RESUMEN
Glioblastoma multiforme (GBM) is the most malignant glioma type with diffuse borders due to extensive tumor cell infiltration. Therefore, understanding the mechanism of GBM cell dispersal is critical for developing effective therapies to limit infiltration. We identified neuropilin-1 as a mediator of cancer cell invasion by a functional proteomic screen and showed its role in GBM cells. Neuropilin-1 is a receptor for semaphorin3A (Sema3A), a secreted chemorepellent that facilitates axon guidance during neural development. Although neuropilin-1 expression in GBMs was previously shown, its role as a Sema3A receptor remained elusive. Using fluorophore-assisted light inactivation and RNA interference , we showed that neuropilin-1 is required for GBM cell migration. We also showed that GBM cells secrete Sema3A endogenously, and RNA interference-mediated downregulation of Sema3A inhibits migration and alters cell morphology that is dependent on Rac1 activity. Sema3A depletion also reduces dispersal, which is recovered by supplying Sema3A exogenously. Extracellular application of Sema3A decreases cell-substrate adhesion in a neuropilin-1-dependent manner. Using immunohistochemistry, we showed that Sema3A is overexpressed in a subset of human GBMs compared with the non-neoplastic brain. Together, these findings implicate Sema3A as an autocrine signal for neuropilin-1 to promote GBM dispersal by modulating substrate adhesion and suggest that targeting Sema3A-neuropilin-1 signaling may limit GBM infiltration.
Asunto(s)
Comunicación Autocrina/fisiología , Neoplasias Encefálicas/patología , Glioblastoma/patología , Semaforina-3A/fisiología , Química Encefálica , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Humanos , Invasividad Neoplásica , Neuropilina-1/fisiología , Proteómica , Semaforina-3A/análisis , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
The nerve growth cone binds to a complex array of guidance cues in its local environment that influence cytoskeletal interactions to control the direction of subsequent axon outgrowth. How this occurs is a critical question and must certainly involve signal transduction pathways. The paper by Suter and Forscher (2001)(this issue) begins to address how one such pathway, an Src family tyrosine kinase, enhances cytoskeletal linkage to apCAM, a permissive extracellular cue for Aplysia growth cones. Interestingly, they show that applied tension increases this kinase's localized phosphorylation that in turn further strengthens linkage. This suggests a potential positive feedback mechanism for amplifying and discriminating guidance information to guide growth cone motility.
Asunto(s)
Axones/fisiología , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismo , Animales , Aplysia , Axones/efectos de los fármacos , Fosforilación , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Insects bear a stereotyped set of limbs, or ventral body appendages. In the highly derived dipteran Drosophila melanogaster, the homeodomain transcription factor encoded by the Distal-less (Dll) gene plays a major role in establishing distal limb structures. We have isolated the Dll orthologue (TcDll) from the beetle Tribolium castaneum, which, unlike Drosophila, develops well-formed limbs during embryogenesis. TcDll is initially expressed at the sites of limb primordia formation in the young embryo and subsequently in the distal region of developing legs, antennae and mouthparts except the mandibles. Mutations in the Short antennae (Sa) gene of Tribolium delete distal limb structures, closely resembling the Dll phenotype in Drosophila. TcDll expression is severely reduced or absent in strong Sa alleles. Genetic mapping and molecular analysis of Sa alleles also support the conclusion that TcDll corresponds to the Sa gene. Our data indicate functional conservation of the Dll gene in evolutionarily distant insect species. Implications for evolutionary changes in limb development are discussed.
Asunto(s)
Escarabajos/embriología , Escarabajos/genética , Drosophila melanogaster/genética , Genes de Insecto , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Factores de Transcripción , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Evolución Molecular , Extremidades/embriología , Datos de Secuencia Molecular , Mutación , FenotipoRESUMEN
Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.
