Project Summary/ Abstract The cytoplasm is a crowded subcellular environment that is packed with organelles, proteins, nucleic acids and other large macromolecules, as well as water and small molecules. How cell biological processes function in this milieu remains poorly understood. Macromolecules present in the cytoplasm are thought to exert physical forces that contribute to cytoplasmic organization, phase separation, and osmotic pressure. Cellular density, which is the concentration of cellular components such as proteins and nucleic acids, is a key predictor of these macromolecular crowding effects. Recent evidence from our lab and others reveals that density and macromolecular crowding effects are not constant but actually change during the cell cycle, as well in various physiological and disease states, and during development. However, little is known about how these changes impact cellular physiology and mechanics. Thus, cellular density and the effects of macromolecular crowding represent critical but understudied aspects of cellular physiology that likely impact most cellular processes. The general goals are to elucidate physical- and molecular- based mechanisms responsible for cellular processes responsible for cell growth and division: mitosis, microtubule dynamics, nuclear size control, chromosome mobility and cell wall assembly. A general thrust of the investigations is to determine how the biophysical properties of the cytoplasm and nucleoplasm impact these diverse cellular processes. In particular, our studies will address how intracellular osmotic pressures generated by macromolecules act to dampen microtubule dynamics, inflate the nucleus, modulate the mechanics of the mitotic spindle, and regulate chromosome motility for DNA repair. Approaches include innovative live cell assays for the biophysical properties of living cells (e.g. microrheology and quantitative phase imaging) and quantitative cell biology approaches in the fission yeast Schizosaccharomyces pombe. These studies will establish a foundation for the emerging field of cellular density and will contribute to our understanding of a fundamental but understudied aspect of cell biology. This work will significantly impact our understanding of mechanisms governing cell growth and division that are relevant for biomedical applications including cancer, aging and fungal pathogenesis.