Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction.

Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called "anaphase problem" is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore-microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear. Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/C(Cdc20) substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/C(Cdc20) activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10-15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/C(Cdc20) in late anaphase helps to keep cyclin B1 levels low.

LePLDbeta1 activation and relocalization in suspension-cultured tomato cells treated with xylanase.

Phospholipase D (PLD) has been implicated in various cellular processes including membrane degradation, vesicular trafficking and signal transduction. Previously, we described a PLD gene family in tomato (Lycopersicon esculentum) and showed that expression of one of these genes, LePLDbeta1, was induced by treatment with the fungal elicitor xylanase. To further investigate the function of this PLD, a gene-specific RNAi construct was used to knock down levels of LePLDbeta1 transcript in suspension-cultured tomato cells. Silenced cells exhibited a strong decrease in xylanase-induced PLD activity and responded to xylanase treatment with a disproportionate oxidative burst. Furthermore, LePLDbeta1-silenced cell-suspension cultures were found to have increased polyphenol oxidase activity, to secrete less of the beta-d-xylosidase LeXYL2 and to secrete and express more of the xyloglucan-specific endoglucanase inhibitor protein XEGIP. Using an LePLDbeta1-green fluorescent protein (GFP) fusion protein for confocal laser scanning microscopy-mediated localization studies, untreated cells displayed a cytosolic localization, whereas treatment with xylanase induced relocalization to punctuate structures within the cytosol. Possible functions for PLDbeta in plant-pathogen interactions are discussed.