Imaging of Mitochondrial and Cytosolic Ca2+ Signals in Cultured Astrocytes. Issue 1 (22nd January 2018)

• Record Type: Journal Article
• Authors: Zhang, Nannan; Ding, Shinghua
• Editors: Gerfen, Charles R.; Holmes, Andrew; Sibley, David; Skolnick, Phil; Wray, Susan
• Is Part Of: Current protocols in neuroscience. Issue 82:Issue 1(2018)
• Page Start: 2.29.1
• Page End: 2.29.11
• Publication Date: 2018-01-22
• Subjects: astrocyte -- ATP -- endoplasmic reticulum (ER) -- GCaMP5G/6s -- mitochondrial Ca2+ uptake -- mitochondrial matrix -- X‐Rhod‐1; Neurosciences -- Laboratory manuals; Neurosciences; Cytological Techniques; Histological Techniques; Nervous System -- anatomy & histology; Neurosciences; Laboratory Manual; Laboratory manuals; 612.8
• DOI: 10.1002/cpns.42
• Languages: English
• ISSNs: 1934-8576

Abstract

Abstract: This unit provides a step‐by‐step protocol for constructing cell type‐ and mitochondria‐targeted GCaMP genetically encoded Ca 2+ indicators (GECIs) for mitochondrial Ca 2+ imaging in astrocytes. Mitochondrial Ca 2+ plays a critical role in controlling cytosolic Ca 2+ buffering, energy metabolism, and cellular signal transduction. Mitochondrial Ca 2+ overload contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Live‐cell mitochondrial Ca 2+ imaging is an important approach to understand mitochondrial Ca 2+ 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 Ca 2+ signals. This protocol provides a novel approach to image mitochondrial Ca 2+ dynamics as well as Ca 2+ interplay between the endoplasmic reticulum and mitochondria. © 2018 by John Wiley & Sons, Inc.