Mitotic entry and exit: quantifying the dynamics of endogenous cyclin B1-EGFP protein in human proliferating cells

Supervisor: Dr. Amit Tzur

Growth of all organisms depend on orderly duplication and distribution of their chromosomes to the newly forming daughter cells in a multi-phase process called the cell cycle, a tightly regulated process, essential to avoid genome instability and other related dysfunctionalities resulting from lack of regulation. The Master regulators of the cell cycle are Cyclins and Cyclin Dependent Kinases (CDK`s) proteins, promoting cell cycle progression. Cyclin B1-CDK1 complex dynamics promotes mitotic (M phase- last phase of the cell cycle) entry and exit.

Cyclin B1 effect on the progression through the M phase has thus far been mostly shown using ectopically overexpressed protein attached to GFP under inducible non-physiological expression. The overexpression might create strong artifacts in intracellular protein trafficking and protein-mediated processes and might not accurately represent the expression pattern of the endogenous protein. Therefore, we have generated human cellular systems that have been genetically edited utilizing CRISPR-Cas9 to express EGFP within the endogenous c-terminal portion of cyclin B1 protein, which is expressed at their physiological level and their regulation over them is preserved. These cells are more likely to retain the characteristic expression profile of the endogenous Cyclin B1 in the cell.

Our purpose is to understand cyclin B1-related processes orchestrating cell cycle decisions, using quantitative single-cell microscopy. since cyclin B1 is essential for mitotic exit, we hope to understand dynamics and threshold values of this cyclin at which cell cycle decisions are orchestrated in mammalian cells.