The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains.

Calcium (Ca 2+ ) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca 2+ concentration ([Ca 2+ ] i ) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca 2+ ] i are ideally suited to modulate Ca 2+ -mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca 2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca 2+ , the introduction and the ongoing development of genetically encoded Ca 2+ indicators (GECI) opened a new era to study Ca 2+ -driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca 2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca 2+ signals at the (sub)-cellular level with pronounced temporal resolution.