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一个基本参数。它是塑造人的基石 组织，细胞大小异常与细胞生物合成中的许多缺陷有关， 组织畸形，组织功能受损。 来自单细胞酵母的研究表明，细胞大小可以通过耦合细胞来控制 从细胞周期的G1期向S期发展，使较小出生的细胞 与较大的出生细胞相比，G1期细胞更长，生长比例更大。然而， 大多数体外动物细胞系的研究没有发现大小耦合的G1/S转变是 尺寸控制的机理。在我的博士后研究中，我开创了一项研究，以解决如何在 活体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偶联其G1/S的时间来控制其大小 过渡到细胞大小，类似于酵母。目前，像元大小信息的大小尚不清楚 给予细胞周期信号网络，以导致细胞大小依赖的G1/S转移率。 在这里，我建议确定细胞大小依赖的分子机制 G1/S通过一套完整的目标过渡。这些目标将检验我的假设，即一个细胞 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 在哺乳动物组织中的控制力。在该奖项的培训阶段，我建议使用 定量活细胞成像结合遗传扰动在两个方面验证了这一假说 哺乳动物上皮细胞模型：(AIM 1)体外肠道有机体；(AIM 2)体内 小鼠的表皮。在本奖项的独立阶段，我提议(目标3)建立 促进基于CRISPR的蛋白质内源标记的实验平台 肠道器官。这将产生进一步研究所需的活细胞成像试剂 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的特征 与组织生理学的其他方面，包括组织张力和细胞骨架动力学。 在一个由导师、合作者和顾问组成的杰出团队的帮助下，我将 培训尖端的活细胞成像，磨练研究技术，并为我的 职业发展。综合起来，提出的科学和训练计划形成了强大的 为了解细胞大小在组织中的作用的独立研究生涯奠定了基础 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.