Stimulation-induced changes in filopodial dynamics determine the action radius of growth cones in the snail Helisoma trivolvis

Filopodia on neuronal growth cones constantly extend and retract, thereby functioning as both sensory probes and structural devices during neuronal pathfinding. To better understand filopodial dynamics and their regulation by encounters with molecules in the environment, we investigated filopodial dynamics of identified B5 neurons from the buccal ganglion of the snail Helisoma trivolvis before and after treatment with nitric oxide (NO). We have previously demonstrated that treatment with several NO-donors caused a transient, cGMP-mediated elevation in [Ca(2+)](i), which was causally related to an increase in filopodial length and a reduction in the number of filopodia on growth cones. We demonstrate here that these effects were the result of distinct changes in filopodial dynamics. The NO-donor SIN-1 induced a general increase in filopodial motility. Filopodial elongation after treatment with SIN-1 resulted from a significant increase in the rate at which filopodia extended, as well as a significant increase in the time filopodia spent elongating. The reduction in filopodial number was caused by a significant decrease in the frequency with which new filopodia were inserted into the growth cone. With the exception of the back where filopodia appeared less motile, filopodial dynamics appeared to be mostly independent of the location on the growth cone. These results suggest that NO can regulate filopodial dynamics on migrating growth cones and might function as a messenger to adjust the action radius of a growth cone during pathfinding.     

Local calcium changes regulate the length of growth cone filopodia

Previous studies have demonstrated that the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones can act as an important regulator of growth cone behavior. Here we investigated whether there is a spatial and temporal correlation between [Ca(2+)](i) and one particular aspect of growth cone behavior, namely the regulation of growth cone filopodia. Calcium was released from the caged compound NP-EGTA (o-nitrophenyl EGTA tetrapotassium salt) to simulate a signaling event in the form of a transient increase in [Ca(2+)](i). In three different experimental paradigms, we released calcium either globally (within an entire growth cone), regionally (within a small area of the lamellipodium), or locally (within a single filopodium). We demonstrate that global photolysis of NP-EGTA in growth cones caused a transient increase in [Ca(2+)](i) throughout the growth cone and elicited subsequent filopodial elongation that was restricted to the stimulated growth cone. Pharmacological blockage of either calmodulin or the Ca(2+)-dependent phosphatase, calcineurin, inhibited the effect of uncaging calcium, suggesting that these enzymes are acting downstream of calcium. Regional uncaging of calcium in the lamellipodium caused a regional increase in [Ca(2+)](i), but induced filopodial elongation on the entire growth cone. Elevation of [Ca(2+)](i) locally within an individual filopodium resulted in the elongation of only the stimulated filopodium. These findings suggest that the effect of an elevation of [Ca(2+)](i) on filopodial behavior depends on the spatial distribution of the calcium signal. In particular, calcium signals within filopodia can cause filopodial length changes that are likely a first step towards directed filopodial steering events seen during pathfinding in vivo.     

Kinase requirement for retinal growth cone motility

Since cytoplasmic Ca²⁺ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantifed over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1–1 μM) and chelerythrin (up to 50 μM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 μM), trifluoperazine (100 μM), and CGS9343B (50 μM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 μM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 μM), produced no effects but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3–5 μM) and ML-9 (5–10 μM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 μM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts. 1994 John Wiley & Sons, Inc.     

Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors

In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]_i). Whole‐cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]_i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP‐induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 487–501, 2013     

Regulation of neuronal growth cone filopodia by nitric oxide

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.     

Nitric oxide release from a single cell affects filopodial motility on growth cones of neighboring neurons

Nitric oxide (NO), a gaseous messenger, has been reported to be involved in a variety of functions in the nervous system, ranging from neuronal pathfinding to learning and memory. We have shown previously that the application of NO via NO donors to growth cones of identified Helisoma buccal neurons B5 in vitro induces an increase in filopodial length, a decrease in filopodial number, and a slowing in neurite advance. It is unclear, however, whether NO released from a physiological source would affect growth cone dynamics. Here we used cell bodies of identified neurons known to express the NO synthesizing enzyme nitric oxide synthase (NOS) as a source of constitutive NO production and tested their effect on growth cones of other cells in a sender-receiver paradigm. We showed that B5 cell bodies induced a rapid increase in filopodial length in NO-responsive growth cones, and that this effect was blocked by the NOS inhibitor 7-NI, suggesting that the effect was mediated by NO. Inhibition of soluble guanylyl cyclase (sGC) with ODQ blocked filopodial elongation induced by B5 somata, confirming that NO acted via sGC. We also demonstrate that the effect of NO was reversible and that a cell releasing NO can affect growth cones over a distance of at least 100 microm. Our results suggest that NO released from a physiological source can affect the motility of nearby growth cones and thus should be considered a signaling molecule with the potential to affect the outcome of neuronal pathfinding in vivo.     

