Browsing by Subject "Cyclin B"

Now showing 1 - 6 of 6
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    Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era.
    (J Cell Sci, 2008-11-01) Wu, Judy Qiju; Kornbluth, Sally
    Vertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.
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    Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.
    (Genes Dev, 1998-07-15) Yang, J; Bardes, ES; Moore, JD; Brennan, J; Powers, MA; Kornbluth, S
    Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.
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    Gene expression disruptions of organism versus organ in Drosophila species hybrids.
    (PLoS One, 2008-08-20) Catron, Daniel J; Noor, Mohamed AF
    Hybrid dysfunctions, such as sterility, may result in part from disruptions in the regulation of gene expression. Studies of hybrids within the Drosophila simulans clade have reported genes expressed above or below the expression observed in their parent species, and such misexpression is associated with male sterility in multigenerational backcross hybrids. However, these studies often examined whole bodies rather than testes or had limited replication using less-sensitive but global techniques. Here, we use a new RNA isolation technique to re-examine hybrid gene expression disruptions in both testes and whole bodies from single Drosophila males by real-time quantitative RT-PCR. We find two early-spermatogenesis transcripts are underexpressed in hybrid whole-bodies but not in assays of testes alone, while two late-spermatogenesis transcripts seem to be underexpressed in both whole-bodies and testes alone. Although the number of transcripts surveyed is limited, these results provide some support for a previous hypothesis that the spermatogenesis pathway in these sterile hybrids may be disrupted sometime after the expression of the early meiotic arrest genes.
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    Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.
    (J Cell Biol, 2005-04-11) Casaletto, Jessica B; Nutt, Leta K; Wu, Qiju; Moore, Jonathan D; Etkin, Laurence D; Jackson, Peter K; Hunt, Tim; Kornbluth, Sally
    Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.
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    Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.
    (J Cell Biol, 1999-01-25) Moore, JD; Yang, J; Truant, R; Kornbluth, S
    Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.
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    Pumilio-mediated Repression of mRNAs in the Early Drosophila Melanogaster Embryo
    (2009) Nomie, Krystle Joli

    Post-transcriptional regulation plays an important role in governing various processes in all organisms. The development of the early embryo of Drosophila melanogaster is governed solely by post-transcriptional mechanisms; therefore, further insights into post-transcriptional regulation can be gained by studying the Drosophila embryo. This thesis addresses the actions of the translational repressor, Pumilio, in regulating two mRNAs during early embryogenesis. First, we examined the ability of Pumilio to regulate the mRNA stability of bicoid, a gene required for Drosophila head development. bicoid mRNA contains the canonical Pumilio recognition site, termed the Nanos response element (NRE), within the 3'UTR. Interestingly, we show that Pumilio binds to the NRE both in vitro and in vivo; however, no physiological significance is associated with this interaction. Furthermore, in pumilio mutant embryos bicoid mRNA stability and translation are unaltered, demonstrating that Pumilio does not regulate bicoid mRNA. Second, Pumilio has been shown to negatively regulate Cyclin B, the cyclin necessary for mitotic entry, in the somatic cytoplasm of the embryo and this repression is alleviated by the PNG Kinase complex through currently unidentified mechanisms. We further investigated the actions of Pumilio in regulating Cyclin B and discovered that the canonical partner of Pumilio, Nanos, is not involved in repressing somatic Cyclin B. Furthermore, we show that the 3'UTR of Cyclin B is not required for the regulation by Pumilio and the PNG Kinase complex. Lastly, through genetic analyses, we conclude that Pumilio may actually act upstream of the PNG Kinase complex to regulate Cyclin B.