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ABSTRACT: The roles of P21-activated kinase (PAK) in the regulation of axon outgrowth downstream of extracellular matrix (ECM) proteins are poorly understood. Here we show that PAKs 1-3 and PIX are expressed in the developing spinal cord and differentially target to point contacts and filopodial tips within motile growth cones. Using a specific interfering peptide called PAK18, we find that axon outgrowth is robustly stimulated on laminin by partial inhibition of PAK-PIX interactions and PAK function, while complete inhibition of PAK function stalls axon outgrowth. Further, modest inhibition of PAK-PIX stimulates the assembly and turnover of growth cone point contacts, while strong inhibition over-stabilizes adhesions. Point mutations within PAK confirm the importance of PIX binding. Together our data suggest that regulation of PAK-PIX interactions in growth cones control neurite outgrowth by influencing the activity of several important mediators of actin filament polymerization and retrograde flow, as well as integrin-dependent adhesion to laminin.
Journal of Cell Science 01/2013; · 6.11 Impact Factor
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ABSTRACT: Intracellular Ca(2+) signals control the development and regeneration of spinal axons downstream of chemical guidance cues, but little is known about the roles of mechanical cues in axon guidance. Here we show that transient receptor potential canonical 1 (TRPC1) subunits assemble mechanosensitive (MS) channels on Xenopus neuronal growth cones that regulate the extension and direction of axon outgrowth on rigid, but not compliant, substrata. Reducing expression of TRPC1 by antisense morpholinos inhibits the effects of MS channel blockers on axon outgrowth and local Ca(2+) transients. Ca(2+) influx through MS TRPC1 activates the protease calpain, which cleaves the integrin adaptor protein talin to reduce Src-dependent axon outgrowth, likely through altered adhesion turnover. We found that talin accumulates at the tips of dynamic filopodia, which is lost upon cleavage of talin by active calpain. This pathway may also be important in axon guidance decisions since asymmetric inhibition of MS TRPC1 is sufficient to induce growth cone turning. Together our results suggest that Ca(2+) influx through MS TRPC1 on filopodia activates calpain to control growth cone turning during development.
Journal of Neuroscience 01/2013; 33(1):273-85. · 7.11 Impact Factor
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ABSTRACT: There is biochemical, imaging and functional evidence that Rho GTPase signaling is a crucial regulator of actin-based structures such as lamellipodia and filopodia. However, although Rho GTPases are believed to serve similar functions in growth cones, the spatiotemporal dynamics of Rho GTPase signaling has not been examined in living growth cones in response to known axon guidance cues. Here we provide the first measurements of Cdc42 activity in living growth cones acutely stimulated with both growth-promoting and growth-inhibiting axon-guidance cues. Interestingly, we find that both permissive and repulsive factors can work by modulating Cdc42 activity, but in opposite directions. We find that the growth-promoting factors laminin and BDNF activate Cdc42, whereas the inhibitor Slit2 reduces Cdc42 activity in growth cones. Remarkably, we find that regulation of focal adhesion kinase (FAK) activity is a common upstream modulator of Cdc42 by BDNF, laminin and Slit. These findings suggest that rapid modulation of Cdc42 signaling through FAK by receptor activation underlies changes in growth cone motility in response to permissive and repulsive guidance cues.
Journal of Cell Science 03/2012; 125(Pt 12):2918-29. · 6.11 Impact Factor
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ABSTRACT: The ability of extending axons to navigate using combinations of extracellular cues is essential for proper neural network formation. One intracellular signaling molecule that integrates convergent signals from both extracellular matrix (ECM) proteins and growth factors is focal adhesion kinase (FAK). Analysis of FAK function shows that it influences a variety of cellular activities, including cell motility, proliferation, and differentiation. Recent work in developing neurons has shown that FAK and Src function downstream of both attractive and repulsive growth factors, but little is known about the effectors or cellular mechanisms that FAK controls in growth cones on ECM proteins. We report that FAK functions downstream of brain-derived neurotrophic factor (BDNF) and laminin in the modulation of point contact dynamics, phosphotyrosine signaling at filopodial tips, and lamellipodial protrusion. BDNF stimulation accelerates paxillin-containing point contact turnover and formation. Knockdown of FAK function either with a FAK antisense morpholino or by expression of FRNK, a dominant-negative FAK isoform, blocks all aspects of the response to BDNF, including the acceleration of point contact dynamics. On the other hand, expression of specific FAK point mutants can selectively disrupt distinct aspects of the response to BDNF. We also show that growth cone turning depends on both signaling cascades tested here. Finally, we provide the first evidence that growth cone point contacts are asymmetrically regulated during turning to an attractive guidance cue.
