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

E2F 和 APC/CCdh1 协调的单细胞动力学调节增殖-静止决策

• 批准号: 10676530
• 负责人: Samsara Upadhya
• 金额: $ 4.77万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2026-04-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676530
• 关键词: Binding; Biological; CCNE1 gene; CDC2 gene; CDC7 gene; CDK2 gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cell division; Cells; Cellular biology; Chromatin; Cyclin A; DNA; DNA Damage; DNA biosynthesis; DNA replication origin; Data; Decision Making; Defect; Development; Ensure; Equilibrium; G1 Phase; GMNN gene; Genes; Genetic; Genetic Transcription; Goals; Growth; Heterogeneity; Human; Immunofluorescence Immunologic; Knowledge; Learning; Licensing; Maintenance; Malignant Neoplasms; Mammals; Measures; Methods; Mitosis; Modeling; Molecular; Monitor; Pathway interactions; Phosphotransferases; Population; Positioning Attribute; Proliferating; Proteins; Regulation; Regulatory Pathway; Replication Initiation; Replication Origin; Reporter; Research Personnel; Resolution; Role; S phase; Signal Pathway; Signal Transduction; Stains; Testing; Time; Tissues; Work; cancer cell; cell regeneration; chemotherapy; helicase; human embryonic stem cell; inhibitor; insight; live cell imaging; loss of function; novel; pathogen; pharmacologic; premature; prevent; progenitor; programs; single cell analysis; small molecule; stem; synergism; targeted treatment; tissue regeneration; tissue repair; ubiquitin-protein ligase; wound healing

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.

项目摘要/摘要 人类大约有40万亿个细胞，其中约0.1％每天分裂以维持组织，伤口 修复和病原体防御。这些细胞包括茎，祖细胞和分化细胞，通常花费 他们的大部分时间都处于非增殖状态（静止，G0），但是当受到刺激时，可能会经历一个或多个 细胞分裂（增殖）的回合。做出了静止和增殖之间的基本决定 在细胞周期的G1阶段。重要的是，在决定增殖的决定之后，细胞必须加载 G1期间复制的足够起源（原点许可）可以在S阶段复制其DNA而不会误差。 细胞如何做出进入静止的决定尚未完全理解。关于如何和E2F的关键问题 APC/CCDH1活性在时间上整合以协调静止和增殖之间的调节 复制起源的许可也尚未得到答复。 Meyer Lab专门研究实时成像数据的单细胞分析。通过最近利用 开发的荧光记者用于关键细胞周期蛋白，实验室可以通过 非常高的分辨率。该项目将利用荧光活动记者用于CDK1/2，APC/CCDH1， CRL4CDT2和E2F了解G1中的S相输入，G0输入和原点许可的信号动力学。 该建议的目的是比较E2F活动与APC/CCDH1活动之间的动态协同作用 静止和循环单细胞，并确定细胞如何维持原点许可周期。这个目的 建议是显示由E2F之间协同作用的两个S相进入信号通路的存在 单个细胞中的APC/CCDH1活性以及该协同作用如何控制原点许可和静止输入。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器 增殖与静止之间的决定，并确保适当的起源许可以防止DNA s阶段的损坏。我计划以以下特定目的检验这一假设： 1。了解E2F和APC/CCDH1活性之间的相互作用在调节增殖质量方面 决定。 2。确定原点许可和DNA复制保真度中E2F和APC/CCDH1活性时间的功能。 预计该项目的成功完成将显示APC/CCDH1和E2F的多方面角色 协同作用。该项目将解决细胞如何忠实地协调许可和DNA复制的历史性谜团 通过利用细胞种群中存在的显着自然异质性。此外，完成此操作 项目将为癌细胞如何逃避靶向DNA复制的化学疗法，提供至关重要的见解 通过进入休眠状态以及健康细胞如何能够维持组织的静态种群 维修和生长。