Determining the molecular mechanism controlling cell size in mammalian epithelia

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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.