Single-cell dynamics of E2F and APC/CCdh1 coordination that regulate the proliferation-quiescence decision

项目摘要/摘要 人类大约有40万亿个细胞，其中约0.1%的细胞每天分裂用于组织维护、创伤 修复和病原体防御。这类细胞包括干细胞、祖细胞和分化的细胞，这些细胞通常 它们大部分时间处于非增殖状态(静止期，G0)，但当受到刺激时，可以经历一个或多个 几轮细胞分裂(增殖)。在停滞和扩散之间做出了这一根本决定 处于细胞周期的G1期。重要的是，在做出增殖决定的同时，细胞必须加载 在G1期有足够的复制起始点(起始点许可)，以便在S期无差错地复制它们的DNA。 细胞是如何做出进入静止状态的决定的，目前还不完全清楚。E2F和E2F如何 APC/CCdh1活性在时间上整合，以协调静止和增殖之间的调节 而复制起源的许可问题也仍然没有答案。 迈耶实验室专门使用活细胞成像数据的单细胞分析。通过利用最近 开发了关键细胞周期蛋白的荧光记者，该实验室可以回答各种生物学问题 非常高的分辨率。本项目将使用CDK1/2，APC/CCdh1， CRL4Cdt2和E2F，以了解G1期进入S、G0进入和原产地许可的信号动力学。 该提案的目标是比较E2F活动和APC/CCdh1活动之间的动态协同作用 静止和循环单个细胞，并确定细胞如何维持原产地许可期。这样做的目的是 建议证明存在两条受E2F协同作用控制的S相进入信号通路 以及单细胞中的APC/CCdh1活性，以及这种协同作用如何控制起源许可和静止进入。我的 中心假设是E2F和APC/CCdh1活动之间的时间相互作用是主要调节因素 在增殖和静止之间做出决定，并确保适当的原产地许可以防止DNA S阶段的破坏。我计划用以下具体目标来检验这一假设： 1.了解E2F和APC/CCdh1活性在调节细胞增殖停滞中的相互作用 决定。 2.确定E2F和APC/CCdh1活动时序在原产地许可和DNA复制保真度中的作用。 该项目的成功完成预计将显示APC/CCdh1和E2F的多方面作用 协同效应。这个项目将解开一个关于细胞如何忠实地协调许可和DNA复制的历史谜团 通过利用细胞群体中存在的显著的自然异质性。此外，这项工作的完成 该项目将为癌细胞如何逃避针对DNA复制的化疗提供关键的见解 通过进入休眠的静止状态以及健康的细胞如何能够维持组织的静止群体 修复和成长。

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.