Understanding the structural mechanism of spontaneous ubiquitin cargo clustering on the cell plasma membrane

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Abstract The PI proposes to study the role of spontaneous Ubiquitin (UB) cargo clustering on the plasma membranes in vesicular trafficking using the model membrane reconstitution. Vesicular trafficking is a process of material transport by lipid vesicles between membrane bound organelles. The process is one of the very basic cellular functions and thus is involved in most life processes of higher organisms. However, its molecular mechanism is still poorly understood. Ubiquitylation, an enzymatic attachment of UB, is involved in many trafficking processes, but canonically, UB cargo has been considered only as a passive binder to the downstream trafficking proteins. Polymeric ubiquitylation of cargos and their multivalent interaction with binder proteins are very common in living cells leading to spontaneous protein condensate domain formation on the cell membranes. These cargo cluster- ing domains are also known to very closely interact with lipid raft domains. Understanding the spontaneous clustering of UB cargo as an active spatial organizer of the vesicular trafficking process can advance our current understanding of the vesicular trafficking mechanism by providing insight into the traditionally overlooked roles of UB cargo in the process. The main methods of this investigation are fluorescence microscopy (FM) and atomic force microscopy (AFM). Image data are analyzed by the deep-learning-assisted analysis software. The goals of this proposal are: (1) Identifying the structural organization of UB cargo clusters created by proteins with different structures; (2) Understanding the lipid composition dependence of the UB cluster organization. The PI places an emphasis on structural understanding of the cargo clustering domain formation in this proposal. FM is used for high throughput imaging of many samples with different protein structures to understand the structural cues that lead to clustering domain formation. AFM is used to study the structural organization of the clusters at the molecular level to obtain direct structural snapshots to test hypothetical models. The proposal will enhance the research environment at Montclair State University by synergistically enabling the use of the high-sensitivity AFM instrument the university recently obtained. Moreover, the proposal will address the current high demand of undergraduate students to participate in research while preparing them to advance to graduate programs in health-related sciences. In collaboration with the GS-LSAMP program, students of underserved population will be actively recruited. Students are expected to acquire experimental skills, learn data analysis, and present in conferences to meet and communicates with scientists in the field. 1