Much effort has been devoted in the last decade to elucidate signal transduction mechanisms initiated by receptor tyrosine kinases. For example, these receptors mediate phosphoinositide hydrolysis and phosphorylation by modulating the activities of phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3-kinase (PI 3-kinase), respectively, and they stimulate the activation of the p21 ras GTPase by assembling multiprotein complexes with Ras-guanine nucleotide exchange activity. Given the current appreciation of complex feedback and crosstalk interactions within and among these signaling cascades, quantitative experimental and analytical methodologies are lacking. In the proposed study, I will develop a relatively high-throughput array platform for imaging membrane recruitment of fluorescent biosensors, allowing two signaling pathways to be monitored simultaneously in living cells. This imaging setup, coupled with an engineering analysis of the data, will allow multiple signaling variables to be related.
