phorbols (TPA, 1 μM), and (3) calphostin C (1 μM), a specific and irreversible PKC inhibitor. The mature projection on the opposite tectum, however, when examined was not unsharpened. Lack of sharpening was reflected in multiunit fields at each tectal point that averaged 27°–30° versus 11° in Ringers and inactive phorbol control regenerates. Intraocular injections of either TPA (1 μM), or calphostin C (1 μM) also prevented sharpening (26° and 32° multiunit fields), suggesting action on PKC axonally transported to the presynaptic terminals. Calphostin C had no noticeable effect on the firing patterns of retinal ganglion cells. The endogenous activator of PKC, arachidonic acid (AA), disrupted sharpening at 20 μM or higher (IC injection, 32° multiunit fields), while a control fatty acid, elaidic acid, had no effect. Although AA at 5 μM showed no effect, and diacylglycerol at 5 μM exhibited only small effects, together they produced a large synergistic effect (32° multiunit fields). Such synergy mirrors the synergy in the activation of several isoforms of PKC. Actual concentrations in the extradural fluid around the brain were assayed via injections of ³H-AA. Levels fell about sixfold after a day and by an additional fivefold the second day before the next injection. The results confirm that activity-driven retinotopic sharpening is very sensitive to manipulations of kinases, especially PKC. © 1994 John Wiley & Sons, Inc.     

Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo

The role of myosins in Xenopus retinal ganglion cell growth cone motility in the optic tract was studied using two pharmacologic inhibitors with different specificities. 2,3-Butanedione monoxime (BDM) disrupts myosin—actin interactions of all myosins, and ML-7 specifically inhibits activation of myosin II. Both inhibitors caused growth cones to assume a collapsed morphology and decreased growth cone speed. Similar effects were observed in vitro. Interestingly, the effects of the two inhibitors, while similar, were clearly distinguishable, raising the possibility that different myosins may have different functional roles in growth cone motility. BDM caused growth cones to withdraw lamellipodia and some filopodia and eventually to freeze, whereas ML-7 caused total collapse and retraction. Concentrations of BDM and ML-7 that had no effect when applied independently stopped growth cones when applied simultaneously, suggesting that these inhibitors act synergistically on myosin function, thus providing evidence of specificity. These results imply that normal growth cone motility in the molecularly and spatially complex environment of the living brain requires myosin function. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 567–578, 1997     

Localization of voltage-sensitive calcium channels along developing neurites: their possible role in regulating neurite elongation

Calcium action potentials were extracellularly recorded from growth cones of differentiated N1E-115 neuroblastoma cells maintained in monolayer cultures. Extracellular recordings along the neurites suggest that voltage-activated Ca²⁺ channels are less abundant in the processes than in the growth cones. In order to investigate if Ca²⁺ entry into the growth cone plays a role in the regulation of neurite growth, we studied the morphological changes induced by experimental conditions which permit calcium entry. Cells were depolarized either by 30 mM potassium (for 10–60 min) or by stimulating the soma (for 20–120 min) with an intracellular electrode. Morphological changes in individual cells were followed by means of time-lapse video recordings. In more than 60% of the experiments, steady-state potassium depolarization induced a pronounced increase of 20–120% in the area of the growth cone. This was frequently associated with neurite elongation. However, such changes could not be detected in the presence of Cd²⁺ concentrations which block the Ca²⁺ channels. Similar results were obtained in the presence of 2 μM of the Ca²⁺ ionophore A-23187 or when the cells were repetitively stimulated (0.2 Hz) in a medium containing 10^?6M TTX and 15 mM TEA. Local microapplication, directly onto single growth cones, of a depolarizing solution containing 5 mM Ca²⁺ also led to similar observations. Scanning electron microscopy indicated that the depolarized growth cone membranes were flattened and contained markedly more rounded protuberances relative to control cultures. Our results indirectly suggest that Ca²⁺ entry might be a trigger in the process of neurite elongation.     

Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain.

Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM-catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML-9, nor by the activators of MLCK, Ca2+ and calmodulin.     

Distribution of phosphorylated GAP-43 (neuromodulin) in growth cones directly reflects growth cone behavior

Phosphorylation of GAP-43 (neuromodulin) by protein kinase C (PKC) occurs at a single site, serine⁴¹. In vivo, phosphorylation is induced after initiation of axonogenesis and is confined to distal axons and growth cones. Within individual growth cones, phosphorylation is nonuniformly distributed. Here, we have used high-resolution video-enhanced microscopy of cultured dorsal root ganglia neurons together with immunocytochemistry with a monoclonal antibody that recognizes PKC-phosphorylated GAP-43 to correlate the distribution of phosphorylated GAP-43 with growth cone behavior. In “quiescent,” nontranslocating growth cones, phosphorylated GAP-43 was confined to the proximal neurite and the central organelle-rich region, and was low in organelle-poor lamellae. However, levels in lamellae were elevated when they became motile. Conversely, levels of phosphorylated GAP-43 were low in either lamellae that were actively retracting or in the central organelle-rich region and proximal neurite of growth cones that had totally collapsed. The results suggest a mechanism whereby phosphorylation of GAP-43 by PKC, potentially in response to extracellular signals, could direct the functional behavior of the growth cone. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 287–299, 1998     

