This is a request for a Zeiss Laser TIRF 3 microscope and live cell imaging system that will permit a group of 5 researchers at the Albany Medical College and 1 at the SUNY School of Public Health to selectively image molecular events that occur within or very close to the plasma membrane of cells as they lie close to a glass coverslip surface. In TIRF microscopy, a very thin, sub-resolution optical volume about 100 nm in depth is illuminated by the evanescent wave and only fluorescent molecules located within this space are excited, while fluorophores located outside this space do not contribute to the background or the fluorescence signal, resulting in the production of high-contrast, high-resolution fluorescence images. The members of the user group will apply this capability in several diverse areas of study: understanding how integrins regulate centrosome function and microtubule assembly during interphase and mitosis;the regulation of the assembly and disassembly of focal contact between cells and molecules in the extracellular substrate by following the dynamic interaction of fluorescent-protein components in live cells during smooth muscle cell migration and tumor cell invasion;use of the FRET based biosensor, Raichu, to assess the distribution of Rac, CDC42 and RhoA in migrating cells;the assembly of the phagocytic cup as vesicles bringing new membrane to the assembly site, approach and fuse into phagosomes;the interaction of signaling molecules and the actin cytoskeleton during phagocytosis by macrophages as detected with FRET-based biosensors;the interaction of adapter proteins with the human FSHR that leads to stimulation of several signaling cascades and receptor desensitization via endocytosis following binding of its ligand FSH use of the FRET based biosensor, Raichu, to assess the distribution of Rac, CDC42 and RhoA in migrating cells;the optimization of TIRF-FRET methodology. Acquisition of this instrument will dramatically enhance the research endeavors of members of the user group that are in progress. Currently they have no access to a TIRF microscope. The presence of the TIRF microscope will become a central resource in the institutional research community and lead to initiation of new projects as success of the user group using this technology becomes widely known.