Asunto(s)
Diferenciación Celular/fisiología , Laminina/fisiología , Priones/fisiología , Animales , Anticuerpos/inmunología , Adhesión Celular/fisiología , Adhesión Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Rayos Láser , Células PC12 , Priones/inmunología , Priones/efectos de la radiación , RatasRESUMEN
We seek to understand how the nerve growth cone acts as a sensory motile machine to respond to chemical cues in the developing embryo. This review focuses on filopodial protrusion and F-actin-based motility because there is good evidence that these processes are required for axon guidance. The clutch hypothesis, which states that filopodial protrusion occurs by actin assembly when an actin filament is fixed with respect to the substrate (i.e., a clutch is engaged), was postulated by Mitchison and Kirscher to link protrusion to actin dynamics. Protrusion would require functional modules for movement of material into filopodia, clutching the F-actin, F-actin assembly at the tip, and retrograde flow. In this review, recent studies of actin-associated proteins involved in filopodial protrusion will be summarized, and their roles will be assessed in the context of the clutch hypothesis. The large number of proteins involved in filopodial motility and their complex interactions make it difficult to understand how these proteins act in protrusion. Recently, we have used microscale chromophore-assisted laser inactivation (micro-CALI) for the focal and acute inactivation of specific actin-associated proteins during filopodial protrusion to address their in situ roles. Our findings suggest that myosin V functions in moving membranes or other material forward in extending filopodia, that talin acts in the clutch module, and that zyxin acts in actin assembly at the tip during filopodial protrusion, perhaps by recruiting Ena/VASP family members to promote actin elongation at this site.
Asunto(s)
Actinas/metabolismo , Conos de Crecimiento/fisiología , Proteínas de Microfilamentos/metabolismo , Seudópodos/fisiología , Animales , Conos de Crecimiento/química , Conos de Crecimiento/ultraestructura , Seudópodos/químicaRESUMEN
Loss of the tumour-suppressor gene TSC1 is responsible for hamartoma development in tuberous sclerosis complex (TSC), which renders several organs susceptible to benign tumours. Hamartin, the protein encoded by TSC1, contains a coiled-coil domain and is expressed in most adult tissues, although its function is unknown. Here we show that hamartin interacts with the ezrin-radixin-moesin (ERM) family of actin-binding proteins. Inhibition of hamartin function in cells containing focal adhesions results in loss of adhesion to the cell substrate, whereas overexpression of hamartin in cells lacking focal adhesions results in activation of the small GTP-binding protein Rho, assembly of actin stress fibres and formation of focal adhesions. Interaction of endogenous hamartin with ERM-family proteins is required for activation of Rho by serum or by lysophosphatidic acid (LPA). Our data indicate that disruption of adhesion to the cell matrix through loss of hamartin may initiate the development of TSC hamartomas and that a Rho-mediated signalling pathway regulating cell adhesion may constitute a rate-limiting step in tumour formation.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endotelio Vascular/citología , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Células 3T3 , Actinas/metabolismo , Animales , Proteínas Sanguíneas/farmacología , Adhesión Celular/fisiología , Endotelio Vascular/enzimología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Lisofosfolípidos/farmacología , Ratones , Fragmentos de Péptidos/farmacología , Transducción de Señal/fisiología , Estrés Mecánico , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos Híbridos , Venas Umbilicales/citologíaRESUMEN
Directed growth cone movement is crucial for the correct wiring of the nervous system. This movement is governed by the concerted actions of cell surface receptors, signaling proteins, cytoskeleton-associated molecules, and molecular motors. In order to investigate the molecular basis of growth cone motility, we applied a new technique to functionally inactivate proteins: micro-scale Chromophore-Assisted Laser Inactivation [Diamond et al. (1993) Neuron 11:409-421]. Micro-CALI uses laser light of 620 nm, focused through microscope optics into a 10-microm spot. The laser energy is targeted via specific Malachite green-labeled, non-function-blocking antibodies, that generate short-lived protein-damaging hydroxyl radicals [Liao et al. (1994) Proc Natl Acad Sci USA 91:2659-2663]. Micro-CALI mediates specific loss of protein function with unachieved spatial and temporal resolution. Combined with time-lapse video microscopy, it offers the possibility to induce and observe changes in growth cone dynamics on a real time base. We present here the effects of the acute and localized inactivation of selected growth cone molecules on growth cone behavior and morphology. Based on our observations, we propose specific roles for these proteins in growth cone motility and neurite outgrowth.