RESUMO
Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Potássio Shaw/metabolismo , Ataxias Espinocerebelares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Canais de Potássio Shaw/química , Canais de Potássio Shaw/genética , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.
Assuntos
Actinas/fisiologia , Aplysia/fisiologia , Cones de Crescimento/fisiologia , Microtúbulos/fisiologia , Pseudópodes/fisiologia , Animais , Aplysia/ultraestrutura , Células Cultivadas , Citocalasina B/farmacologia , Miosina Tipo II/fisiologiaRESUMO
Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.
Assuntos
Actinas/fisiologia , Miosina Tipo II/fisiologia , Neurônios/fisiologia , Animais , Aplysia , Axônios/fisiologia , Células Cultivadas , Corantes Fluorescentes , Cones de Crescimento/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis , Imuno-Histoquímica , Microscopia de Fluorescência , Neurônios/ultraestrutura , Pseudópodes/fisiologiaRESUMO
A growth cone is a motile structure at the tips of axons that is driven by the actin network and guides axon extension. Low actin adhesion to the substrate creates a stationary actin treadmill that allows leading-edge protrusion when adhesion increases in response to guidance cues. We use experimental measurements in the Aplysia bag growth cone to develop and constrain a simple mechanical model of the actin treadmill. We show that actin retrograde flow is primarily generated by myosin contractile forces, but when myosin is inhibited, leading-edge membrane tension increases and drives the flow. By comparing predictions of the model with previous experimental measurements, we demonstrate that lamellipodial and filopodial filament breaking contribute equally to the resistance to the flow. The fully constrained model clarifies the role of actin turnover in the mechanical balance driving the actin treadmill and reproduces the recent experimental observation that inhibition of actin depolymerization causes retrograde flow to slow exponentially with time. We estimate forces in the actin treadmill, and we demonstrate that measured G-actin distributions are consistent with the existence of a forward-directed fluid flow that transports G-actin to the leading edge.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Cones de Crescimento/metabolismo , Fenômenos Mecânicos , Modelos Biológicos , Miosinas/metabolismo , Actinas/química , Fenômenos Biomecânicos , Cinética , Multimerização Proteica , Estrutura Quaternária de Proteína , Pseudópodes/metabolismoRESUMO
Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.
Assuntos
Citoesqueleto de Actina/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Eucarióticas/metabolismo , Microtúbulos/metabolismo , Animais , Divisão Celular/fisiologia , Proteínas Contráteis/metabolismo , Células Eucarióticas/ultraestrutura , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Humanos , Cicatrização/fisiologiaRESUMO
We describe open-loop and closed-loop multiplexed force measurements using holographic optical tweezers. We quantify the performance of our novel video-based control system in a driven suspension of colloidal particles. We demonstrate our system's abilities with the measurement of the mechanical coupling between Aplysia bag cell growth cones and beads functionalized with the neuronal cell adhesion molecule, apCAM. We show that cells form linkages which couple beads to the underlying cytoskeleton. These linkages are intermittent, stochastic and heterogeneous across beads distributed near the leading edge of a single growth cone.
Assuntos
Aplysia/citologia , Aplysia/fisiologia , Cones de Crescimento/fisiologia , Cones de Crescimento/ultraestrutura , Holografia/instrumentação , Micromanipulação/instrumentação , Pinças Ópticas , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estresse MecânicoRESUMO
We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin-microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at approximately 1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.
Assuntos
Actinas/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Pseudópodes/metabolismo , Animais , Aplysia/citologia , Cones de Crescimento/ultraestrutura , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia de Fluorescência , Microtúbulos/ultraestrutura , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de TempoRESUMO
Serotonin (5-HT) is known to increase the rate of growth cone advance via cofilin-dependent increases in retrograde actin network flow and nonmuscle myosin II activity. We report that myosin II activity is regulated by PKC during 5-HT responses and that PKC activity is necessary for increases in traction force normally associated with these growth responses. 5-HT simultaneously induces cofilin-dependent decreases in actin network density and PKC-dependent increases in point contact density. These reciprocal effects facilitate increases in traction force production in domains exhibiting decreased actin network density. Interestingly, when PKC activity was up-regulated, 5-HT treatments resulted in myosin II hyperactivation accompanied by catastrophic cofilin-dependent decreases in actin filament density, sudden decreases in traction force, and neurite retraction. These results reveal a synergistic relationship between cofilin and myosin II that is spatiotemporally regulated in the growth cone via mechanocatalytic effects to modulate neurite growth.
