[Show abstract][Hide abstract] ABSTRACT: Chromosome lagging at anaphase and migration of both sister chromatids to the same pole, i.e. nondisjunction, are two chromosome-segregation errors producing aneuploid cell progeny. Here, we developed an assay for the simultaneous detection of both chromosome-segregation errors in the marsupial PtK1 cell line by using multiplex fluorescence in situ hybridization with specific painting probes obtained by chromosome flow sorting. No differential susceptibility of the six PtK1 chromosomes to undergo nondisjunction and/or chromosome loss was observed in ana-telophase cells recovering from a nocodazole- or a monastrol-induced mitotic arrest, suggesting that the recurrent presence of specific chromosomes in several cancer types reflects selection effects rather than differential propensities of specific chromosomes to undergo missegregation. Experiments prolonging metaphase duration during drug recovery and inhibiting Aurora-B kinase activity on metaphase-aligned chromosomes provided evidence that some type of merotelic orientations was involved in the origin of both chromosome-segregation errors. Visualization of mero-syntelic kinetochore-microtubule attachments (a merotelic kinetochore in which the thicker microtubule bundle is attached to the same pole to which the sister kinetochore is connected) identified a peculiar malorientation that might participate in the generation of nondisjunction. Our findings imply random missegregation of chromosomes as the initial event in the generation of aneuploidy in mammalian somatic cells.
[Show abstract][Hide abstract] ABSTRACT: Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores.
Molecular biology of the cell 06/2008; 19(5):1873-82. DOI:10.1091/mbc.E07-10-1050 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.
[Show abstract][Hide abstract] ABSTRACT: Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.
Molecular Biology of the Cell 10/2003; 14(9):3821-33. DOI:10.1091/mbc.E03-01-0860 · 4.47 Impact Factor