Recent work on Drosophila oogenesis has uncovered connections between cell-cycle checkpoints and pattern formation. Genes of the spindle class, which encode double-strand break repair enzymes and RNA helicases, affect oocyte polarity and the decision whether to differentiate as an oocyte or a nurse cell. (+info)
C-myc overexpression and p53 loss cooperate to promote genomic instability.
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p53 monitors genomic integrity at the G1 and G2/M cell cycle checkpoints. Cells lacking p53 may show gene amplification as well as the polyploidy or aneuploidy typical of many tumors. The pathways through which this develops, however, are not well defined. We demonstrate here that the combination of p53 inactivation and c-myc overexpression in diploid cells markedly accelerates the spontaneous development of tetraploidy. This is not seen with either N-myc or L-myc. Tetraploidy is accompanied by significantly higher levels of cyclin B and its associated cdc2 kinase activity. Mitotic spindle poisons accelerate the appearance of tetraploidy in cells either lacking functional p53 or overexpressing c-myc whereas the combination is additive. Restoration of p53 function in cells overexpressing c-myc causing rapid apoptosis, indicating that cells yet to become tetraploid have nonetheless suffered irreversible genomic and/or mitotic spindle damage. In the face of normal p53 function, such damage would either be repaired or trigger apoptotis. We propose that loss of p53 and overexpression of c-myc permits the emergence and survival of cells with increasingly severe damage and the eventual development of tetraploidy. (+info)
Defects in Saccharomyces cerevisiae protein phosphatase type I activate the spindle/kinetochore checkpoint.
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A conditional allele of type 1 protein phosphatase (glc7-129) in Saccharomyces cerevisiae causes first cycle arrest in G2/M, characterized by cells with a short spindle and high H1 kinase activity. Point-of-execution experiments indicate Glc7p function is required in G2/M just before anaphase for the completion of mitosis. Loss of the spindle/kinetochore checkpoint in glc7-129 cells abolishes the G2/M cell cycle arrest with a concomitant increase in chromosome loss and reduced viability. These results support a role for Glc7p in regulating kinetochore attachment to the spindle, an event monitored by the spindle/kinetochore checkpoint. (+info)
The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast.
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Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. We isolated mutants in IPL1, which encodes a protein kinase, in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and we propose that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors. (+info)
Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers.
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The product of the abnormal spindle (asp) gene was found to be an asymmetrically localized component of the centrosome during mitosis, required to focus the poles of the mitotic spindle in vivo. Removing Asp protein function from Drosophila melanogaster embryo extracts, either by mutation or immunodepletion, resulted in loss of their ability to restore microtubule-organizing center activity to salt-stripped centrosome preparations. This was corrected by addition of purified Asp protein. Thus, Asp appears to hold together the microtubule-nucleating gamma-tubulin ring complexes that organize the mitotic centrosome. (+info)
Influence of centriole behavior on the first spindle formation in zygotes of the brown alga Fucus distichus (Fucales, Phaeophyceae).
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The influence of centrioles, derived from the sperm flagellar basal bodies, and the centrosomal material (MTOCs) on spindle formation in the brown alga Fucus distichus (oogamous) was studied by immunofluorescence microscopy using anti-centrin and anti-beta-tubulin antibodies. In contrast to a bipolar spindle, which is formed after normal fertilization, a multipolar spindle was formed in polyspermic zygote. The number of mitotic poles in polyspermic zygotes was double the number of sperm involved in fertilization. As an anti-centrin staining spot (centrioles) was located at these poles, the multipolar spindles in polyspermic zygotes were produced by the supplementary centrioles. When anucleate egg fragments were fertilized, chromosome condensation and mitosis did not occur in the sperm nucleus. Two anti-centrin staining spots could be detected, microtubules (MTs) radiated from nearby, but the mitotic spindle was never produced. When a single sperm fertilized multinucleate eggs (polygyny), abnormal spindles were also observed. In addition to two mitotic poles containing anti-centrin staining spots, extra mitotic poles without anti-centrin staining spots were also formed, and as a result multipolar spindles were formed. When karyogamy was blocked with colchicine, it became clear that the egg nucleus proceeded independently into mitosis accompanying chromosome condensation. A monoastral spindle could be frequently observed, and in rare cases a barrel-shaped spindle was formed. However, when a sperm nucleus was located near an egg nucleus, the two anti-centrin staining spots shifted to the egg nucleus from the sperm nucleus. In this case, a normal spindle was formed, the egg chromosomes arranged at the equator, and the associated MTs elongated from one pole of the egg spindle toward the sperm chromosomes which were scattered. From these results, it became clear that paternal centrioles derived from the sperm have a crucial role in spindle formation in the brown algae, such as they do during animal fertilization. However, paternal centrioles were not adequate for the functional centrosome during spindle formation. We speculated that centrosomal materials from the egg cytoplasm aggregate around the sperm centrioles and are needed for centrosomal activation. (+info)
Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice.
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NuMA is a nuclear matrix protein in interphase and relocates to the spindle poles in mitotis. Different NuMA constructs, in which either N- or C-terminal domains were deleted, and the full-length construct were expressed in Escherichia coli, and the NuMA polypeptides were purified to homogeneity and allowed to assemble in vitro. Electron microscopy showed that NuMA can build multiarm oligomers by interaction of the C-terminal globular domains. Each arm of the oligomer corresponds to a NuMA dimer. Oligomers with up to 10 or 12 arms have been observed for both full-length NuMA and for constructs that still contain the proximal part of the C-terminal tail domain. Other results from this laboratory have shown that transient overexpression of NuMA in HeLa cells induces a nuclear scaffold with a quasi-hexagonal organization that can fill the nuclei. Here we show that computer modelling of the three-dimensional packing of NuMA into such scaffolds can explain the different spacing of the hexagons seen when constructs with different coiled-coil lengths are used. Thus, the 12 arm oligomer, for which we have in vitro evidence, may be the structural unit from which the nuclear scaffold in transfected cells is built. (+info)
Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p.
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Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Delta cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation. (+info)