RESUMO
During the development of multicellular organisms, cellular diversity is often achieved through asymmetric cell divisions that produce two daughter cells having different developmental potentials. Prior to an asymmetric cell division, cellular components segregate to opposite ends of the cell defining an axis of polarity. The mitotic spindle rotationally aligns along this axis of polarity, thereby ensuring that the cleavage plane is positioned such that segregated components end up in individual daughter cells. Here we report our characterization of a novel gene required for spindle alignment in Caenorhabditis elegans. During the first mitosis in spd-3(oj35) embryos the spindle failed to align along the anterior/posterior axis, leading to abnormal cleavage configurations. spd-3(oj35) embryos had additional defects reminiscent of dynein/dynactin loss-of-function possibly caused by the mislocalization of dynactin. Surprisingly, we found that SPD-3GFP localized to mitochondria. Consistent with this localization, spd-3(oj35) worms exhibited slow growth and increased ATP concentrations, which are phenotypes similar to those described for other mitochondrial mutants in C. elegans. To our knowledge, SPD-3 is the first example of a link between mitochondria and spindle alignment in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Clonagem Molecular , Primers do DNA/genética , DNA de Helmintos/genética , Complexo Dinactina , Dineínas/metabolismo , Feminino , Genes de Helmintos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Meiose/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fenótipo , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fuso Acromático/metabolismoRESUMO
Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double-strand breaks, where it functions in repair and triggers cell-cycle checkpoints via activation of the ataxia-telangiectasia mutated kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These preneoplastic B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53. Moreover, Mre11 deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B-cell lymphomas. Our findings indicate that MRN cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends. Cancer Res; 77(19); 5327-38. ©2017 AACR.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/patologia , Enzimas Reparadoras do DNA/fisiologia , Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Hidrolases Anidrido Ácido , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Instabilidade Genômica , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Proteína Homóloga a MRE11 , Camundongos , Mutação , Proteínas Nucleares/fisiologia , Oncogenes , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.