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/fisiología , Rayos Láser , Proteínas del Tejido Nervioso/fisiología , Actinas/fisiología , Animales , Calcineurina/fisiología , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/fisiología , Moléculas de Adhesión Celular Neuronal/efectos de la radiación , Colorantes , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/efectos de la radiación , Daño del ADN , Conos de Crecimiento/química , Humanos , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/fisiología , Microscopía por Video/instrumentación , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas Asociadas a Microtúbulos/efectos de la radiación , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/fisiología , Proteínas Motoras Moleculares/efectos de la radiación , Miosinas/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/efectos de la radiación , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Colorantes de Rosanilina , Transducción de Señal , Talina/fisiología , Vinculina/fisiologíaRESUMEN
Chromophore-assisted laser inactivation (CALI) is a new technology for acute protein inactivation in living cells. It targets laser energy to specific proteins via non-function-blocking antibodies that are labeled with the dye malachite green. Excitation of the dye generates short-lived free radicals that damage the bound protein without affecting other cellular components. The wavelength of laser light used (620 nm) is not readily absorbed by cells such that non-specific light damage does not occur. CALI provides an alternative to other inactivation strategies and has the advantages of high spatial and temporal resolution. The ultimate value of this technology for cancer research will be assessed by how effective CALI is in ascribing in situ function during cancer-relevant processes and in identifying and validating protein targets for drug discovery. Recent work using CALI on ezrin and pp60-c-src, two proteins that may be involved in cancer, suggests its potential. Further application of CALI will likely be of utility for understanding cellular mechanisms of cancer and developing cancer therapeutics.
Asunto(s)
Proteínas Bacterianas , Proteínas de Drosophila , Rayos Láser , Microscopía por Video/métodos , Proteínas/química , Factores de Transcripción , Animales , Anticuerpos/química , Colorantes , Proteínas del Citoesqueleto/química , Proteínas de Homeodominio/química , Humanos , Immunoblotting , Proteínas de la Membrana/química , Proteínas de Neoplasias/química , Fosfoproteínas/química , Fotoquímica , Proteínas/inmunología , Colorantes de Rosanilina/químicaRESUMEN
The formation of neurocircuitry depends on the control of neurite outgrowth that, in turn, can be divided into two processes: nerve growth cone protrusion and neurite extension. It has long been known that the neural cell adhesion molecules L1 and NCAM-180 promote neurite outgrowth, but how they function in growth cones is unclear. We addressed the roles of L1 and NCAM-180 in neurite outgrowth by using microscale chromophore-assisted laser inactivation (micro-CALI) of these proteins to perturb their functions at precise times in single growth cones of embryonic chick dorsal root ganglion neurons grown in culture. Micro-CALI of L1 causes neurite retraction after a 10 min lag period but does not affect growth cone protrusion. In contrast, micro-CALI of NCAM-180 causes rapid growth cone retraction but does not affect neurite extension. The simultaneous inactivation of both these molecules resulted in both distinct effects that were segregated in time. The behavior of growth cones after these micro-CALI treatments resemble the drug-induced perturbation of microtubules for L1 and F-actin for NCAM-180. These findings suggest distinct roles in the growth cone for L1 and NCAM-180 in different steps of neurite outgrowth: L1 functions in neurite extension,whereas NCAM-180 functions in growth cone protrusion.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Neuronas/citología , Neuronas/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Embrión de Pollo , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Rayos Láser , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/inmunología , Movimiento , Moléculas de Adhesión de Célula Nerviosa/inmunología , Neuritas/ultraestructuraRESUMEN
The role of the microtubule-associated protein (MAP) tau in axon growth remains controversial. Antisense experiments have suggested that tau is required for axon outgrowth, whereas genetic knockout and immunodepletion studies have suggested that tau plays no role in this process. To investigate the role of tau in both neurite outgrowth and growth cone motility, we have used a different approach, the chromophore-assisted laser inactivation (CALI) of tau in chick dorsal root ganglion (DRG) neurons in culture. This approach generates an acute loss of tau function that is not subject to compensation by other MAPs. Inactivation of tau in whole DRG neurons (including cell body and neurites) reduced neurite number and length. Inactivation of tau within regions of growth cones using micro-scale CALI caused a decrease in neurite extension rate by approximately 2-fold. Surprisingly, it also caused a approximately 20% decrease in the lamellipodial size within the inactivation region, whereas the filopodial motility was not affected. These results suggest that tau is required in neurite outgrowth and that tau also functions in lamellipodial motility at the growth cone leading edge.
