RESUMO
Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Morfogênese/fisiologia , Miosina Tipo II/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases Associadas a rho/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Citoesqueleto/metabolismo , Humanos , Dados de Sequência Molecular , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transgenes , Quinases Ativadas por p21/genética , Quinases Associadas a rho/genéticaAssuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Humanos , Proteínas de Membrana/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Embryonic morphogenesis involves the coordinate behaviour of multiple cells and requires the accurate balance of forces acting within different cells through the application of appropriate brakes and throttles. In C. elegans, embryonic elongation is driven by Rho-binding kinase (ROCK) and actomyosin contraction in the epidermis. We identify an evolutionary conserved, actin microfilament-associated RhoGAP (RGA-2) that behaves as a negative regulator of LET-502/ROCK. The small GTPase RHO-1 is the preferred target of RGA-2 in vitro, and acts between RGA-2 and LET-502 in vivo. Two observations show that RGA-2 acts in dorsal and ventral epidermal cells to moderate actomyosin tension during the first half of elongation. First, time-lapse microscopy shows that loss of RGA-2 induces localised circumferentially oriented pulling on junctional complexes in dorsal and ventral epidermal cells. Second, specific expression of RGA-2 in dorsal/ventral, but not lateral, cells rescues the embryonic lethality of rga-2 mutants. We propose that actomyosin-generated tension must be moderated in two out of the three sets of epidermal cells surrounding the C. elegans embryo to achieve morphogenesis.