Project Summary/Abstract Humans have approximately 40 trillion cells and ~ 0.1% of them divide every day for tissue maintenance, wound repair, and pathogen defense. Such cells include stem, progenitor and differentiated cells that typically spend most of their time in a non-proliferative state (quiescence, G0) but when stimulated can undergo one or more rounds of cell division (proliferation). This fundamental decision between quiescence and proliferation is made in the G1 phase of the cell cycle. Importantly, along with making the decision to proliferate, cells must load sufficient origins of replication (origin licensing) during G1 to replicate their DNA without error during S phase. How cells make the decision to enter quiescence is not fully understood. Critical questions of how E2F and APC/CCdh1 activities are temporally integrated to coordinate the regulation between quiescence and proliferation and the licensing of origins of replication also remain unanswered. The Meyer lab specializes in the use of single-cell analysis of live-cell imaging data. By utilizing recently developed fluorescent reporters for key cell cycle proteins, the lab can answer various biological questions with very high resolution. This project will employ the use of fluorescent activity reporters for CDK1/2, APC/CCdh1, CRL4Cdt2 and E2F to understand signaling dynamics of S phase entry, G0 entry and origin licensing in G1. The goal of this proposal is to compare the dynamic synergy between E2F activity and APC/CCdh1 activity in quiescent and cycling single cells and identify how cells maintain an origin licensing period. The objective of this proposal is to show the existence of two S phase entry signaling pathways controlled by synergy between E2F and APC/CCdh1 activity in single cells and how this synergy controls origin licensing and quiescence entry. My central hypothesis is that the temporal interplay between E2F and APC/CCdh1 activities is the primary regulator of the decision between proliferation and quiescence and, ensures proper origin licensing to prevent DNA damage in S phase. I plan to test this hypothesis with the following specific aims: 1. Understand the interplay between E2F and APC/CCdh1 activities in regulating the proliferation-quiescence decision. 2. Determine the function of E2F and APC/CCdh1 activity timing in origin licensing and DNA replication fidelity. The successful completion of this project is expected to show the multifaceted roles of APC/CCdh1 and E2F synergy. This project will resolve a historic enigma of how cells faithfully coordinate licensing and DNA replication by utilizing the remarkable natural heterogeneity that exists in cell populations. Further, the completion of this project will provide crucial insights into how cancer cells may evade chemotherapies that target DNA replication by entering a dormant quiescent state and how healthy cells are able to maintain quiescent populations for tissue repair and growth.

Project Narrative The maintenance of quiescent cell populations in the body is essential to repair wounds, maintain healthy tissue and for pathogen defense, and once cells enter quiescence, they must also be able to re-enter the cell cycle and divide again. This project will investigate multifaceted cellular pathways regulated by E2F and APC/CCdh1 activity and how they synergize to create two pathways into S phase that must be suppressed to allow cells to enter quiescence, and how this synergy allows for the licensing of origins of DNA replication in G1. Completion of this study in single cells will support the identification of potential treatment targets for quiescent cancer cell populations and pro-proliferative treatments for increased cellular regeneration.