Imaging of Mitochondrial and Cytosolic Ca2+ Signals in Cultured Astrocytes

This unit provides a step-by-step protocol for constructing cell type- and mitochondria-targeted GCaMP genetically encoded Ca²⁺ indicators (GECIs) for mitochondrial Ca²⁺ imaging in astrocytes. Mitochondrial Ca²⁺ plays a critical role in controlling cytosolic Ca²⁺ buffering, energy metabolism, and cellular signal transduction. Mitochondrial Ca²⁺ overload contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Live-cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics and thus cell physiology and pathology. We implement astrocyte-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s). By loading X-Rhod-1 into astrocytes, we can simultaneously image mitochondrial and cytosolic Ca²⁺ signals. This protocol provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria. © 2018 by John Wiley & Sons, Inc.