Journal of Neuroscience 09/2011; 31(38):13585-95. · 7.11 Impact Factor
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Timothy M Gomez
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ABSTRACT: Axon outgrowth and pathfinding occurs through a complex series of interacting biochemical signaling pathways that regulate the motility of neuronal growth cones. Over the past 30 years, Paul Letourneau and his students have explored the molecular basis of growth cone motility and have contributed immensely to this field. In celebration of his 65th birthday, this essay is written in gratitude for Paul's many contributions and training.
Developmental Neurobiology 09/2011; 71(9):780-4. · 3.55 Impact Factor
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ABSTRACT: Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, because classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.
Developmental Neurobiology 06/2011; 71(11):901-23. · 3.55 Impact Factor
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ABSTRACT: Xenopus laevis provides a robust model system to study cellular signaling and downstream processes during development both in vitro and in vivo. Intracellular signals must function within highly restricted spatial and temporal domains to activate specific downstream targets and cellular processes. Combining the versatility of developing Xenopus neurons with advances in fluorescent protein biosensors and imaging technologies has allowed many dynamic cellular processes to be visualized. This review will focus on the techniques we use to visualize and measure cell signaling, motility and adhesion by quantitative fluorescence microscopy in vitro and in vivo.
Developmental Neurobiology 04/2011; 72(4):585-99. · 3.55 Impact Factor
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ABSTRACT: We have investigated the role of Vav2, a reported Rac1/Cdc42 GEF, on the development of Xenopus spinal neurons in vitro and in vivo. Both gain and loss of Vav2 function inhibited the rate neurite extension on laminin (LN), while only GFP-Vav2 over-expression enhanced process formation and branching. Vav2 over-expression protected neurons from RhoA-mediated growth cone collapse, similar to constitutively active Rac1, suggesting that Vav2 activates Rac1 in spinal neurons. Enhanced branching on LN required both Vav2 GEF activity and N-terminal tyrosine residues, but protection from RhoA-mediated collapse only required GEF activity. Interestingly, wild-type spinal neurons exhibited increased branching on the cell adhesion molecule L1, which required Vav2 GEF function, but not N-terminal tyrosine residues. Finally, we find that Vav2 differentially affects the Rohon-Beard peripheral and central process extension but promotes neurite branching of commissural interneurons near the ventral midline. Together, we suggest that balanced Vav2 activity is necessary for optimal neurite outgrowth and promotes branching by targeting GEF activity to branch points.
Molecular and Cellular Neuroscience 03/2010; 44(2):118-28. · 3.66 Impact Factor
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ABSTRACT: Adhesion controls growth cone motility, yet the effects of axon guidance cues on adhesion site dynamics are poorly understood. Here we show that ephrin-A1 reduces retinal ganglion cell (RGC) axon outgrowth by stabilizing existing adhesions and inhibiting new adhesion assembly. Ephrin-A1 activates focal adhesion kinase (FAK) in an integrin- and Src-dependent manner and the effects of ephrin-A1 on growth cone motility require FAK activation. We also find that FAK is expressed in a high temporal to low nasal gradient in RGCs, similar to EphA receptors, and that balanced FAK activation is necessary for optimal axon outgrowth. Last, we find that FAK is required for proper topographic positioning of retinal axons along the anterior-posterior axis of the optic tectum in both Xenopus and zebrafish, a guidance decision mediated in part by A-type ephrins. Together, our data suggest that ephrin-A1 controls growth cone advance by modulating adhesive point contacts through FAK activation and that graded FAK signaling is an important component of ephrin-A-mediated retinotopic mapping.
Journal of Neuroscience 11/2009; 29(44):13981-91. · 7.11 Impact Factor
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ABSTRACT: Extracellular matrix (ECM) components regulate neurite outgrowth in tissue culture and in vivo. Live imaging of phosphotyrosine (PY) signals revealed that Xenopus laevis growth cones extending on permissive ECM substrata assemble adhesive point contacts containing enriched levels of tyrosine-phosphorylated proteins. Whereas focal adhesion kinase (FAK) signaling is dispensable for the assembly of focal adhesions in non-neuronal cells, FAK activity is required for the formation of growth cone point contacts. FAK-dependent point contacts promote rapid neurite outgrowth by stabilizing lamellipodial protrusions on permissive ECM substrata. Moreover, local FAK activity is required for ECM-dependent growth cone turning in vitro, suggesting that FAK may control axon pathfinding in vivo. Consistent with this possibility, proper growth and guidance of Rohon-Beard sensory neurons and spinal commissural interneurons requires FAK activity. These findings identify FAK as a key regulator of axon growth and guidance downstream of growth cone-ECM interactions.
