Development and Application of Sub-Mitochondrial Targeted Ca2 + Biosensors
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Date
2019-10-04
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Journal Article
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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.
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published
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Volume
13
Pages / Article No.
449
Publisher
Frontiers Media
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Subject
Ca2+ measurements; Biosensors; Mitochondrial Ca2+; Mitochondrial cristae; Ca2+; Live cell imaging; Mitochondria