title: Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store.
abstract: Mitochondrial Ca(2+) plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca(2+) imaging is an important approach to understand mitochondrial Ca(2+) dynamics. Recently developed GCaMP genetically-encoded Ca(2+) indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca(2+) imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca(2+) dynamics in culture, revealing Ca(2+) uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca(2+) indicators, our results show that mitochondrial Ca(2+) uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca(2+) dynamics on cytosolic Ca(2+) changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca(2+) increase using mito-GCaMP5G and a synthetic organic Ca(2+) indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca(2+) signaling pathway, our results demonstrated that the mitochondrial Ca(2+) uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca(2+) dynamics as well as Ca(2+) interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.
authors.affiliation: Dalton Cardiovascular Research Center, Columbia, MO 65211, United States; Department of Bioengineering, University of Missouri, Columbia, MO 65211, United States.
authors.forename: Hailong
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Bioengineering, University of Missouri, Columbia, MO 65211, United States.
authors.forename: Xiaowan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Dalton Cardiovascular Research Center, Columbia, MO 65211, United States.
authors.forename: Nannan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Neurobiology, University of Alabama, Birmingham, AL 35294, United States.
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Gottipati
authors.affiliation: Department of Neurobiology, University of Alabama, Birmingham, AL 35294, United States; Department of Biotechnology, University of Rijeka, 51000 Rijeka, Croatia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Parpura
authors.affiliation: Dalton Cardiovascular Research Center, Columbia, MO 65211, United States; Department of Bioengineering, University of Missouri, Columbia, MO 65211, United States. Electronic address: dings@missouri.edu.
authors.forename: Shinghua
authors.initials: S
authors.lastname: Ding
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000494:pharmacology; D008164:Luminescent Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D002118:Calcium
keywords: ATP; ER-mitochondrial communication; IP(3) 5-phosphatase; Mitochondrial Ca(2+) uptake; Mitochondrial matrix