application) Research: The goal of this proposal is to elucidate the itinerary and mechanisms of endocytosis and intracellular trafficking mediated by ganglioside GM1 in polarized intestinal epithelia. We recently demonstrated that ganglioside GM1 drives the association of cholera toxin (CT) with Detergent Insoluble Membrane Domains (DIGs) and that this association is necessary for toxin action in T84 cells. Preliminary studies now suggest that GM1-mediated signal transduction depends on membrane cholesterol and the ceramide structure of GM1. The studies outlined in this research proposal will use CT and related toxins to further examine the mechanisms by which GM1 targets ligands into and across the intestinal epithelial cell. S12.
Aim 1 will identify structural features of GM1 which determine association with DIGs and subsequent trafficking into intestinal cells. Specifically, we will examine the effects on trafficking of altering: 1) GM1 ceramide or oligosaccharides structure; 2) membrane cholesterol; and 3) ganglioside clustering. Sp.
Aim 2 will demonstrate the specificity of GM1 as a targeting motif for 1) endocytosis, 2) retrograde trafficking into the Golgi and ER, and 3) transcytosis. Sp.
Aim 3 will compare GM1-dependent endocytosis via apical and basolateral cell membranes. In particular, we will compare 1) how GM1-binding at apical and basolateral cell surfaces affects the efficiency of toxin uptake, and 2) whether toxin bound to apical or basolateral GM1 eventually colocalize in the apical recycling endosome. Candidate: Dr. Wolf is a Board Certified pediatric gastroenterologist and is an Instructor of Pediatrics at Children's Hospital. She has been involved in basic research since high school and is dedicated to a career in academic medicine. Her initial findings have resulted in a first-author publication in The Journal of Cell Biology. Studies for two other manuscripts are near completion. Dr. Wolf will continue to train under the mentorship of Dr. Lencer, in a supportive and productive research environment, as she transitions to independent work. Environment: Dr. Wolf will conduct her research in Dr. Wayne Lencer's laboratory at Children's Hospital. She has full 75% protected time, assigned desk and bench space, and access to all supplies and equipment necessary to complete the proposed research program. She will continue to participate in regularly scheduled lab meetings and joint seminars in cell biology. The richness of educational opportunities and individuals available to Dr. Wolf on campus at Harvard Medical School will foster her development as an independent investigator capable of determining her own future directions.
