Regulators of Calcium Homeostasis Identified by Inference of Kinetic Model Parameters from Live Single Cells Perturbed by siRNA

Assigning molecular functions and revealing dynamic connections between large numbers of partially characterized proteins in regulatory networks are challenges in systems biology. We showed that functions of signaling proteins can be discovered with a differential equations model of the underlying signaling process to extract specific molecular parameter values from single-cell, time-course measurements. By analyzing the effects of 250 small interfering RNAs on Ca2+ signals in single cells over time, we identified parameters that were specifically altered in the Ca2+ regulatory system. Analysis of the screen confirmed known functions of the Ca2+ sensors STIM1 (stromal interaction molecule 1) and calmodulin and of Ca2+ channels and pumps localized in the endoplasmic reticulum (ER) or plasma membrane. Furthermore, we showed that the Alzheimer's disease-linked protein presenilin-2 and the channel protein ORAI2 prevented overload of ER Ca2+ and that feedback from Ca2+ to phosphatidylinositol 4-kinase and PLC delta (phospholipase C delta) may regulate the abundance of the plasma membrane lipid PI(4,5)P-2 (phosphatidylinositol 4,5-bisphosphate) to control Ca2+ extrusion. Thus, functions of signaling proteins and dynamic regulatory connections can be identified by extracting molecular parameter values from single-cell, time-course data.