RESUMO
Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.
Assuntos
Axônios/fisiologia , Cones de Crescimento/fisiologia , Pinocitose/fisiologia , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Cones de Crescimento/efeitos dos fármacos , Proteínas Hedgehog/farmacologia , Técnicas In Vitro , Miosina Tipo II/metabolismo , Pinocitose/efeitos dos fármacos , Células Ganglionares da Retina/citologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Transfecção , Transferrina/metabolismo , Alcaloides de Veratrum/farmacologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The ocular motility disorder "Congenital fibrosis of the extraocular muscles type 1" (CFEOM1) results from heterozygous mutations altering the motor and third coiled-coil stalk of the anterograde kinesin, KIF21A. We demonstrate that Kif21a knockin mice harboring the most common human mutation develop CFEOM. The developing axons of the oculomotor nerve's superior division stall in the proximal nerve; the growth cones enlarge, extend excessive filopodia, and assume random trajectories. Inferior division axons reach the orbit but branch ectopically. We establish a gain-of-function mechanism and find that human motor or stalk mutations attenuate Kif21a autoinhibition, providing in vivo evidence for mammalian kinesin autoregulation. We identify Map1b as a Kif21a-interacting protein and report that Map1bâ»/â» mice develop CFEOM. The interaction between Kif21a and Map1b is likely to play a critical role in the pathogenesis of CFEOM1 and highlights a selective vulnerability of the developing oculomotor nerve to perturbations of the axon cytoskeleton.
Assuntos
Axônios/patologia , Oftalmopatias Hereditárias/genética , Fibrose/genética , Cinesinas/genética , Cinesinas/metabolismo , Mutação/genética , Transtornos da Motilidade Ocular/genética , Nervo Oculomotor/patologia , Fatores Etários , Animais , Animais Recém-Nascidos , Axônios/ultraestrutura , Contagem de Células , Modelos Animais de Doenças , Embrião de Mamíferos , Oftalmopatias Hereditárias/patologia , Oftalmopatias Hereditárias/fisiopatologia , Movimentos Oculares/genética , Movimentos Oculares/fisiologia , Fibrose/patologia , Fibrose/fisiopatologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Vias Neurais/metabolismo , Vias Neurais/patologia , Vias Neurais/ultraestrutura , Transtornos da Motilidade Ocular/patologia , Transtornos da Motilidade Ocular/fisiopatologia , Nervo Oculomotor/ultraestruturaRESUMO
Axon pathfinding is essential for the establishment of proper neuronal connections during development. Advances in neuroimaging and genomic technologies, coupled with animal modeling, are leading to the identification of an increasing number of human disorders that result from aberrant axonal wiring. In this review, we summarize the recent clinical, genetic and molecular advances with regard to three human disorders of axon guidance: Horizontal gaze palsy with progressive scoliosis, Congenital mirror movements, and Congenital fibrosis of the extraocular muscles, Type III.
Assuntos
Axônios/fisiologia , Doenças dos Nervos Cranianos/patologia , Oftalmoplegia/patologia , Escoliose/patologia , Animais , Axônios/patologia , Doenças dos Nervos Cranianos/genética , Receptor DCC , Modelos Animais de Doenças , Humanos , Oftalmoplegia/genética , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Escoliose/genética , Tubulina (Proteína)/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The function and mechanism of macropinocytosis in cells outside of the immune system remain poorly understood. We used a neuroblastoma cell line, Neuro-2a, to study macropinocytosis in neuronal cells. We found that phorbol 12-myristate 13-acetate (PMA) and insulin-like growth factor 1 (IGF-1) induced two distinct types of macropinocytosis in the Neuro-2a cells. IGF-1-induced macropinocytosis occurs mostly around the cell bodies and requires phosphoinositide 3-kinase (PI3K), while PMA-induced macropinocytosis occurs predominantly in the neurites and is independent of PI3K activity. Both types of macropinocytosis were inhibited by a specific inhibitor of nonmuscle myosin II, blebbistatin. siRNA knockdown of nonmuscle myosin II isoforms, -IIA and -IIB, resulted in opposite effects on macropinocytosis induced by PMA or IGF. Myosin IIA knockdown significantly increased, whereas myosin IIB knockdown significantly decreased, macropinocytosis with correlating changes in membrane ruffle formation.