SIGNAL TRANSDUCTION CORE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this core is to provide technical assistance in designing, making vectors expressing fluorescent fusion proteins for intracellular localization and protein trafficking to investigators interested in studying cellular transduction and signaling pathways. Although the use of molecular biology is indispensable, cell biology studies offer a link that connect in vitro and in vivo data and is useful to confirm hypothesis or models proposed. Observations from studies with fluorescently tagged proteins typically show the steady state distribution of a protein, but they do not directly provide information about the kinetic properties of molecules. To determine the kinetic properties of proteins in vivo, the movement of the protein of interest must be made visible. The most commonly used technique for this is FRAP (fluorescence recovery after photobleaching). In this method, a small area of a cell is rapidly bleached using a high-intensity laser pulse. The movement of unbleached molecules from the neighboring areas is then recorded by time-lapse microscopy as the recovery of fluorescence in the bleached area. The microscope will also allow for time lapse movie caption to monitor fluorescently labeled protein movement in the cell in response to different drug treatment altering a specific pathway, ligand-receptor downstream targets and so on. In vivo molecular interactions could also be investigated by fluorescent energy transfer (FRET).