PLA2 and secondary metabolites of arachidonic acid control filopodial behavior in neuronal growth cones

The neuronal growth cone provides the sensory and motor structure that guides neuronal processes to their target. The ability of a growth cone to navigate correctly depends on its filopodia, which sample the environment by continually extending and retracting as the growth cone advances. Several second messengers systems that are activated upon contact with extracellular cues have been reported to affect growth cone morphology by changing the length and number of filopodia. Because recent studies have suggested that guidance cues can signal via G-protein coupled receptors to regulate phospholipases, we here investigated whether phospholipase A2 (PLA2) may control filopodial dynamics and could thereby affect neuronal pathfinding. Employing identified Helisoma neurons in vitro, we demonstrate that inhibition of PLA2 with 2 microM BPB caused a 40.3% increase in average filopodial length, as well as a 37.3% reduction in the number of filopodia on a growth cone. The effect of PLA2 inhibition on filopodial length was mimicked by the inhibition of G-proteins with 500 ng/ml pertussis toxin and was partially blocked by the simultaneous activation of PLA2 with 50 nM melittin. We provide evidence that PLA2 acts via production of arachidonic acid (AA), because (1) the effect of inhibition of PLA2 could be counteracted by supplying AA exogenously, and (2) the inhibition of cyclooxygenase, which metabolizes AA into prostaglandins, also increased filopodial length. We conclude that filopodial contact with extracellular signals that alter the activity of PLA2 can control growth cone morphology and may affect neuronal pathfinding by regulating the sensory radius of navigating growth cones.     

Eicosanoid activation of protein kinase C epsilon: involvement in growth cone repellent signaling

Exposure of growing neurons to thrombin or semaphorin 3A stimulates a receptor-mediated signaling cascade that results in collapse of their growth cones. This collapse response necessitates eicosanoid production, as we have shown earlier. The present report investigates whether and which protein kinase C (PKC) isoforms may be activated by such eicosanoids. To examine these questions, we isolated growth cones from fetal rat brain and tested whether thrombin or the eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), could activate endogenous growth cone PKC. We show that both thrombin and 12(S)-HETE stimulate the phosphorylation of the myristoylated alanine-rich protein kinase C substrate, an 87-kDa adhesion site protein. Furthermore, we show both with immunoprecipitated and with recombinant PKC that 12(S)-HETE activation is selective for the epsilon isoform and does not require accessory proteins. Last, we demonstrate that PKC activation is necessary for thrombin-induced growth cone collapse. These data indicate that eicosanoid-mediated repellent effects result from the direct and selective activation of PKCepsilon and suggest the involvement of myristoylated alanine-rich protein kinase C substrate phosphorylation in growth cone collapse.     

Signaling Pathway Downstream of GABAA Receptor in the Growth Cone

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABA_A receptor appear even in early stages of CNS development. We investigated the GABA_A receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABA_A binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl⁻ flux (not influx but efflux) and increased the intracellular Ca²⁺ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABA_A receptor localized in the growth cone membrane → Cl⁻ efflux induction through the GABA_A-associated Cl⁻ channel → Ca²⁺ influx through an L-type voltage-sensitive Ca²⁺ channel → Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.     