Filopodial behavior is dependent on the phosphorylation state of neuronal growth cones

Several lines of evidence suggest that phosphorylation events play an important role in transducing neurite outgrowth signals. Here we tested if such phosphorylation events altered filopodial dynamics on neuronal growth cones and thereby might affect pathfinding decisions. The general protein kinase inhibitor K252a caused an increase in the overall length of filopodia, thereby increasing the action radius of a growth cone. Application of specific kinase inhibitors demonstrated that myosin light chain kinase, Ca/calmodulin-dependent kinase II, and protein kinase A were likely not involved in this filopodial response. Inhibition of protein kinase C (PKC) with calphostin C or cerebroside, however, induced filopodial elongation similar to that seen with K252a. Activation of PKC with the phorbol ester PMA produced the opposite effect, namely filopodial shortening. Consistent with this finding, the protein phosphatase activator C(2)-ceramide resulted in a significant increase in filopodial length, whereas application of the protein phosphatase inhibitor okadaic acid caused the opposite effect, filopodial shortening. Lastly, the tyrosine kinase inhibitor genistein also caused filopodial elongation, and this effect could be negated by the tyrosine phosphatase inhibitor sodium ortho-vanadate. Using the calcium indicator fura-2, we further showed that these drugs did not cause a measurable change in the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones. Taken together, these results suggest that the action radius of a growth cone and its resulting pathfinding abilities could be rapidly altered by contact with extracellular cues, leading to changes in the activity of protein kinases and phosphatases.     

Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.     

Astrocyte process growth induction by actin breakdown

cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F-actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.     

The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones

Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin. We report that these collapsing factors had different effects. ML-7 induced F-actin reorganization consistent with debundling whereas serotonin and TPA predominately depolymerized and possibly debundled F-actin. Additionally, these collapsing factors induced the formation of a dense actin-ring around the central domain, the thicker proximal area of growth cones [Zhou and Cohan, 2001: J. Cell Biol. 153:1071-1083]. The formation of the actin-ring occurred subsequent to the loss of actin bundles. The ML-7-induced actin-ring was found to inhibit microtubule extension into the P-domain. Thus, ML-7, serotonin, and TPA induce growth cone collapse associated with a decline in radially aligned F-actin bundles through at least two mechanisms involving debundling of actin filaments and/or actin depolymerization.     

Evidence that Protein Kinase C Activities Involved in Regulating Neurite Growth Are Localized to Distal Neurites

Abstract: Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by >50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca²⁺-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 µ M PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca²⁺-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC₅₀ value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.     

Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons

This study focuses on the effects of K⁺ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K⁺ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K⁺ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K⁺ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K⁺ solution. Identified neurons differed in the magnitude of their response to K⁺ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K⁺, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K⁺ application evoked a brief period (5–10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K⁺ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca²⁺ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca²⁺ change during depolarization, reflecting their different sensitivities to depolarization.     

Evidence that Prostaglandin E2 Can Block Calcium-Activated 86Rb Efflux from Rat Brain Synaptosomes via a Protein Kinase C-Dependent Mechanism

Abstract: The effects of prostaglandin E₂ (PGE₂) on ⁸⁶Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K⁺) channel modulation. A selective dose-dependent inhibition of the calcium-activated charybdotoxin-sensitive component of efflux was found upon application of PGE₂. No significant effect was seen on basal and voltage-dependent components over the concentration range of 10–⁸ to 10–⁵M. The protein kinase C (PKC) inhibitors H-7 (10 μM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 40-phorbol 12, 13-dibutyrate, which has been reported to down-regulate PKC, abolished the PGE₂-in- duced inhibition, whereas HA1004 (10 μM) and Rp-3′,5’cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4β-Phorbol 12, 13-dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca²⁺-dependent component of ⁸⁶Rb efflux but also had no effect on the basal and voltage-dependent components. These data suggest that PGE₂ can inhibit rat brain nerve ending calcium-activated ⁸⁶Rb efflux, and this inhibition may involve PKC activation.     