Nature Neuroscience 11/2006; 9(10):1274-83. · 15.53 Impact Factor
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Saumen Pal,
Jing Wu,
Justin K Murray,
Samuel H Gellman,
Michele A Wozniak,
Patricia J Keely,
Meghan E Boyer, Timothy M Gomez,
Sean M Hasso,
John F Fallon,
Emery H Bresnick
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ABSTRACT: Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However, the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a "neuropeptide," neurokinin-B (NK-B), reversibly inhibits endothelial cell vascular network assembly and opposes angiogenesis in the chicken chorioallantoic membrane. Disruption of endogenous NK-B signaling promoted angiogenesis. Mechanistic analyses defined a multicomponent pathway in which NK-B signaling converges upon cellular processes essential for angiogenesis. NK-B-mediated ablation of Ca2+ oscillations and elevation of 3'-5' [corrected] cyclic adenosine monophosphate (cAMP) reduced cellular proliferation, migration, and vascular endothelial growth factor receptor expression and induced the antiangiogenic protein calreticulin. Whereas NK-B initiated certain responses, other activities required additional stimuli that increase cAMP. Although NK-B is a neurotransmitter/ neuromodulator and NK-B overexpression characterizes the pregnancy-associated disorder preeclampsia, NK-B had not been linked to vascular remodeling. These results establish a conserved mechanism in which NK-B instigates multiple activities that collectively oppose vascular remodeling.
The Journal of Cell Biology 10/2006; 174(7):1047-58. · 10.26 Impact Factor
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ABSTRACT: Ca2+ signals are known to be important regulators of neurite outgrowth and steering. Here we show that inhibiting Ca2+ influx through stretch-activated channels using various compounds, including a highly specific peptide isolated from Grammostola spatulata spider venom (GsMTx4), strongly accelerates the rate of neurite extension on diverse substrata and within the intact spinal cord. Consistent with the presence of stretch-activated channels, we show that Ca2+ influx is triggered by hypotonic solutions, which can be partially blocked by GsMTx4. Finally, chelating local, but not global, Ca2+ signals prevents the acceleration that is normally produced by GsMTx4. Blocking Ca2+ influx through other channel types has little or opposite effects, but release from intracellular stores is required for maximal acceleration. Together, our data suggest that Ca2+ functions at distinct microdomains in growth cones, with influx through mechanosensitive channels acting to inhibit outgrowth in opposition to influx through other plasma membrane channels and release from stores.
Journal of Neuroscience 06/2006; 26(21):5656-64. · 7.11 Impact Factor
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ABSTRACT: Ca(2+) signals have profound and varied effects on growth cone motility and guidance. Modulation of Ca(2+) influx and release from stores by guidance cues shapes Ca(2+) signals, which determine the activation of downstream targets. Although the precise molecular mechanisms that underlie distinct Ca(2+)-mediated effects on growth cone behaviours remain unclear, recent studies have identified important players in both the regulation and targets of Ca(2+) signals in growth cones.
Nature reviews. Neuroscience 03/2006; 7(2):115-25. · 30.44 Impact Factor
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ABSTRACT: Growth cone advance depends on coordinated membrane protrusion and adhesion to the extracellular matrix. Although many studies have addressed the mechanisms responsible for membrane protrusion, the assembly of integrin-dependent adhesion sites known as point contacts remains poorly understood in growth cones. We show balanced Rac1 activity controls both leading edge protrusion and point contact dynamics during neurite outgrowth. Immunocytochemistry and live imaging of paxillin-green fluorescent protein (GFP) showed that inhibiting Rac1 blocked point contact formation, whereas Rac1 overactivation produced small, unstable point contacts. Both inhibition and overactivation of Rac1 reduced the persistence of lamellar protrusions and neurite outgrowth. Inhibition of ROCK (Rho kinase), a RhoA effector, perturbed protrusion and point contact dynamics similar to Rac1 overactivation. Moreover, the repulsive guidance cue Semaphorin 3A, which signals through Rac1, destabilizes point contacts. Together, our data suggest that coordinated Rho GTPase activities regulate neurite outgrowth through point contact formation and stabilization of membrane protrusion.