Autonomous regulation of growth cone filopodia

The fan-shaped array of filopodia is the first site of contact of a neuronal growth cone with molecules encountered during neuronal pathfinding. Filopodia are highly dynamic structures, and the “action radius” of a growth cone is strongly determined by the length and number of its filopodia. Since interactions of filopodia with instructive cues in the vicinity of the growth cone can have effects on growth cone morphology within minutes, it has to be assumed that a large part of the signaling underlying such morphological changes resides locally within the growth cone proper. In this study, we tested the hypothesis that two important growth cone parameters namely, the length and number of its filopodiaare regulated autonomously in the growth cone. We previously demonstrated in identified neurons from the snail Helisoma trivolvis that filopodial length and number are regulated by intracellular calcium. Here, we investigated filopodial dynamics and their regulation by the second-messenger calcium in growth cones which were physically isolated from their parent neuron by neurite transection. Our results show that isolated growth cones have longer but fewer filopodia than growth cones attached to their parent cell. These isolated growth cones, however, are fully capable of undergoing calcium-induced cytoskeletal changes, suggesting that the machinery necessary to perform changes in filopodial length and number is fully intrinsic to the growth cone proper. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 179–192, 1998     

Negative regulation of phosphatidylinositol 3-kinase and Akt signalling pathway by PKC

Although substantial studies have begun to explore the regulation of phosphatidylinositol 3-kinase/Akt cascade by different signalling pathways, whether protein kinase C (PKC) activity plays a crucial role remains as yet unclear. In this study, we found that in A549 and HEK293 cells non-selective PKC inhibitors Ro 31-8220 and bisindolylmaleimide VIII, and PKCbeta inhibitor LY 379196, caused Akt/PKB phosphorylation at Ser 473 and increased the upstream activator, integrin-linked kinase (ILK) activity. The increased Akt phosphorylation was blocked by phosphatidylinositol 3-kinase inhibitor wortmannin and the newly identified PIP(3)-dependent kinases (PDK) inhibitor SB 203580. In contrast to the Akt stimulation caused by PKC inhibitors, PMA attenuated Akt/PKB phosphorylation. We also found that this stimulating effect on Akt phosphorylation by PKC inhibitors was not the result of phosphatase inhibition, since treatment with PP2A, PP2B and tyrosine phosphatase inhibitors (okadaic acid, FK506 and sodium orthovanadate, respectively) had no effect. We conclude that phosphatidylinositol 3-kinase/Akt signalling pathway is regulated by PKC in a negative manner.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

The actin-binding protein UNC-115/abLIM controls formation of lamellipodia and filopodia and neuronal morphogenesis in Caenorhabditis elegans

The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

L1 and N-CAM Antibodies Trigger Protein Phosphatase Activity in Growth Cone-Enriched Membranes

Abstract: Triggering of the cell adhesion molecules L1 or N-CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N-CAM or specific antibodies decreases the steady-state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N-CAM in the growth cone-enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N-CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule-activated phosphatase. The most prominent substrates were a membrane-associated 200-kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N-CAM triggering. The antibody-induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N-CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N-CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N-CAM intracellular signaling pathways in growth cones.     

Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.     

Phosphorylation of the Casein Kinase II Domain of B-50 (GAP-43) in Rat Cortical Growth Cones

Abstract: Growth-associated phosphoprotein B-50 is a neural protein kinase C (PKC) substrate enriched in nerve growth cones that has been implicated in growth cone plasticity. Here we investigated whether B-50 is a physiological substrate for casein kinase II (CKII) in purified rat cortical growth cone preparations. Using site-specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spectrometry, and phosphoamino acid analysis, we demonstrate that endogenous growth cone B-50 is phosphorylated at multiple sites, on both serine and threonine residues. Consistent with previous reports, stimulation of PKC activity increased the phosphorylation of only those proteolytic fragments containing Ser⁴¹. Under basal conditions, however, phosphorylation was predominantly associated with fragments not containing Ser⁴¹. Mass spectrometry of tryptic digests of B-50, which had been immunoprecipitated from untreated growth cones, revealed that in situ phosphorylation occurs within peptides B-50_181–198 and B-50_82–98. These peptides contain the major and minor in vitro CKII phosphosites, respectively. In addition, cyanogen bromide digestion of immunoprecipitated chick B-50 generated a 4-kDa C-terminal B-50 phosphopeptide, confirming that phosphorylation of the CKII domain occurs across evolutionary diverse species. We conclude that B-50 in growth cones is not only a substrate for PKC, but also for CKII.     

RACK-1 acts with Rac GTPase signaling and UNC-115/abLIM in Caenorhabditis elegans axon pathfinding and cell migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.     

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.