Development and Application of Sub-Mitochondrial Targeted Ca^{2 +} Biosensors

Mitochondrial Ca²⁺ uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca²⁺ 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- Ca²⁺ 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 Ca²⁺-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 Ca²⁺ levels inside the CL but not the IMS in response to IP₃-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca²⁺ through the OMM while the cytosolic Ca²⁺ signal remained unchanged. The novel sub-mitochondrially targeted Ca²⁺ biosensors proved to be suitable for Ca²⁺ 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 Ca²⁺ signaling under (patho)physiological conditions.