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.

项目总结/摘要 人类有大约40万亿个细胞，其中约0.1%每天分裂，用于组织维护，伤口愈合， 修复和病原体防御这样的细胞包括干细胞、祖细胞和分化细胞，其通常花费 大多数时间处于非增殖状态（静止，G 0），但当受到刺激时，可以经历一个或多个 细胞分裂（增殖）。在静止和增殖之间做出的这个基本决定 处于细胞周期的G1期。重要的是，沿着决定增殖，细胞必须加载 在G1期有足够的复制起点（起点许可），以在S期无错误地复制它们的DNA。 细胞是如何决定进入静止期的还不完全清楚。关于E2 F和 APC/CCDh 1活性在时间上整合以协调静止和增殖之间的调节 而复制源的许可也仍然没有答案。 Meyer实验室专门从事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报告基因，实验室可以回答各种生物学问题， 非常高的分辨率。该项目将使用CDK 1/2，APC/CCDh 1， CRL 4Cdt 2和E2 F理解S期进入、G 0进入和G1中的起源许可的信令动态。 该提议的目标是比较E2 F活性和APC/CCDh 1活性之间的动态协同作用， 静态和循环单细胞，并确定细胞如何维持一个起源许可期。的目的 我们的建议是显示两个S期进入信号通路的存在，由E2 F之间的协同作用控制。 和APC/CCDh 1活性以及这种协同作用如何控制起源许可和静止进入。我 中心假设是E2 F和APC/CCDh 1活性之间的时间相互作用是主要的调节因子 在扩散和静止之间的决定，并确保适当的原产地许可，以防止DNA S期损伤。我计划用以下具体目标来检验这一假设： 1.了解E2 F和APC/CCDh 1活性在调节增殖-静止中的相互作用 决定 2.确定E2 F和APC/CCDh 1活性时序在起始许可和DNA复制保真度中的功能。 预计该项目的成功完成将显示APC/CCDh 1和E2 F的多方面作用 协同作用。这个项目将解决细胞如何忠实地协调许可和DNA复制的历史之谜 通过利用细胞群体中存在的显著的天然异质性。此外，完成这一 该项目将为癌细胞如何逃避靶向DNA复制的化疗提供重要见解 通过进入休眠的静止状态以及健康细胞如何能够维持组织的静止群体 修复与成长