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

项目摘要 调节成体干细胞的细胞周期是维持组织完整性的关键。单元格 不可逆转地致力于在G1/S过渡时进行划分；然而，G1/S过渡是如何调节的 成体干细胞周期目前尚不清楚。我建议研究分子基础是如何 G1/S转换是在肠道干细胞中调节的，具有生理上较高的 扩散。我将使用肠道器官培养作为ISC增殖的体外模型和 肠道内环境平衡。有机化合物特别适合于这个问题，因为它们是 易于长期进行活细胞成像，同时仍保留了许多干细胞生理学 它们在体内的对应物。我的初步研究表明，ISCs对 抑制Cyclin D/CDK4/6，这是一组Cyclin/CDK复合体，是细胞周期的关键调节因子 G1/S过渡。我建议使用活体三维成像肠道器官来 研究G1/S转变如何调节间质干细胞细胞周期和器官稳态。(目标1) 与巴塞尔弗里德里希·米歇尔研究所的Prisca Liberali博士合作，我将学习 并使用光片显微镜对表达的有机物进行长期活细胞成像 细胞周期和细胞干性的报告者。这使我能够直接测量细胞周期动力学 ICSS中CDK4/6被抑制。为此，我还将开发计算图像分析 分析大规模四维图像数据的工具。(目标2)我将研究分子 ISCs中G1/S转变的机制。为了测试细胞周期表型 CDK4/6抑制中的ISCS是由其已知底物--视网膜母细胞瘤蛋白(Rb)--介导的 会特异性地干扰Rb中Cyclin D结合的基序，看看Rb是否磷酸化 特别是通过Cyclin D/CDK4/6调控ISC G1/S是必要的。此外，我会 测试Cyclin D和Rb的表达水平是否也将G1/S转变与ISCs的细胞大小挂钩， 通过干扰CDK4/6、Cyclin D和Rb，I的表达水平，并检测ISC细胞 大小分布发生变化。最后，我将调查下游细胞的命运是否受到影响 通过经历CDK4/6抑制引起的细胞周期中断的ISCs，来了解细胞类型如何 动态平衡也可能受到G1/S转变的影响。这个项目将阐明 成体干细胞用于调节其G1/S转换的机制是 包括发育生物学和癌症。

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.