Assuntos
Aplysia/crescimento & desenvolvimento , Axônios/metabolismo , Cones de Crescimento , Miosina Tipo II/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animais , Aplysia/genética , Catálise , Cofilina 1/genética , Fenômenos Mecânicos , Proteínas dos Microfilamentos/genética , Neuritos/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Fosforilação/genética , Proteína Quinase C/genética , Serotonina/genética , Serotonina/metabolismoRESUMO
Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. We have used multimode fluorescent speckle microscopy (FSM) to directly address this problem. We report that actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, LPA mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration.
Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Cones de Crescimento/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Aplysia , Células CultivadasRESUMO
The membranes of all eukaryotic motile (9 + 2) and immotile primary (9 + 0) cilia harbor channels and receptors involved in sensory transduction (reviewed by). These membrane proteins are transported from the cytoplasm onto the ciliary membrane by vesicles targeted for exocytosis at a point adjacent to the ciliary basal body. Here, we use time-lapse fluorescence microscopy to demonstrate that select GFP-tagged sensory receptors undergo rapid vectorial transport along the entire length of the cilia of Caenorhabditis elegans sensory neurons. Transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 move in ciliary membranes at rates comparable to the intraflagellar transport (IFT) machinery located between the membrane and the underlying axonemal microtubules. OSM-9 motility is disrupted in certain IFT mutant backgrounds. Surprisingly, motility of transient receptor potential polycystin (TRPP) channel PKD-2 (polycystic kidney disease-2), a mechano-receptor, was not detected. Our study demonstrates that IFT, previously shown to be necessary for transport of axonemal components, is also involved in the motility of TRPV membrane protein movement along cilia of C. elegans sensory cells.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Membrana Celular/metabolismo , Cílios/metabolismo , Canais Iônicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Transporte Biológico/fisiologia , Proteínas de Fluorescência Verde , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Neurônios Aferentes/fisiologia , Canais de Cátion TRPP , Canais de Cátion TRPVRESUMO
Dynamic microtubules explore the peripheral (P) growth cone domain using F actin bundles as polymerization guides. Microtubule dynamics are necessary for growth cone guidance; however, mechanisms of microtubule reorganization during growth cone turning are not well understood. Here, we address these issues by analyzing growth cone steering events in vitro, evoked by beads derivatized with the Ig superfamily cell adhesion protein apCAM. Pharmacological inhibition of microtubule assembly with low doses of taxol or vinblastine resulted in rapid clearance of microtubules from the P domain with little effect on central (C) axonal microtubules or actin-based motility. Early during target interactions, we detected F actin assembly and activated Src, but few microtubules, at apCAM bead binding sites. The majority of microtubules extended toward bead targets after F actin flow attenuation occurred. Microtubule extension during growth cone steering responses was strongly suppressed by dampening microtubule dynamics with low doses of taxol or vinblastine. These treatments also inhibited growth cone turning responses, as well as focal actin assembly and accumulation of active Src at bead binding sites. These results suggest that dynamic microtubules carry signals involved in regulating Src-dependent apCAM adhesion complexes involved in growth cone steering.
Assuntos
Cones de Crescimento/fisiologia , Microtúbulos/fisiologia , Neurônios/fisiologia , Quinases da Família src/metabolismo , Actinas/metabolismo , Animais , Aplysia , Western Blotting , Moléculas de Adesão Celular Neuronais/metabolismo , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Microesferas , Neurônios/citologia , Paclitaxel , Proteínas Recombinantes de Fusão , Vimblastina , Quinases da Família src/fisiologiaRESUMO
Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion molecule (apCAM) leads to actin filament assembly near nascent adhesion sites. Such actin assembly can generate significant localized forces that have not been characterized in the larger context of axon growth and guidance. We used apCAM-coated bead substrates applied to the surface of neuronal growth cones to characterize the development of forces evoked by varying stiffness of mechanical restraint. Unrestrained bead propulsion matched or exceeded rates of retrograde network flow and was dependent on Arp2/3 complex activity. Analysis of growth cone forces applied to beads at low stiffness of restraint revealed switching between two states: frictional coupling to retrograde flow and Arp2/3-dependent propulsion. Stiff mechanical restraint led to formation of an extensive actin cup matching the geometric profile of the bead target and forward growth cone translocation; pharmacological inhibition of the Arp2/3 complex or Rac attenuated F-actin assembly near bead binding sites, decreased the efficacy of growth responses, and blocked accumulation of signaling molecules associated with nascent adhesions. These studies introduce a new model for regulation of traction force in which local actin assembly forces buffer nascent adhesion sites from the mechanical effects of retrograde flow.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Aplysia/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Cones de Crescimento/metabolismo , Neurônios/metabolismo , Tração , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Growth cones of elongating neurites exert force against the external environment, but little is known about the role of force in outgrowth or its relationship to the mechanical organization of neurons. We used traction force microscopy to examine patterns of force in growth cones of regenerating Aplysia bag cell neurons. We find that traction is highest in the peripheral actin-rich domain and internal stress reaches a plateau near the transition between peripheral and central microtubule-rich domains. Integrating stress over the area of the growth cone reveals that total scalar force increases with area but net tension on the neurite does not. Tensions fall within a limited range while a substantial fraction of the total force can be balanced locally within the growth cone. Although traction continuously redistributes during extension and retraction of the peripheral domain, tension is stable over time, suggesting that tension is a tightly regulated property of the neurite independent of growth cone dynamics. We observe that redistribution of traction in the peripheral domain can reorient the end of the neurite shaft. This suggests a role for off-axis force in growth cone turning and neuronal guidance.
