Quantitative fluorescence imaging provides powerful measurement opportunities for understanding cellular signaling pathways and networks. I will discuss in my talk different currently useful fluorescence biosensors and imaging strategies and will present some of our recent results in applying fixed cell and live cell imaging to investigate the spatial and temporal control in second messenger signaling systems. I will first focus on a strategy that we developed to rapidly perturb phosphoinositide signaling pathways using chemically-induced enzyme activation that led to new insights into the roles of PI(45)P2 and PI(345)P3 lipid second messengers in cell migration and chemotaxis. I will then focus on fluorescence-imaging based siRNA screens that led to the discovery of STIM1 proteins that sense Ca2+ in the endoplasmic reticulum and control Ca2+ influx into cells.

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