Journal of Neuroscience 03/2006; 26(5):1418-28. · 7.11 Impact Factor
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ABSTRACT: Commissural interneurons (CI) of the vertebrate spinal cord are guided ventrally toward the floor plate, but subsequently cross the midline and select a longitudinal fascicle at specific dorsal-ventral (D-V) positions. We examined at high resolution the detailed behaviors of individual pathfinding CI growth cones on the ipsilateral and contralateral sides of the spinal cord of living Xenopus embryos. We find that pre-crossing CI growth cones exhibit distinct pathfinding behaviors compared to post-crossing axons and that the behavioral switch occurs immediately upon crossing to the contralateral side. Groups of pioneer commissural axons typically extend simultaneously toward the ventral midline following discrete paths with separation between adjacent commissurals apparently maintained through contact inhibition. In contrast, shortly after crossing the midline, commissural axons turn longitudinally and begin to fasciculate with other crossed CIs. However, growth cones of crossed commissurals often select their final D-V longitudinal track through a series of rapid step-like dorsal adjustments that may be due to differential fasciculation with longitudinal axons. Together, our results suggest that guidance of commissural axons is controlled in part through interactions among CIs that switch rapidly from avoidance to fasciculation after midline crossing.
Developmental Biology 01/2006; 288(2):474-86. · 4.07 Impact Factor
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ABSTRACT: Extracellular cues guide axon outgrowth by activating intracellular signaling cascades that control the growth cone cytoskeleton. However, the spatial and temporal coordination of signaling intermediates remains essentially unknown. Live imaging of tyrosine phosphorylation in growth cones revealed dynamic phospho-tyrosine (PY) signals in filopodia that directly correlate with filopodial behavior. Local PY signals are generated at distal tips of filopodia during extension and are lost during retraction. Active Src family kinases localize to the tips of filopodia, and Src activity regulates both filopodial dynamics and local PY signaling. Positive guidance cues stimulate filopodial motility by locally increasing tyrosine phosphorylation in a cell division cycle 42 (Cdc42)-dependent manner. Locally reduced Src activity on one side of the growth cone generates an asymmetry in filopodial motility and PY signaling that promotes repulsive turning, suggesting that local changes in filopodial PY levels may underlie growth cone pathfinding decisions. p21-activated kinase (PAK), a Cdc42 effector whose activity is regulated by Src phosphorylation, also localizes to the tips of extending filopodia and controls filopodial motility. Coordinated activation of cytoskeletal effector proteins by GTPase binding and Src-mediated tyrosine phosphorylation may function to produce specific growth cone behaviors in response to guidance cues.
Journal of Neuroscience 09/2005; 25(33):7669-81. · 7.11 Impact Factor
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ABSTRACT: The Ena/VASP family of proteins consists of adaptor molecules that localize to subcellular sites of actin polymerization. The role of Ena/VASP proteins in the regulation of cell motility and axon outgrowth has been controversial. Recently, these proteins have been proposed to function as "anticapping" factors, which may have differential effects on filopodial versus lammelipodial actin-based protrusions. A study by Lebrand et al. in this issue of Neuron supports this model and identifies PKA as a key regulator of Ena/VASP function downstream of the chemoattractant Netrin.
Neuron 05/2004; 42(1):1-3. · 14.74 Impact Factor
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ABSTRACT: Spontaneous intracellular calcium ([Ca2+](i)) transients in growth cone filopodia reduce filopodial motility, slow neurite outgrowth, and promote turning when generated asymmetrically; however, the downstream effectors of these Ca2+ -dependent behaviors are unknown. We report that Ca2+ transients in filopodia activate the intracellular protease calpain, which slows neurite outgrowth and promotes repulsive growth cone turning upon local activation. Active calpain alters the balance between tyrosine kinase and phosphatase activities in filopodia, resulting in a net decrease in tyrosine phosphorylation, which mediates both filopodial stabilization and reduced lamellipodial protrusion. Our findings indicate that locally generated Ca2+ signals repel axon outgrowth through calpain-dependent regulation of phosphotyrosine signaling at integrin-mediated adhesion sites.
Neuron 06/2003; 38(4):597-609. · 14.74 Impact Factor
