Development and Application of Sub-Mitochondrial Targeted Ca2 + Biosensors

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.

Sigma-1 Receptor Promotes Mitochondrial Bioenergetics by Orchestrating ER Ca2+ Leak during Early ER Stress

“…In S1R depleted and BD1047 treated cells, tunicamycin treatment did not elevate IMS Ca 2+ after 2 h and 8 h (Figure 4i,j and Figure S5b,c), thus supporting our assumption of a specific role of S1R as an orchestrator of the enhanced ER Ca 2+ leak during early ER stress, directing the Ca 2+ leak towards mitochondrial Ca 2+ uptake sites. inner membrane, we measured Ca 2+ levels in the mitochondrial inter-membrane space (IMS) using a recently developed IMS-targeted genetically encoded ratiometric Ca 2+ sensor, IMS-GEM-GECO [19,20]. IMS Ca 2+ closely followed the same trends as the mitochondrial Ca 2+ , with an increase after 2 h of tunicamycin treatment, which was normalized after 8 h of treatment (Figure 4h and Figure S5a).…”

“…To test whether the observed differences in mitochondrial Ca 2+ were due to the direction of the ER Ca 2+ leak and not associated with Ca 2+ “permeability” of the mitochondrial inner membrane, we measured Ca 2+ levels in the mitochondrial inter-membrane space (IMS) using a recently developed IMS-targeted genetically encoded ratiometric Ca 2+ sensor, IMS-GEM-GECO [ 19 , 20 ]. IMS Ca 2+ closely followed the same trends as the mitochondrial Ca 2+ , with an increase after 2 h of tunicamycin treatment, which was normalized after 8 h of treatment ( Figure 4 h and Figure S5a ).…”

“…IMS Ca 2+ was measured using genetically encoded IMS targeted Ca 2+ sensor IMS-GEM-GECO [ 19 , 20 ]. The sensor was excited with 385 nm LED and emission collected at 480 nm and 530 nm using a CFP/YFP/RFP filter set and 505dcxr beam-splitter.…”

Recent Advances in Chemical Biology of Mitochondria Targeting

“…Its overload can lead to a variety of pathological conditions, including neuronal apoptosis and death in nervous system diseases ( Li et al, 2014 ). Therefore, targeting Ca 2+ inside mitochondria to treat related diseases, most of which are nervous system diseases, has emerged as a promising mitochondrial targeting strategy ( Waldeck-Weiermair et al, 2019 ).…”

Mitochondrial Ion Channels of the Inner Membrane and Their Regulation in Cell Death Signaling

“…As illustrated above, considerable new information allowed to link pathways/proteins that regulate IMM channels to cell death/survival signaling during the last decade. Since factors that regulate mitochondrial calcium accumulation play a crucial role in this context, understanding of the relation between channel modulation and localized [Ca 2+ ] changes, thanks to novel, sub-mitochondrial targeted Ca 2+ sensors ( Waldeck-Weiermair et al, 2019 ), may further widen our view. Similarly, measurement of simultaneous, real-time dynamics of ATP and ROS in mitochondria in vivo ( van Hameren et al, 2019 ) may provide important insights to integrate IMM (and OMM) channels into death signaling pathways.…”