RESUMO
Rac GTPases are key regulators of cell shape and cytoskeletal organization. While some regulators of Rac activity are known, such as GTPase-activating proteins (GAPs) that repress Rac activity, other Rac regulators remain to be identified. The novel Caenorhabditis elegans WD-repeat protein SWAN-1 was identified in a yeast two-hybrid screen with the LIM domains of the Rac effector UNC-115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases. The swan-1(ok267) loss-of-function mutation suppressed defects caused by the hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and filopodia formation induced by constitutively active Rac in C. elegans neurons. Furthermore, SWAN-1(+) transgenic expression suppressed the effects of overactive Rac, including ectopic lamellipodia and filopodia formation in C. elegans neurons, ectopic lamellipodia formation in cultured mammalian fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular morphogenesis and cytoskeletal organization. While broadly conserved across species, SWAN-1 family members show no sequence similarity to previously known Rac inhibitors.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Citoesqueleto/metabolismo , Feminino , Imunoprecipitação , Proteínas de Membrana , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Morfogênese , Neurônios/citologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas , Pseudópodes , Homologia de Sequência de Aminoácidos , Supressão Genética , Transgenes/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas rac de Ligação ao GTP/genéticaRESUMO
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.