Funds are requested for the purchase of a Zeiss LSM 510 confocal microscope and Eppendorf micro-injection apparatus. Cell biology is entering a new phase in which fimctional studies in vitro are being complemented by investigations of their dynamics in the living cell. One important new approach is to photobleach fluorescentlytagged macromolecules within user-defined areas of a cell. The rate at which molecules from unbleached areas then diffuse into the bleached area provides quantitative information on macromolecular dynamics. Variants of this approach include FRAP (fluorescence recovery after photobleaching), FLIP (fluorescent label iterative photobleaching) and CALl (chromophore-assisted laser inactivation). A second new approach is ICS (imaging correlation spectroscopy), which uses random motions of fluorescent molecules within living cells to determine diffusion coefficients. This method can reveal protein-protein interactions and aggregation states in situ that are unobtainable by other techniques. These studies require access to a confocal microscope such as the Zeiss LSM 510, equipped with a UV laser, aconsto-optical tunable lifters (AOTFs) to constrain the laser to user-defined areas of the cell, multitracking and dual direction scanning for simultaneous detection of multiple fluorophores, rapid scan rates, sensitive photomultipliers, plus a micro-injection apparatus, and heated stage, that are essential to the development and implementation of the assays needed for our projects. No such instrument is available at the University of Virginia. The requested microscope will be housed in the Cancer Center, and be administered by the Central Electron Microscope Facility of the School of Medicine. Users include faculty from multiple departments and centers (6 major users, 4 minor). Research projects cover the dynamics of nucleocytoplasmic transport, nuclear organization, vesicle transport, and cell motility. The advisory committee will comprise 3 of the major users, plus the Director and the Assoc. Director of the Central EM Facility, and the Director of the Keck Center for Cell Imaging. Substantial financial support from the University and School of Medicine will permit low user fees, ongoing maintenance and training. This instrument will significantly enhance the research mission of the School of Medicine.
| Kremer, Brandon E; Adang, Laura A; Macara, Ian G (2007) Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7. Cell 130:837-50 |
