Applications of Genetically Targeted and Optimized Calcium Sensors

Abstract

We have recently developed a series of FRET-based genetically encoded sensors for calcium. These sensors (dubbed “cameleons”) are made from cyan and yellow fluorescent proteins combined with engineered calcium sensing elements. These optimized cameleon sensors have now been genetically targeted to specific locations within cells including: the ER, mitochondria, Golgi, nucleus, plasma membrane, and cytosol. In the present work we put these sensors to the test in both standard tissue culture cells and in primary hippocampal neurons. We have now used these sensors to define how ER and mitochondrial calcium handling is altered by mutations in the calcium regulatory protein presenilin. Presenilin is an integral membrane protein that localizes to the ER, secretory pathway, and plasma membrane. There is emerging evidence from a number of research groups that presenilin plays a critical role in modulating ER calcium signaling. Using a combination of genetically targeted and small molecule sensors we have identified how mutations in presenilin alter calcium homeostasis in the ER and calcium release through the IP3R. In another line of research aimed at testing the sensitivity and versatility of our sensors, we have identified localized calcium signals generated upon invasion of a host mammalian cell by bacteria. In this work we will discuss the strengths and weaknesses of genetically encoded calcium sensors and potential avenues for further improvement and optimization.