How do eukaryotic cells sense and move along a concentration gradient of chemical attractants? Chemoattractants such as chemokines are detected by G-protein coupled receptors (GPCR). Using a combination of molecular genetic, biochemical and cell biological approaches, we study chemoattractant elicited signal transduction in both mammalian cells and the model system Dictyostelium discoideum. We employ live cell imaging technology to visualize signaling events in single cells. During the last fiscal year, we established two fluorescence microscope systems: an Olympus IX70 equipped with two filter wheels that is linked to a coo-CCD digital camera and a Zeiss confocal Laser Scanning Microscope LSM 510 META. Using these systems, we are able to monitor the distribution of signaling proteins, tagged with various fluorescence proteins, in single living cells. In the mammalian system we determined the spatial distribution and visualized the signaling cascades of the chemokine receptor CXCR1 at the single cell level. In the D. discoideum system we monitored the temporal and spatial activities of GPCRs in the membranes of chemotaxing cells. Interactions between the subunits of the G proteins were detected as was GPCR-induced dissociation of G proteins in single cells.
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