INVESTIGADORES
COLMAN LERNER Alejandro Ariel
congresos y reuniones científicas
Título:
Quantitative measurement of scaffold recruitment to the plasma membrane by confocal microscopy
Autor/es:
ALAN BUSH; ALEJANDRO COLMAN-LERNER
Lugar:
Kiental, Suiza
Reunión:
Workshop; MAP Kinase-based signal transduction in yeast and mammalian cells; 2009
Institución organizadora:
ETH, Zurich.
Resumen:
We are interested in understanding how cells process external information and use it to make decisions. We study the budding yeast (Saccharomyces cerevisiae) mating pheromone response, a prototypic signaling pathway comprised of two modules; a G protein coupled receptor, and a MAP kinase cascade which activates downstream targets necessary for mating. At the interface between these modules lies the scaffold protein Ste5, which simultaneously binds all three MAPK cascade components and localizes them to the membrane. Here we developed a quantitative method to measure membrane recruitment of YFP tagged Ste5 protein. The method relies on confocal imaging of pheromone treated cells and the later high-throughput analysis of the acquired images with our software, Cell-ID. The distribution of fluorescence in single cells is quantified and, through a calibration, the absolute fraction of membrane recruited molecules can be obtained. Cares are taken to deal with focus-drift and photobleaching. The technique can be used to measure the dynamics and dose response curve of this activation step of the pathway.