AUTHOR=Waldeck-Weiermair Markus , Gottschalk Benjamin , Madreiter-Sokolowski Corina T. , Ramadani-Muja Jeta , Ziomek Gabriela , Klec Christiane , Burgstaller Sandra , Bischof Helmut , Depaoli Maria R. , Eroglu Emrah , Malli Roland , Graier Wolfgang F. TITLE=Development and Application of Sub-Mitochondrial Targeted Ca2 + Biosensors JOURNAL=Frontiers in Cellular Neuroscience VOLUME=13 YEAR=2019 URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2019.00449 DOI=10.3389/fncel.2019.00449 ISSN=1662-5102 ABSTRACT=

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.