Determining the molecular mechanism controlling cell size in mammalian epithelia

项目摘要 细胞大小是组织生理学的基本参数。它是塑造 组织，并且异常的细胞大小与细胞生物合成中的许多缺陷有关， 组织畸形和受损的组织功能。 单细胞酵母的工作表明，细胞大小可以通过偶联细胞来控制 生长到从细胞周期的G1期到S期的进展，使得较小的出生细胞花费 更长，在G1期生长更成比例。但 大多数体外动物细胞系的研究并没有将大小偶联的G1/S转变确定为细胞周期的一个重要特征。 尺寸控制机制。在我的博士后研究中，我开创了一项研究， 体内小鼠上皮控制其细胞大小。与大多数体外研究形成鲜明对比的是， 我发现体内的表皮干细胞通过结合G1/S的时间来控制它们的大小 转变成细胞大小，类似于酵母。目前，尚不清楚细胞大小信息是如何被 细胞周期信号传导网络，导致细胞大小依赖性的G1/S转换速率。 在这里，我建议确定细胞大小依赖的分子机制， 通过一套综合目标实现G1/S过渡。这些目标将验证我的假设， 视网膜母细胞瘤蛋白（RB）通路的大小依赖性调节是G1/S大小的基础 控制哺乳动物组织。在这个奖项的培训阶段，我建议使用 定量活细胞成像结合遗传扰动，以检验这一假设在两个 哺乳动物上皮细胞模型：（目的1）离体肠类器官;和（目的2）体内 小鼠表皮在这个奖项的独立阶段，我建议（目标3）建立 一个实验平台，以促进基于CRISPR的蛋白质内源性标记， 肠类器官这将产生进一步研究所需的活细胞成像试剂 表征细胞周期和细胞大小如何耦合，以及细胞大小如何相互作用 组织生理学的其他方面，包括组织张力和细胞骨架动力学。 在优秀的导师、合作者和顾问团队的帮助下，我将 培训尖端的活细胞成像，磨练研究技术，并获得技能，我 职业发展。共同提出的科学和培训计划，形成了一个强大的 在理解细胞大小在组织中的作用的独立研究生涯的基础 形态发生和维持

Project Summary Cell size is a fundamental parameter of tissue physiology. It is the building block in shaping tissues, and aberrant cell size is associated with numerous defects in cellular biosynthesis, tissue malformation, and impaired tissue function. Work from unicellular yeast showed that cell size can be controlled by coupling cell growth to progression from G1 to S phase of the cell cycle, so that smaller-born cells spend longer and grow proportionately more in G1 phase compared to larger-born cells. However, the majority of studies of in vitro animal cell lines did not identify size coupled G1/S transition as the mechanism of size control. In my postdoctoral studies, I pioneered a study to address how an in vivo mouse epithelium controls its cell size. In striking contrast to the majority of studies in vitro, I found that epidermal stem cells in vivo control their size by coupling the timing of their G1/S transition to cell size, similar to yeast. Currently, it is unknown how cell size information is imparted to cell cycle signaling network to result in a cell size-dependent G1/S transition rate. Here, I propose to determine the molecular mechanism underlying cell size-dependent G1/S transition through an integrated set of aims. These aims will test my hypothesis that a cell size-dependent modulation of the retinoblastoma protein (RB) pathway underlies G1/S size control in mammalian tissues. During the training phase of this award, I propose to use quantitative live-cell imaging combined with genetic perturbation to test this hypothesis in two models of mammalian epithelia: (Aim 1) ex vivo intestinal organoids; and (Aim 2) the in vivo mouse epidermis. During the independent phase of this award, I propose to (Aim 3) establish an experimental platform to facilitate CRISPR-based endogenous tagging of proteins in intestinal organoids. This will generate live-cell imaging reagents necessary for further characterization of how cell cycle and cell size are coupled, as well as how cell size interacts with other aspects of tissue physiology, including tissue tension and cytoskeletal dynamics. With the help of an outstanding team of mentors, collaborators, and consultants, I will train in cutting-edge live-cell imaging, hone research techniques, and acquire skills for my career development. Together, the proposed scientific and training program form a strong foundation for an independent research career in understanding the role of cell size in tissue morphogenesis and maintenance.