Development and Application of Sub-Mitochondrial Targeted Ca Biosensors.

Mitochondrial Ca uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca levels inside the CL but not the IMS in response to IP-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca through the OMM while the cytosolic Ca signal remained unchanged. The novel sub-mitochondrially targeted Ca biosensors proved to be suitable for Ca imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca signaling under (patho)physiological conditions.