This proposal constitutes a 5-year plan to transition a nephrologist and epithelial cell biologist trainee to an academic career as an independent research investigator. The candidate's long-term career goal is to be an academic physician-scientist who is a leader in the field of mechanisms that control epithelial cell polarity. Her immediate goals are designed to facilitate her successful transition to independence: 1) learn to analyze glycosylation modifications of proteins which she will apply to her study of mechanisms controlling polarity, 2) (which will serve to) separate her research goals from her primary mentor's, 3) learn an in vivo model of lumenogenesis, 4) learn advanced microscopic techniques so that she can study the behavior of polarity proteins in live cells, and 5) capitalize on her recent technological advancements on her current project and develop a publication portfolio. Her achievement of these immediate goals will place her in good position to successfully compete for funding to launch her career as an independent researcher. The candidate is exceptionally committed to a career as a physician-scientist. She has sound basic science research training and several years of experience in the laboratory of Dr. Ben Margolis, a renowned expert on epithelial cell polarity. She recently overcame several technological barriers that hampered her research progress on her protein of interest, the apical membrane determinant protein Crumbs3a. Her recent work revealed that differential glycosylation of Crumbs3a appears to be involved in the polarization state of epithelial cells, suggesting a novel potential mechanism for control of cell polarity which she will explore in this proposal. Her research hypothesis is based on two independent discoveries that she made during her course of research. She will test her hypothesis that generation of epithelial polarity and lumenogenesis require both 1) proper placement of Crumbs3a, which depends on its FERM-binding domain, and 2) specific glycosylation modifications of Crumbs3a.
In Aim #1, she will examine the role of Crumbs3a's FERM-binding domain in controlling Crumbs3a's position on the cell membrane, thereby dictating the position of an apical lumen. She will use a combination of protein knockdown and mutagenesis, 3-dimensional cyst assay, custom cell surface trafficking assay, and fluorescence recovery after photobleaching.
In Aim #2, she will evaluate an as yet unexplored mechanism of controlling polarity, that cells use differential glycosylation to control genesis of the apical membrane and lumen. She will use a combination of mass spectrometric analysis of glycosylation modifications of Crumbs3a in different states of polarity, protein mutagenesis, and lectin binding arrays. She will also examine delivery of Crumbs3a and glycoproteins to the apical lumen surface during organogenesis of lumen-forming organs in mice using lectin immunohistochemistry and protein immuno-isolation techniques. Aspects of this project will serve as the basis for an independent R01 research proposal, which she will write during the latter half of the award period. She has compiled a team of knowledgeable and successful experts at her institution to function as mentors, collaborators, and consultants to facilitate her training and assist her in achieving her research goals. Since the study of glycosylation modifications is outside Dr. Margolis'expertise, she will be co-mentored by Dr. Subramaniam Pennathur, a proteomics expert, with support of Dr. David Lubman, a physical chemist with extensive experience in mass spectrometric evaluation of glycosylation modifications. Center for Organogenesis senior faculty members Drs. Greg Dressler and Deb Gumucio are embryologists with extensive experience in organogenesis of mice will aid in developing her mouse model of lumenogenesis. Her training progress will be closely monitored by her Mentorship Committee;it is comprised of three physician-scientists, each with a successful research program, providing ideal mentorship for the candidate. The candidate's institutional environment is ideal to support this training plan. She is at a major research institution and has wide access to the necessary resources and equipment to achieve the goals of her project. She is well supported by a Division that has a strong nephrologic-based basic science research program, and that has experience mentoring and transitioning physician-scientists to successful academic careers in research. The candidate's Career Development Plan and Research Proposal are well designed and take advantage of the numerous intellectual and facility resources the candidate has available. The candidate is ideally positioned to achieve her immediate training and research goals, which are appropriate milestones for her successful transition to independence as a researcher.

Public Health Relevance

Establishing and maintaining epithelial cell polarity is critical for proper function of tubule-based organs such as kidneys and intestine. Polarity is frequently pathogenically perturbed in cancer and polycystic kidney disease but necessary for recovery from injury, such as in acute kidney injury caused by acute tubular necrosis. The goal of this project is to elucidate mechanisms cells use to control epithelial polarity. Such knowledge of basic cell biology will have wide impact on understanding the pathogenesis of such diseases, and be the foundation for advancement in associated diagnostic tools and therapies.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Clinical Investigator Award (CIA) (K08)
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Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
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University of Michigan Ann Arbor
Internal Medicine/Medicine
Schools of Medicine
Ann Arbor
United States
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Czerniecki, Stefan M; Cruz, Nelly M; Harder, Jennifer L et al. (2018) High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping. Cell Stem Cell 22:929-940.e4
Stoops, Emily H; Hull, Michael; Olesen, Christina et al. (2015) The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135. J Cell Biol 211:287-94
Harder, Jennifer L; Hodgin, Jeffrey B; Kretzler, Matthias (2015) Integrative Biology of Diabetic Kidney Disease. Kidney Dis (Basel) 1:194-203
Whiteman, Eileen L; Fan, Shuling; Harder, Jennifer L et al. (2014) Crumbs3 is essential for proper epithelial development and viability. Mol Cell Biol 34:43-56
Harder, Jennifer L; Whiteman, Eileen L; Pieczynski, Jay N et al. (2012) Snail destabilizes cell surface Crumbs3a. Traffic 13:1170-85