This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Notch pathway is an evolutionary conserved signaling system used extensively throughout embryonic development that continues to function in the maintenance of tissues and stem cells in adults. Notch signaling directs the specification of a variety of cell types and contributes to tissue patterning, and morphogenesis through effects on cellular differentiation, proliferation, survival and death. Our working model for ligand-induced Notch signaling is based on Weinmaster's published and preliminary findings obtained with mammalian cell culture, and incorporates the critical role for forces generated during ligand endocytosis with Notch structural findings. Specifically, we hypothesize that endocytosis of ligand-bound Notch generates a robust mechanical force that physically pulls the Notch receptor apart to expose the ADAM cleavage site and facilitate activating proteolysis. Our model also proposes that furin cleavage to produce the Notch heterodimer predisposes Notch to physical dissociation through mechanical force generated during ligand endocytosis. However, ligand endocytosis has also been proposed to facilitate recycling to generate a high affinity Notch ligand. To address this controversy, we are using optical tweezers to determine roles for ligand endocytosis in regulating binding strength and force generation. SMM laser tweezers used to measure endocytic forces and avidity between Notch coated beads and ligand expressing cells. Correlations between endocytic forces, adhesion strength and Notch transedocytoiss are sought.
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