Determining how the G1/S cell cycle transition regulates the homeostasis of adult intestinal stem cells

项目摘要 成体干细胞的细胞周期调控对维持组织完整性至关重要。细胞 在G1/S转换时不可逆转地承诺分裂;然而，G1/S转换如何调节 成体干细胞周期目前尚不清楚。我打算研究 G1/S转换在肠干细胞（ISC）中受到调节，其具有生理上高的转化率。 增殖我将使用肠类器官培养物作为ISC增殖的离体模型， 肠内稳态类器官特别适合这个问题，因为它们是 适合长期活细胞成像，同时仍然保留大部分干细胞生理功能 它们的体内对应物。我的初步研究表明，ISCs对 抑制细胞周期蛋白D/CDK 4/6，一组细胞周期蛋白/CDK复合物，是细胞周期蛋白的关键调节因子。 G1/S转换。我建议使用活体三维成像肠道类器官， 研究G1/S转换如何调节ISC细胞周期和类器官稳态。(Aim第一章 在与巴塞尔的弗里德里希·米舍尔研究所的普里斯卡·利伯拉里博士的合作中，我将学习 并使用光片显微镜对表达 细胞周期和细胞干细胞报告基因。这让我可以直接测量细胞循环动力学 在ICSs中，CDK 4/6抑制期间。为了这个目标，我也将开发计算图像分析 分析大规模四维图像数据的工具。(Aim（2）研究分子 这是ISC中G1/S转换的基础机制。为了测试在细胞周期中观察到的细胞表型， CDK 4/6抑制期间的ISCs由其已知底物视网膜母细胞瘤蛋白（RB）介导， 将特异性干扰RB中与细胞周期蛋白D结合的基序，以观察RB磷酸化 特别是通过细胞周期蛋白D/CDK 4/6是必要的适当ISC G1/S控制。此外，我将 测试细胞周期蛋白D和RB的表达水平是否也将G1/S转变与ISCs中的细胞大小相关联， 通过干扰CDK 4/6、Cyclin D和RB，I的表达水平， 尺寸分布变化。最后，我将研究下游细胞命运是否受到影响， 由于CDK 4/6抑制而经历细胞周期破坏的ISC，以观察细胞类型 体内平衡也可能受到G1/S转换的影响。该项目将阐明 成体干细胞用于调节其G1/S转换的机制，这是成体干细胞研究中的一个重要问题。 发育生物学和癌症。

Project Summary Regulation of the cell cycle of adult stem cells is crucial to maintaining tissue integrity. Cells irreversibly commit to division at the G1/S transition; however, how G1/S transition regulates adult stem cell cycles is currently unknown. I propose to study the molecular basis of how the G1/S transition is regulated in intestinal stem cell (ISC), which have physiologically high rates of proliferation. I will use intestinal organoid cultures as ex vivo models of ISC proliferation and intestinal homeostasis. Organoids are especially suited to this question because they are amenable to long-term live-cell imaging, while still retaining much of the stem cell physiology of their in vivo counterparts. My preliminary studies suggest that ISCs are especially sensitive to inhibition of Cyclin D/CDK4/6, a group of cyclin/CDK complexes that are key regulators of the G1/S transition. I propose to use live three-dimensional imaging intestinal organoids to investigate how the G1/S transition regulates ISC cell cycle and organoid homeostasis. (Aim 1) In collaboration with Dr. Prisca Liberali in the Friedrich Miescher Institute in Basel, I will learn and use light-sheet microscopy to perform long-term live-cell imaging on organoids expressing cell cycle and cell stemness reporters. This allows me to directly measure cell cycling dynamics in ICSs during CDK4/6 inhibition. For this aim, I will also develop computational image analysis tools to analyze large-scale 4-dimensional image data. (Aim 2) I will investigate the molecular mechanism that underlies G1/S transition in ISCs. To test that the cell cycle phenotype seen in ISCs during CDK4/6 inhibition is mediated by its known substrate, retinoblastoma protein (RB), I will specifically interfere with a motif in RB bound by Cyclin D, to see if RB phosphorylation specifically by Cyclin D/CDK4/6 is necessary for proper ISC G1/S control. Furthermore, I will test if expression levels of Cyclin D and RB also couple the G1/S transition to cell size in ISCs, by perturbing the expression levels of CDK4/6, Cyclin D, and RB, I and examining how ISC cell size distribution changes. Finally, I will investigate whether downstream cell fates are affected by ISCs that experience cell cycle disruption due to CDK4/6 inhibition, to see how cell-type homeostasis may also be affected by the G1/S transition. This project will elucidate the mechanisms used by adult stem cells to regulate their G1/S transition, an important problem in both developmental biology and cancer.