Deciphering the role of MEMO1 during amelogenesis - PROJECT SUMMARY: Amelogenesis imperfecta (AI) is a rare genetic congenital enamel disorder characterized by enamel that is prone to rapid wear and breakage. AI affects approximately 1 in 15,000 children in the United States and poses a significant financial and psychosocial burden for those affected. Moreover, our understanding of the etiology of AI is limited with roughly 50% of cases having no known genetic cause. The long-term goal of this research proposal is to study the function of a novel AI associated gene, Mediator of Cell Motility 1, or MEMO1, to uncover molecular mechanisms of normal and abnormal enamel development—ultimately leveraging these details in regenerating enamel. The applicant previously identified a role for the protein MEMO1 during amelogenesis. Oral ectoderm loss of MEMO1 led to severe enamel hypomineralization and chipping, like that observed in humans afflicted with AI. Despite severe enamel pathology associated with loss of Memo1, several questions remained. For example, what are the spatial and temporal dynamics of MEMO1 during amelogenesis? How does MEMO1 function within the ameloblast? This fellowship aims to tackle these questions with the central hypothesis that MEMO1 is essential within the definitive ameloblast at the earliest onset of enamel mineralization and functions within a network of integrin- based signaling and cytoskeletal dynamics. This hypothesis is based on preliminary data and shared enamel pathology associated with oral epithelial loss of Memo1 and integrins. Two experimental aims will address this central hypothesis. In aim 1, conditional mouse genetics, µCT, nano-indentation, histology, immunofluorescence, and single cell RNA sequencing will assess the spatial and temporal requirements of MEMO1 during amelogenesis. In aim 2, a Memo1 knockout ameloblast cell line will be coupled with immunofluorescence, integrin-activation assays, live-cell imaging, co-immunoprecipitation, proximity-based ligation proteomics, and pull-down assays to assess MEMO1’s ameloblast-specific, cellular function. Insights from the proposed research will enhance our comprehension of MEMO1’s contribution to normal enamel development and mechanisms underlying AI. The resulting insights may pave the way for improved diagnostic and therapeutic strategies for AI. With combined mentorship from Dr. Van Otterloo and Dr. Tootle, the applicant will develop skillsets in single cell bioinformatic analysis, proteomics, live cell imaging, and micro- computed tomography greatly advancing their doctoral training. The comprehensive training plan, cohesive team, and supportive institutional environment outlined in this research proposal will advance treatment of oral and dental disorders and propel the applicant forward along a path of an independent researcher.