Assuntos
Aplysia/fisiologia , Neuritos/fisiologia , Actinas/metabolismo , Animais , Aplysia/metabolismo , Cones de Crescimento/fisiologia , Hemolinfa/metabolismo , Hemolinfa/fisiologia , Microtúbulos/fisiologia , Neuritos/metabolismo , TraçãoRESUMO
We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.
Assuntos
Aplysia/fisiologia , Hemolinfa/metabolismo , Laminina/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Cones de Crescimento/fisiologia , Hemocianinas/metabolismo , Neuritos/fisiologia , Neurônios/citologia , Regeneração , Especificidade por SubstratoRESUMO
Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase-potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.
Assuntos
Actinas/metabolismo , Aplysia/metabolismo , Neurônios/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Proteína Quinase C/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Aplysia/citologia , Aplysia/fisiologia , Movimento Celular/genética , Cones de Crescimento/metabolismo , Contração Muscular/genética , Contração Muscular/fisiologia , Neurogênese/genética , Fosforilação , Transdução de SinaisRESUMO
Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.
Assuntos
Citoesqueleto de Actina/metabolismo , Aplysia/citologia , Moléculas de Adesão Celular/metabolismo , Animais , Aplysia/metabolismo , Adesão Celular , Células CultivadasRESUMO
Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca(2+); however, mechanistic roles for Ca(2+) in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) â inositol trisphosphate â Ca(2+) â calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca(2+) release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT-dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.
Assuntos
Actinas/metabolismo , Calcineurina/metabolismo , Cofilina 1/metabolismo , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Serotonina/farmacologia , Animais , Aplysia , Azepinas/farmacologia , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Fosfatos de Inositol/metabolismo , Laminina/metabolismo , Microscopia Confocal , Microscopia de Fluorescência/métodos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Naftalenos/farmacologia , Neuritos/metabolismo , Neuritos/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II-dependent contractility with consequent effects on growth cone motility.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Aplysia/metabolismo , Miosina Tipo II/metabolismo , Actinas/ultraestrutura , Animais , Aplysia/ultraestrutura , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Microscopia EletrônicaRESUMO
The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.
Assuntos
Actinas/metabolismo , Cones de Crescimento/metabolismo , Neurônios/ultraestrutura , Citoesqueleto de Actina/ultraestrutura , Animais , Antifúngicos , Movimento Celular , Células Cultivadas , Depsipeptídeos/farmacologia , Cones de Crescimento/química , Cones de Crescimento/ultraestrutura , Miosina Tipo II/metabolismo , Miosina Tipo II/fisiologia , PolimerizaçãoRESUMO
The small G protein Rac regulates cytoskeletal protein dynamics in neuronal growth cones and has been implicated in axon growth, guidance, and branching. Intracellular Ca(2+) is another well known regulator of growth cone function; however, effects of Rac activity on intracellular Ca(2+) metabolism have not been well characterized. Here, we investigate how Rac1 activity affects release of Ca(2+) from intracellular endoplasmic reticulum (ER) stores stimulated by application of serotonin (5-hydroxytriptamine). We also address how Rac1 effects on microtubule assembly dynamics affect distribution of Ca(2+) release sites. Multimode fluorescent microscopy was used to correlate microtubule and ER behavior, and ratiometric imaging was used to assess intracellular Ca(2+) dynamics. We report that Rac1 activity both promotes Ca(2+) release and affects its spatial distribution in neuronal growth cones. The underlying mechanism involves synergistic Rac1 effects on microtubule assembly and reactive oxygen species (ROS) production. Rac1 activity modulates Ca(2+) by 1) enhancing microtubule assembly which in turn promotes spread of the ER-based Ca(2+) release machinery into the growth cone periphery, and 2) by increasing ROS production which facilitated inositol 1,4,5-trisphosphate-dependent Ca(2+) release. These results cast Rac1 as a key modulator of intracellular Ca(2+) function in the neuronal growth cone.