Our lab is interested in understanding how cells make fate decisions, such as the decision to enter the cell cycle. Cells are thought to execute the decision to proliferate and commit to the cell cycle in G1 phase. Despite the identification of many proteins involved with cell cycle entry, it has remained a long- standing question how these proteins function together to form a logical commitment mechanism. Major challenges for resolving this question have been a reliance on cell synchronization, bulk cell measurements, endpoint rather than dynamic measurements, and significant cell-to-cell variability in the timing of cell cycle events, all of which can obscure molecular events due to poor temporal resolution.
To address these challenges, we used an automated live, single-cell assay to track asynchronously dividing cells with high time resolution. This approach allowed us to monitor thousands of single cells and watch in real-time as they entered and exited the cell cycle. We developed a novel analysis for measuring the activity of one of the key players in regulating G1 progression, the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C). The APC/C biosensor we characterized is based on the previously reported Geminin FUCCI reporter, which was designed primarily to qualitatively mark the start of S phase. However, we developed a way to use the Geminin reporter for quantitative dynamic measurements of APC/C activity, an approach that can be broadly applied to build live-cell reporters for other ubiquitin ligases in the future. Using the live-cell APC/C reporter in nontransformed human mammary epithelial cells (MCF10A), we found that APC inactivates rapidly and immediately prior to the start of S phase. The rapid inactivation kinetics were nearly identical in every single cell measured across multiple cell lines, suggesting a robust bistable mechanism underlies APC/C inactivation. We further showed that Cyclin E and Cyclin dependent kinase 2 (Cdk2) trigger APC/C inactivation and that the protein Early-mitotic inhibitor 1 (Emi1) greatly accelerates APC/C inactivation and makes it irreversible. Thus, Cdk2 and Emi1 collaborate to make APC/C inactivation reliable and irreversible, two characteristics that are well suited for regulating cell cycle progression.
Given the robust, bistable inactivation mechanism regulating APC/C, and that E3 ligases are well suited for mediating irreversible decisions, we hypothesized that APC/C inactivation may underlie the true cell cycle commitment mechanism. Our live-cell approach allowed us to acutely perturb cells that were mitogen- insensitive but had not yet inactivated the APC/C and entered S phase. Surprisingly, when we introduced the cells to various types of stress we found that cells reversed back to a mitogen-sensitive quiescent state and inactivated Cdk2. APC/C inactivation exhibited hysteresis, as the activation of Cdk2 and inactivation of APC/C both became resistant to stress only after APC/C had been inactivated. These results demonstrated that cells only commit to the cell cycle after inactivating the APC/C just prior to the start of S phase, and imply that throughout G1 phase competition between mitogen signaling and stress signaling can trigger an exit back to quiescence until APC/C is inactivated.
The current focus of the lab is aimed at gaining a detailed understanding of the regulatory circuits generating irreversibility in the G1/S transition in normal cells, how these circuits are rewired in the context of mutations and cancer, and more generally develop and apply tools to understand how E3 ligases underlie irreversible transitions in diverse biological contexts. We are actively recruiting new members of the lab, so if you would like to know more about current research projects, please contact Steve Cappell.