RESUMO
Centrosomes, the major microtubule-organizing centres (MTOCs) of animal cells, are comprised of a pair of centrioles surrounded by pericentriolar material (PCM). Early in the cell cycle, there is a single centrosome, which duplicates during S-phase to direct bipolar spindle assembly during mitosis. Although crucial for proper cell division, the mechanisms that govern centrosome duplication are not fully understood. Here, we identify the Caenorhabditis elegans gene sas-5 as essential for daughter-centriole formation. SAS-5 is a coiled-coil protein that localizes primarily to centrioles. Fluorescence recovery after photobleaching (FRAP) experiments with green fluorescent protein (GFP) fused to SAS-5 (GFP-SAS-5) demonstrated that the protein shuttles between centrioles and the cytoplasm throughout the cell cycle. Analysis of mutant alleles revealed that the presence of SAS-5 at centrioles is crucial for daughter-centriole formation and that ZYG-1, a kinase that is also essential for this process, controls the distribution of SAS-5 to centrioles. Furthermore, partial RNA-interference (RNAi)-mediated inactivation experiments suggest that both sas-5 and zyg-1 are dose-dependent regulators of centrosome duplication.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Mitose/genética , Transporte Ativo do Núcleo Celular/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Dosagem de Genes , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico/genética , Interferência de RNA/fisiologiaRESUMO
The removal of apoptotic cells is essential for the physiological well being of the organism. In Caenorhabditis elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. Here, we show that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, we find that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation.
Assuntos
Actinas/metabolismo , Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Fagocitose/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Citoesqueleto/química , Citoesqueleto/metabolismo , Transtornos do Desenvolvimento Sexual , Genótipo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/genéticaRESUMO
It has been estimated that 15%-30% of the â¼20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole-genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with apparent sperm-specific functions. In total, 58 putative essential genes were identified on chromosomes III-V, of which 52 genes are represented by novel alleles in this collection. Of these 52 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalog of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.