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.
Controlling the adult stem cell's cell cycle is very important to tissue integrity, both during homeostasis and regeneration. I will study how the G1/S transition, an important checkpoint that marks an irreversible decision to enter the cell cycle, is regulated in the adult stem cells of the intestine. The molecular details of how adult stem cells regulate their cell cycle are important not only for understanding how tissues regulate cell turnover, but also how these mechanisms may become dysregulated in cancers where transformed adult stem cells can initiate and sustain cancerous growth.
