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.