Bipolar spindles assembled normally by 5-6 h post-GVBD and chromosome alignment at the spindle equator in Cep55-depleted oocytes was similar to that in mock-depleted controls ( Fig. 1G Fig. S1, Movies 1 and 2). To investigate this, we undertook timelapse imaging of fluorescently labelled spindles and chromosomes in live oocytes ( Wei et al., 2018). Given the requirement of Cep55 for mitotic abscission ( Fabbro et al., 2005 Zhao et al., 2006), we presumed that PBE inhibition following Cep55 knockdown was the result of a late post-anaphase I defect. Results are representative of at least three independent experiments. (H) Chromosome spreads from mock- and Cep55-depleted oocytes prepared at 20 h post-GVBD. (G) Representative timelapse images of spindles and chromosomes in live mock-depleted ( n=54 oocytes) and Cep55-depleted ( n=65 oocytes) oocytes during MI (see Movies 1 and 2).
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PBE rates were determined at 20 h post-GVBD. (F) PBE rates in Cep55-depleted oocytes with or without co-expression of Cep55 from injected Cep55 cRNA. (D,E) Rates of GVBD and polar body extrusion (PBE) in mock- and Cep55-depleted oocytes.
(C) Band intensities of Cep55 on blot were quantified and normalised to values found in controls. (B) Samples (50 oocytes) of Cep55-specific siRNA-injected oocytes were immunoblotted along with control siRNA-injected (mock-depleted) oocytes at 7 h post-GVBD for Cep55 and vinculin. Cep55 levels were low at GV and GVBD, but increased markedly by late MI. (A) Samples (50 oocytes) at the GV stage, GVBD and late MI (7 h post-GVBD) were immunoblotted for Cep55 and vinculin. Thus, by controlling the timing of APC-Cdc20 activation, the mitotic SAC has stringent control over proteolysis and, by extension, anaphase timing, to prevent aneuploidy.Ĭep55 depletion inhibits anaphase I.
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APC-Cdc20-mediated proteolysis is therefore the principal anaphase driver during mitosis ( Meadows and Millar, 2015 Sullivan and Morgan, 2007). Inhibitory Cdk1 phosphorylation reinforces mitotic exit in cellular extracts, but this involves altered proteolysis ( D'Angiolella et al., 2007 Forester et al., 2007). In addition to cyclin B1 availability, Cdk1 activity is controlled by inhibitory Wee1/Myt1 kinase (Wee1B in oocytes)-mediated phosphorylation of Cdk1 residues Thr14 and Tyr15, which are removed by Cdc25 phosphatases (Cdc25B in oocytes) ( Han et al., 2005 Malumbres, 2014 Adhikari and Liu, 2014 Lincoln et al., 2002). As separase is also inhibited by cyclin-dependent kinase 1 (Cdk1) ( Stemmann et al., 2001), anaphase also requires APC-Cdc20-mediated destruction of the Cdk1 activator, cyclin B1 ( Pesin and Orr-Weaver, 2008 Wolf et al., 2006). Activation of separase occurs following release from its inhibitory chaperone, securin ( Waizenegger et al., 2002), brought about through securin proteolysis orchestrated by the anaphase-promoting complex (APC) acting in concert with its cell-division cycle protein 20 (Cdc20) coactivator ( Homer, 2013 Pesin and Orr-Weaver, 2008 Herbert et al., 2003). In both mitosis and meiosis, anaphase occurs when the chromosomal glue, cohesin, is cleaved by the cysteine protease, separase ( Hauf et al., 2001 Kudo et al., 2006 Terret et al., 2003). To prevent aneuploidy, the timing of anaphase must be finely tuned such fine-tuning is the responsibility of the spindle assembly checkpoint (SAC) ( Foley and Kapoor, 2013). Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity.
In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1).
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