Alpha(1)-AR-induced positive inotropic response in heart is dependent on myosin light chain phosphorylation

The possible involvement of different kinases in the alpha(1)-adrenoreceptor (AR)-mediated positive inotropic effect (PIE) was investigated in rat papillary muscle and compared with beta-AR-, endothelin receptor- and phorbol ester-induced changes in contractility. The alpha(1)-AR-induced PIE was not reduced by the inhibitors of protein kinase C (PKC), MAPK (ERK and p38), phosphatidyl inositol 3-kinase, or calmodulin kinase II. However, PKC inhibition attenuated the effect of phorbol 12-myristate 13-acetate (PMA) on contractility. alpha(1)-AR-induced PIE was reduced by approximately 90% during inhibition of myosin light chain kinase (MLCK) by 1-(5-chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine (ML-9). Endothelin-induced PIE was also reduced by ML-9, but ML-9 had no effect on beta-AR-induced PIE. The Rho kinase inhibitor Y-27632 also reduced the alpha(1)-AR-induced PIE. The alpha(1)-AR-induced PIE in muscle strips from explanted failing human hearts was also sensitive to MLCK inhibition. alpha(1)-AR induced a modest increase in (32)P incorporation into myosin light chain in isolated rat cardiomyocytes. This effect was eliminated by ML-9. The PIE of alpha(1)-AR stimulation seems to be dependent on MLCK phosphorylation.     

GAP-43 phosphorylation is dynamically regulated in individual growth cones

In vivo, kinase C phosphorylation of the growth-associated protein GAP-43 is spatially and temproally associated with the proximity of growing axons to their targets. Here we have used dissociated dorsal root ganglia (DRG)s and an antibody specific for the phosphorylated form of GAP-43 to demonstrate that neurite regeneration in culture also begins in the absence of detectable levels of phosphorylated GAP-43. Since the β isoform of kinase C was found to be enriched in growth cones before stably phosphorylated GAP-43 was detected, it may normally be inactive during initial neurite outgrowth; however, premature phosphorylation of GAP-43 could be stimulated in newly dissociated DRGs by plating them on cultures in which phosphorylation had already been initiated; media conditioned by such cultures caused no response suggesting an effect of either cell-cell or cell-substrate contact. Increased GAP-43 phosphorylation correlated with a reduced extent of neurite outgrowth but not with the rate at which individual growth cones translocated so that motile growth cones contained very low levels of phosphorylated GAP-43, whereas stationary growth cones showed much more immunoreactivity. Downregulation of kinase C by phorbol ester prevented increased GAP-43 phosphorylation and led to growth cone collapse. Finally, phosphorylated GAP-43 was found to be differently distributed within growth cones. Increased immunoreactivity was frequently observed in the neck of the growth cone and was heterogeneously distributed in lamellae and filopodia. These results, which demonstrate the dynamic regulation of GAP-43 phosphorylation in individual growth cones, are discussed with reference to the association between changes in growth cone shape and the ability to translocate and change direction. © 1992 John Wiley & Sons, Inc.     

Myosin light chain phosphorylation and growth cone motility

According to the treadmill hypothesis, the rate of growth cone advance depends upon the difference between the rates of protrusion (powered by actin polymerization at the leading edge) and retrograde F-actin flow, powered by activated myosin. Myosin II, a strong candidate for powering the retrograde flow, is activated by myosin light chain (MLC) phosphorylation. Earlier results showing that pharmacological inhibition of myosin light chain kinase (MLCK) causes growth cone collapse with loss of F-actin-based structures are seemingly inconsistent with the treadmill hypothesis, which predicts faster growth cone advance. These experiments re-examine this issue using an inhibitory pseudosubstrate peptide taken from the MLCK sequence and coupled to the fatty acid stearate to allow it to cross the membrane. At 5-25 microM, the peptide completely collapsed growth cones from goldfish retina with a progressive loss of lamellipodia and then filopodia, as seen with pharmacological inhibitors, but fully reversible. Lower concentrations (2.5 microM) both simplified the growth cone (fewer filopodia) and caused faster advance, doubling growth rates for many axons (51-102 microm/h; p <.025). Rhodamine-phalloidin staining showed reduced F-actin content in the faster growing growth cones, and marked reductions in collapsed ones. At higher concentrations, there was a transient advance of individual filopodia before collapse (also seen with the general myosin inhibitor, butanedione monoxime, which did not accelerate growth). The rho/rho kinase pathway modulates MLC dephosphorylation by myosin-bound protein phosphatase 1 (MPP1), and manipulations of MPP1 also altered motility. Lysophosphatidic acid (10 microM), which causes inhibition of MPP1 to accumulate activated myosin II, caused a contracted collapse (vs. that due to loss of F-actin) but was ineffective after treatment with low doses of peptide, demonstrating that the peptide acts via MLC phosphorylation. Inhibiting rho kinase with Y27632 (100 microM) to disinhibit the phosphatase increased the growth rate like the MLCK peptide, as expected. These results suggest that: varying the level of MLCK activity inversely affects the rate of growth cone advance, consistent with the treadmill hypothesis and myosin II powering of retrograde F-actin flow; MLCK activity in growth cones, as in fibroblasts, contributes strongly to controlling the amount of F-actin; and the phosphatase is already highly active in these cultures, because rho kinase inhibition produces much smaller effects on growth than does MLCK inhibition.     

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     

Rac1-dependent actin filament organization in growth cones is necessary for β1-integrin–mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine–phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine–phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D -lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine–phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for β1 integrin–mediated growth cone advance, but not for growth on poly-D lysine. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 524–540, 1998