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 Ca2+ 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 Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics. Recently developed GCaMP genetically-encoded Ca2+ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca2+ 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 Ca2+ dynamics in culture, revealing Ca2+ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca2+ indicators, our results show that mitochondrial Ca2+ 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 Ca2+ dynamics on cytosolic Ca2+ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca2+ increase using mito-GCaMP5G and a synthetic organic Ca2+ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca2+ signaling pathway, our results demonstrated that the mitochondrial Ca2+ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.

Probing the Complexities of Astrocyte Calcium Signaling

“…The latest GCaMP6 series developed by the Janelia GENIE Project performs as well as some organic Ca 2+ indicator dyes [24]. The properties of GCaMPs are further leveraged by targeting them to sub cellular compartments such as the plasma membrane [28-31], mitochondria [32-34] and endoplasmic reticulum [33, 35]. Ratiometric FRET based GECIs are also widely used and have been improved to tune Ca 2+ affinity to desirable levels [36].…”

“…The data were indiscernible. Furthermore, GECIs have been deployed in several studies and no untoward effects have been reported [26, 28-32, 37, 38, 42-44, 47-55]. In future astrocyte studies, appropriate controls will still be needed.…”

Imaging of Mitochondrial and Cytosolic Ca2+ Signals in Cultured Astrocytes

“…Insert mitochondrial matrix (MM)-targeting sequence (mito-) ATGT CCGTCCTGAC GCCGCTGCTG CTGCGGGGCT TGACAGGCTC GGCCCGGCGG CTCCCAGTGC CGCGCGCCAA GATCCATTCG TTG (Rizzuto et al, 1995) in the backbone of AAV plasmid pZac2.1 containing astrocytic gfaABC 1 D promoter (Lee et al, 2008;Li et al, 2014;Xie et al, 2010). …”

“…Subclone GCaMP5G/6s or monomeric red fluorescence protein (mRFP) as a control into the cloning sites BamH I and Not 1 using PCR to obtain new expressing plasmids that can target transgene expression in mitochondria of astrocytes (Li et al, 2014) (Figure 2). …”

“…In this protocol, we employed astrocyte- and mitochondria-specific targeting of GCaMP5G/6s to assess mitochondrial Ca 2+ dynamics in primary cultured astrocytes. ATP stimulates astrocytic Ca 2+ increase through Ca 2+ release from ER via activation of IP 3 Rs (Figure 1)(Ding et al, 2007;Ding, 2013;Li et al, 2014). Large increases in Ca 2+ signals can be observed in individual mitochondria in astrocytes with stimulation of ATP.…”