RESUMO
In the developing nervous system, axons are guided to their targets by the growth cone. Lamellipodial and filopodial protrusions from the growth cone underlie motility and guidance. Many molecules that control lamellipodia and filopodia formation, actin organization, and axon guidance have been identified, but it remains unclear how these molecules act together to control these events. Experiments are described here that indicate that, in Caenorhabditis elegans, two WH2-domain-containing activators of the Arp2/3 complex, WVE-1/WAVE and WSP-1/WASP, act redundantly in axon guidance and that GEX-2/Sra-1 and GEX-3/Kette, molecules that control WAVE activity, might act in both pathways. WAVE activity is controlled by Rac GTPases, and data are presented here that suggest WVE-1/WAVE and CED-10/Rac act in parallel to a pathway containing WSP-1/WASP and MIG-2/RhoG. Furthermore, results here show that the CED-10/WVE-1 and MIG-2/WSP-1 pathways act in parallel to two other molecules known to control lamellipodia and filopodia and actin organization, UNC-115/abLIM and UNC-34/Enabled. These results indicate that at least three actin-modulating pathways act in parallel to control actin dynamics and lamellipodia and filopodia formation during axon guidance (WASP-WAVE, UNC-115/abLIM, and UNC-34/Enabled).
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Axônios/fisiologia , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Animais , Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Movimento Celular , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Many genes that affect axon pathfinding and cell migration have been identified. Mechanisms by which these genes and the molecules they encode interact with one another in pathways and networks to control developmental events are unclear. Rac GTPases, the cytoskeletal signaling molecule Enabled, and NIK kinase have all been implicated in regulating axon pathfinding and cell migration. Here we present evidence that, in Caenorhabditis elegans, three Rac GTPases, CED-10, RAC-2, and MIG-2, define three redundant pathways that each control axon pathfinding, and that the NIK kinase MIG-15 acts in each Rac pathway. Furthermore, we show that the Enabled molecule UNC-34 defines a fourth partially redundant pathway that acts in parallel to Rac/MIG-15 signaling in axon pathfinding. Enabled and the three Racs also act redundantly to mediate AQR and PQR neuronal cell migration. The Racs and UNC-34 Ena might all control the formation of actin-based protrusive structures (lamellipodia and filopodia) that mediate growth cone outgrowth and cell migration. MIG-15 does not act with the three Racs in execution of cell migration. Rather, MIG-15 affects direction of PQR neuronal migration, similar to UNC-40 and DPY-19, which control initial Q cell polarity, and Wnt signaling, which acts later to control Q cell-directed migration. MIG-2 Rac, which acts with CED-10 Rac, RAC-2 Rac, and UNC-34 Ena in axon pathfinding and cell migration, also acts with MIG-15 in PQR directional migration.
Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Movimento Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Axônios/enzimologia , Sequência de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Movimento Celular/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/enzimologia , Neurônios/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/genética , Quinase Induzida por NF-kappaBRESUMO
This chapter is concerned with a method of analysis and quantification of cell migration defects in mutants of the nematode worm Caenorhabditis elegans. The method takes advantage of transgenic expression of the green fluorescent protein to visualize migrating cells. By following these protocols, one will be able to analyze cell migration defects in new mutant strains for comparison to wild-type and to other mutants. Techniques described include obtaining wild-type and mutant worm strains as well as strains harboring green fluorescent protein transgenes; maintenance and manipulation of C. elegans in the laboratory; introducing transgenes into different genetic backgrounds; mounting worms for fluorescence microscopy; and scoring and analysis of cell migration defects.
Assuntos
Caenorhabditis elegans/fisiologia , Movimento Celular/fisiologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Cruzamentos Genéticos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Masculino , Microscopia de Fluorescência/métodos , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Transfecção/métodosRESUMO
Mutations in the unc-39 gene of C. elegans lead to migration and differentiation defects in a subset of mesodermal and ectodermal cells, including muscles and neurons. Defects include mesodermal specification and differentiation as well a neuronal migration and axon pathfinding defects. Molecular analysis revealed that unc-39 corresponds to the previously named gene ceh-35 and that the UNC-39 protein belongs to the Six4/5 family of homeodomain transcription factors and is similar to human Six5, a protein implicated in the pathogenesis of type I myotonic dystrophy (DM1). We show that human Six5 and UNC-39 are functional homologs, suggesting that further characterization of the C. elegans unc-39 gene might provide insight into the etiology of DM1.