Asunto(s)
Conos de Crecimiento/fisiología , Neuritas/fisiología , Neuronas Aferentes/fisiología , Proteínas tau/fisiología , Animales , Unión Competitiva/efectos de la radiación , Recuento de Células/efectos de la radiación , Movimiento Celular/efectos de la radiación , Tamaño de la Célula/efectos de la radiación , Embrión de Pollo , Pollos , Ganglios Espinales/citología , Ganglios Espinales/efectos de la radiación , Rayos Láser , Microtúbulos/metabolismo , Neuritas/efectos de la radiación , Neuronas Aferentes/citología , Proteínas tau/metabolismo , Proteínas tau/efectos de la radiaciónRESUMEN
Immunocytochemistry and in vitro studies have suggested that the ERM (ezrin-radixin-moesin) protein, radixin, may have a role in nerve growth cone motility. We tested the in situ role of radixin in chick dorsal root ganglion growth cones by observing the effects of its localized and acute inactivation. Microscale chromophore-assisted laser inactivation (micro-CALI) of radixin in growth cones causes a 30% reduction of lamellipodial area within the irradiated region whereas all control treatments did not affect lamellipodia. Micro-CALI of radixin targeted to the middle of the leading edge often split growth cones to form two smaller growth cones during continued forward movement (>80%). These findings suggest a critical role for radixin in growth cone lamellipodia that is similar to ezrin function in pseudopodia of transformed fibroblasts. They are consistent with radixin linking actin filaments to each other or to the membrane during motility.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Movimiento Celular/fisiología , Proteínas del Citoesqueleto , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Conos de Crecimiento/fisiología , Proteínas de la Membrana/metabolismo , Animales , Especificidad de Anticuerpos , Proteínas Sanguíneas/inmunología , Embrión de Pollo , Rayos Láser , Proteínas de la Membrana/inmunología , Biología Molecular/métodos , Neuritas/metabolismoRESUMEN
The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.
Asunto(s)
Proteínas Bacterianas , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Genes de Insecto/efectos de la radiación , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Factores de Transcripción , Tribolium/genética , Animales , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Genes de Insecto/efectos de los fármacos , Proteínas de Homeodominio/fisiología , Proteínas de Insectos/fisiología , Rayos Láser , Morfogénesis/genética , Fármacos Sensibilizantes a Radiaciones/farmacología , Colorantes de Rosanilina/farmacología , Tribolium/embriologíaRESUMEN
BACKGROUND: Changes in cell shape and motility are important manifestations of oncogenic transformation, but the mechanisms underlying these changes and key effector molecules in the cytoskeleton remain unknown. The Fos oncogene induces expression of ezrin, the founder member of the ezrin/radixin/moesin (ERM) protein family, but not expression of the related ERM proteins, suggesting that ezrin has a distinct role in cell transformation. ERM proteins have been suggested to link the plasma membrane to the actin-based cytoskeleton and are substrates and anchoring sites for a variety of protein kinases. Here, we examined the role of ezrin in cellular transformation. RESULTS: Fos-mediated transformation of Rat-1 fibroblasts resulted in an increased expression and hyperphosphorylation of ezrin, and a concomitant increased association of ezrin with the cortical cytoskeleton. We tagged ezrin with green fluorescent protein and examined its distribution in normal and Fos-transformed fibroblasts: ezrin was concentrated at the leading edge of extending pseudopodia of Fos-transformed Rat-1 cells, and was mainly cytosolic in normal Rat-1 cells. Functional ablation of ezrin by micro-CALI (chromophore-assisted laser inactivation) blocked plasma-membrane ruffling and motility of Fos-transformed fibroblasts. Ablation of ezrin in normal Rat-1 cells caused a marked collapse of the leading edge of the cell. CONCLUSIONS: Ezrin plays an important role in pseudopodial extension in Fos-transformed Rat-1 fibroblasts, and maintains cell shape in normal Rat-1 cells. The increased expression, hyperphosphorylation and subcellular redistribution of ezrin upon fibroblast transformation coupled with its roles in cell shape and motility suggest a critical role for ezrin in oncogenic transformation.
