Abstract PR04: Characterizing the sequence of cell-cycle events during proliferation and quiescence

Abstract

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR04) of the Conference Proceedings. Although the cell-cycle field has identified most of the key biochemical events involved in cell-cycle progression, we do not have a clear picture of overall cell-cycle dynamics. The prevalence of cell-to-cell variability is increasingly appreciated, but this heterogeneity is blurred when bulk analysis approaches are used, potentially resulting in incorrect interpretations of biological data. Using a live-cell sensor for CDK2 activity, we recently discovered that unperturbed cycling cells bifurcate into two subpopulations as they exit mitosis: one subpopulation begins to immediately increase CDK2 activity (CDK2inc cells), whereas the other subpopulation turns off CDK2 activity and enters a period of quiescence (CDK2low cells). These cells can later re-enter the cell cycle by building up CDK2 activity (CDK2low->inc). Here we use single-cell time-lapse microscopy followed immediately by immunofluorescence to determine the dynamics of over 15 cell-cycle proteins in unperturbed cycling mammary epithelial cells. By sorting individual cells by their CDK2inc versus CDK2low trajectory, and aligning them as a function of time-since-anaphase, we identified several unexpected differences in protein levels (or modification states) in the two subpopulations. Moreover, we identified certain proteins whose concentrations steadily decrease the longer the cells are in quiescence, suggesting the existence of multiple levels of quiescence depth. Cells can also emerge from quiescence by engaging CDK2 activity. When we align these CDK2low->inc cells to the time of initial increase in CDK2 activity, we recapitulate the cell-cycle dynamics seen in the CDK2inc cells. These results argue that cell-cycle commitment is not a point in time, but rather a molecular event (CDK2 activation) that varies temporally across cells. In summary, our single-cell measurements provide a critical resource for the field by describing the dynamics and sequence of many key cell-cycle proteins in unperturbed proliferating and quiescent cells. This abstract is also being presented as Poster A05. Citation Format: Sara Gookin, Sabrina Leigh Spencer. Characterizing the sequence of cell-cycle events during proliferation and quiescence. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR04.