Effects of Suppressed Cyclin B1 Protein Accumulation on Timing of Cell Cycle Events in Xenopus laevis Cell-Free Extract System

"The cyclin-dependent kinase (Cdk1) oscillator has been widely characterized in bulk xenopus egg extract experiments1,2. It was established in a published study that hysteresis, the toggle-like switching of Cdk1 activity by phosphorylation reactions with cyclin B1, drives the cell cycle irreversibly forward2. Cyclin B1 is shown to have a minimum concentration threshold that is reached before the progression from interphase into mitosis. Yet the timing of cell cycle events under conditions of suppressed cyclin B1 synthesis have not been hitherto uncovered. Bulk cytosolic extracts have been a key tool for understanding the early embryonic cell cycle. However, they are far from an in vivo model of Cdk1 temporal dynamics. As a result, water-in-oil microemulsion droplets containing Xenopus laevis cytosolic egg extract served as a simplified model of early embryos in this study. This cell-free extract system was applied in conjunction with a Cdk1-EV Forster Resonance Energy Transfer (FRET) Biosensor3 to uncover the characteristics of Cdk1 dynamics and the progression of cell cycle in the presence of a PEST-tagged cyclin B1. It is strongly suggested from the data collected that the suppression of cyclin B1 accumulation by PEST tagging leads to a delay in the switch from inactive to active Cdk1, thereby prolonging interphase and delaying mitotic entry. Furthermore, the study employed two separate DNA vector systems, pCS2+ and pcDNA3.1(+), for the purposes of molecular cloning. After the initial round of experimentation, it was noted that the timing of cell cycle events in droplets differs depending on the vector system used to produce the exogenous cyclin B1 mRNA. After a synchronous first peak in Cdk1 activity corresponding to mitosis, pcDNA3.1(+) mRNA-derived droplets exhibited delayed, asynchronous second peaks and drastically increased periods compared to the pCS2+ mRNA-derived counterparts. The inconsistency between vector systems indicates that the pcDNA3.1(+) mRNA is not well-suited for the xenopus egg extract environment and is likely less stable than pCS2+ mRNA.HonorsBiophysicsUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/191226/1/oblanch_-_Owen_Blanchard.pd