Asunto(s)
Transformación Celular Neoplásica , Fibroblastos/fisiología , Fosfoproteínas/fisiología , Animales , Membrana Celular/metabolismo , Movimiento Celular , Tamaño de la Célula , Proteínas del Citoesqueleto , Electroforesis en Gel Bidimensional , Fibroblastos/citología , Rayos Láser , Microscopía por Video , Proteínas Oncogénicas v-fos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , RatasRESUMEN
BACKGROUND: The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons. RESULTS: To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum. CONCLUSIONS: These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.
Asunto(s)
Axones/fisiología , Colorantes/química , Rayos Láser , Proteínas/metabolismo , Retina/fisiología , Colorantes de Rosanilina/química , Proteínas de Saccharomyces cerevisiae , Animales , Anticuerpos Monoclonales , Embrión de Pollo , Quitina Sintasa , Efrina-A2 , Proteínas Fúngicas/metabolismo , Ratones , Ratones Endogámicos BALB C , Retina/efectos de la radiaciónAsunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/fisiología , Anemia Hemolítica Congénita/sangre , Anemia Hemolítica Congénita/etiología , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/deficiencia , Dióxido de Carbono/sangre , Bovinos , Tamaño de la Célula/fisiología , Eritrocitos/citología , Eritrocitos/metabolismo , Homeostasis/fisiología , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico , RatonesRESUMEN
Filopodial motility is critical for many biological processes, particularly for axon guidance. This motility is based on altering the F-actin-based cytoskeleton, but the mechanisms of how this occurs and the actin-associated proteins that function in this process remain unclear. We investigated two of these proteins found in filopodia, talin and vinculin, by inactivating them in subregions of chick dorsal root ganglia neuronal growth cones and by observing subsequent behavior by video-enhanced microscopy and quantitative morphometry. Microscale chromophore-assisted laser inactivation of talin resulted in the temporary cessation of filopodial extension and retraction. Inactivation of vinculin caused an increased incidence of filopodial bending and buckling within the laser spot but had no effect on extension or retraction. These findings show that talin acts in filopodial motility and may couple both extension and retraction to actin dynamics. They also suggest that vinculin is not required for filopodial extension and retraction but plays a role in the structural integrity of filopodia.
Asunto(s)
Neuronas/fisiología , Seudópodos/fisiología , Talina/fisiología , Vinculina/fisiología , Animales , Anticuerpos Monoclonales , Axones/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Pollo , Ganglios Espinales/citologíaRESUMEN
The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.
Asunto(s)
Axones/fisiología , Proteínas de Unión a Calmodulina/fisiología , Dendritas/fisiología , Cadenas Ligeras de Miosina/fisiología , Miosina Tipo V , Proteínas del Tejido Nervioso/fisiología , Seudópodos/fisiología , Adenosina Trifosfato/farmacología , Animales , Axones/ultraestructura , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/inmunología , Células Cultivadas , Embrión de Pollo , Dendritas/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Ganglios Espinales/citología , Rayos Láser , Microinyecciones , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/inmunología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/inmunologíaRESUMEN
Establishing molecular mechanisms of axon guidance presents one of the greatest challenges in understanding the development of the nervous system. There are many neurons, and each neuron by virtue of its location, biochemistry, and time of development, may generate a unique axon morphology in its response to environmental cues that may also change during development. The context dependence and combinatorial nature of these interactions make analysis of axon guidance particularly difficult. This article will focus on the neuronal growth cone as axon guidance is controlled by interaction of the growth cone with its environment. I present here an overview of growth cone motility from the perspective of cytoskeletal dynamics. I conclude with a discussion of our application of regional laser inactivation of growth cone proteins to address what proteins might be involved in locally modulating the cytoskeleton and how they affect growth cone motility.