Project Summary Abstract The focus of our current NIGMS funded R01 proposal (R01GM134231) is to study the role of deubiquitinating enzymes in proliferation, cell cycle control and genome maintenance. The ubiquitin proteasome system is the major regulator of protein degradation in all eukaryotes. Ubiquitination is implicated in virtually all aspects of cell physiology and human disease. The post-translational conjugation of ubiquitin onto specific substrates requires the concerted action of three enzymes, termed E1, E2 and E3. However, substrate ubiquitination and degradation is determined not only by the addition of ubiquitin onto substrates, but also by the action of catalytic proteases termed deubiquitinases (DUBs), which remove ubiquitin from substrates proteins, thereby antagonizing degradation. The major goals of the parent R01 are to determine substrates and mechanisms of deubiquitination during cell division, mechanisms of DUB action in the cell cycle, and the identity of additional DUBs involved in cell cycle. The unifying theme of this research is the study of protein dynamics in cell cycle progression. Determining the abundance of proteins in specific phases of the cell cycle is therefore vital to determining the mechanisms underlying proteome remodeling and connecting enzymes with cognate substrates. This analysis has significant technical challenges. First, many enzymes that control cell cycle progression are themselves dynamically regulated, and their manipulation (e.g., knockdown or knockout) can perturb cell cycle progression. Since the abundance of many cell cycle proteins, and particularly cell cycle regulated ubiquitin substrates, is regulated as cells proliferate, their abundance can be altered by alterations to the cell cycle. Thus, the ability to examine the abundance of specific proteins in specific cell cycle phases, without the need for synchronization, is a tremendous advantage. We address these challenges through single cell imaging. This current proposal requests support for the purchase of a Revolve R4 Echo Imaging System. This instrument would allow our lab to perform imaging analysis in-house and would significantly improve the capabilities of our current day-to-day tissue culture microscope, which lacks the ability of capture images, precluding on-the-fly, quantitative analysis of transfection efficiencies or cell morphology. This purchase would expand our current capabilities, facilitate orthogonal analysis methods that are easy to use and accessible, and provide everyday access to improved cell culture processes.