RESUMO
Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP's possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1-cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.
Assuntos
Proteína Quinase CDC2/metabolismo , Centrossomo/metabolismo , Ciclina B/metabolismo , Proteínas do Citoesqueleto/metabolismo , Mitose , Animais , Ciclo Celular , Segregação de Cromossomos , Proteínas do Citoesqueleto/genética , DNA Complementar/genética , Células HEK293 , Humanos , Camundongos , Mutação , Células NIH 3T3 , FosforilaçãoRESUMO
Spindle bipolarity is crucial for segregating chromosome during somatic cell division. Previous studies have suggested that cytoskeleton associated protein 2 (CKAP2) is involved in spindle assembly and chromosome segregation. In this study, we show that CKAP2-depleted primary hepatocytes exhibit over-duplicated centrosomes with disjoined chromosomes from metaphase plate. These cells proceed to apoptosis or multipolar cell division and subsequent apoptotic cell death. In addition, a mouse liver regeneration experiment showed a marked decrease in efficiency of hepatic regeneration in CKAP2-depleted liver. These data suggest a physiological role of CKAP2 in the formation of spindle bipolarity, which is necessary for maintaining chromosomal stability.
Assuntos
Divisão Celular/genética , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/citologia , Fuso Acromático/genética , Animais , Células Cultivadas , Centrossomo/fisiologia , Segregação de Cromossomos/genética , Diploide , Pontos de Checagem da Fase M do Ciclo Celular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , DNA Helicases/fisiologia , Endodesoxirribonucleases/fisiologia , Instabilidade Genômica , Longevidade , Morfogênese , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , DNA Helicases/genética , Replicação do DNA , Endodesoxirribonucleases/genética , Masculino , Mutação , Cauda/anormalidadesRESUMO
Human mitofilin is a mitochondrial protein that controls cristae formation. Here, we investigated the role of the Caenorhabditis elegans mitofilin homologs, IMMT-1 and -2, in reproduction, physiology, and mitochondrial cristae formation. Mutation of either immt-1 or immt-2 produced defects in germline development and egg-laying. These defects were exacerbated by the double mutation, which greatly reduced motility, increased levels of reactive oxygen species, decreased mitochondrial mass, and imparted resistance to oxidative stress. Cryo-electron microscopy and electron tomography revealed that each of the single mutations resulted in curved and stacked mitochondrial crista tubules as well as a reduced number of crista junctions. The immt-2 mutation was also associated with the presence of outer mitochondrial membrane pores, which were larger in the double mutant. IMMT-1 and IMMT-2 proteins were localized to the inner mitochondrial membrane, as seen by immunoelectron microscopy, and they behaved as oligomers or large complexes with F(1)F(0) ATP synthase in native polyacrylamide gel electrophoresis. These findings suggest that the two C. elegans mitofilin isoforms have non-overlapping functions in controlling mitochondrial cristae formation.
