Cyclin B1 regulation and APC/C processivity in mouse oocyte meiosis I

Abstract

Chromosome alignment is orchestrated by the activity of CDK1 bound to its coactivator cyclin B1. Equally important, chromosome segregation is orchestrated by termination of CDK1 activity, driven by cyclin B1 destruction. Both events must be precisely timed by APC/C (Anaphase Promoting Complex/Cyclosome) activity, an E3 ligase that targets cell cycle proteins for destruction via the ubiquitin-proteasome pathway. In mitosis, this system is rapid, robust, and well-studied. Critically, through oocyte chromosome alignment, CDK1 and APC/C activities must be regulated differently, over substantially longer time frames. Specifically, the APC/C must first be dampened to prevent cyclin B1 destruction and CDK1 activity loss as chromosomes begin aligning. This is followed by a period of non-CDK1-bound cyclin B1 destruction during final alignment stages, followed by CDK1-bound-cyclin B1 destruction once fully aligned. This ordering is vital to minimise errors that otherwise cause the cell to arrest (infertility) or produce an embryo that is incompatible with life (miscarriage). However, little is published regarding the molecular mechanism of cyclin B1 targeting in oocytes, or indeed the wider landscape of any APC/C substrate ordering. To address this, I have expressed fluorescent versions of cyclin B1 and APC/C substrates in live mouse oocytes, utilising time-lapse fluorescence microscopy to map their levels and movements through meiosis I. Additionally, I mutated short linear motifs within these substrates, as well as APC/C subunit knockdowns to obtain mechanistic insight into substrate targeting. I reveal that cyclin B1 harbours a motif which boosts its affinity for a specific form of the APC/C during chromosome alignment. This form of the APC/C does not exist globally across the cell, but likely in high priority areas across this spindle zone. This and other insights, describe novel aspects of cell cycle regulation that are critical to produce healthy eggs