Every type of cell maintains a specific size range to best perform its physiological functions. But, how does a cell know how big it is? This is a question fundamental to the biology of all cells and is poorly understood on a mechanistic level. Recently, the Skotheim lab found that, during the first gap phase (G1), the model organism budding yeast dilutes a cell cycle inhibitor Whi5, a protein that prevents the transition from G1 to S phase. Through the dilution of the Whi5 inhibitor, yeast cells ?sense? size by elegantly linking the extent of cell growth in G1 with the decision to irreversibly enter S phase and divide. Critical to its function as a size ?sensor?, Whi5 is not synthesized in proportion cell size, which distinguishes it from nearly every other yeast protein. Therefore, its synthesis must be subjected unique regulation, which is not understood. Moreover, the discovery of inhibitor dilution suggests that a universal feature of some size regulating factors may be that their concentration changes in cells of different sizes. My proposal aims to 1) interrogate the WHI5 locus using genetics and proteomics to determine the mechanism by which it promotes size-independent synthesis, and 2) screen the eukaryotic proteome for other proteins whose concentrations change with changes in cell size.
(Aim 1) Our preliminary data indicate that the size-independent synthesis of Whi5 protein is due to size-independent transcription. Because little is known about transcriptional regulation at the WHI5 locus, I aim to determine the promoter regions responsible for size-independent WHI5 expression using a series of genetic deletions and substitutions within the WHI5 promoter. I will use single cell tracking microscopy to determine how these disruptions impact the size- dependent synthesis of an mCitrine fluorescent reporter. In parallel, I will employ proteomics to identify WHI5 promoter-bound proteins and a Reporter-based Synthetic Genetic Array (R-SGA) to find genes that specifically impact WHI5 transcription. Once identified, candidate regulators will be interrogated to understand the mechanism by which they influence WHI5 transcription.
(Aim 2) We hypothesize that a general feature of some size regulating proteins is that their concentrations change with cell size. I will use proteomics to screen the proteomes of budding yeast and humans to identify proteins or protein groups whose concentrations change with cell size. A pilot experiment performed on a FACS-sorted human fibroblast cells demonstrates the efficacy of our planned proteomic approach. We identify size-dependent concentration changes in individual proteins and protein groups while controlling for confounding changes related to cell cycle phase. By expanding the depth of this analysis and performing similar experiments in budding yeast, we aim to identify candidate regulators of cell size or regulators of other biological process that are influenced by cell size. Moreover, by tracking the behavior of protein groups as cells grow in size, we can quantify how the cellular components scale with size on the macromolecular or organellar level. Because cell size control is a ubiquitous feature of eukaryotic cells, both at the single cell and organismal level, my findings will broadly impact our understanding of eukaryotic cell biology.
The question of how cells actively regulate their size is fundamental to the biology of all organisms and is poorly understood on a mechanistic level. Recently, the Skotheim lab found that, during the first gap phase (G1), budding yeast cells ?sense? size by diluting a cell cycle inhibitor protein Whi5, thereby elegantly linking the extent of cell growth in G1 with the decision to enter S phase. For inhibitor dilution to work in practice, the proteins which ?sense? and regulate cell size must be subjected to unique regulation, which this proposal aims to uncover.
