Imaging Interorganelle Contacts and Local Calcium Dynamics at the ER-Mitochondrial Interface

The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca2+] changes ([Ca2+](ER-mt)) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca2+ transfer. Pericam-tagged linkers revealed IP3-induced ([Ca2+](ER-mt)) signals that exceeded 9 mu M and endured buffering bulk cytoplasmic [Ca2+] increases. Altering linker length to modify the space available for the Ca2+ transfer machinery had a biphasic effect on [Ca2+](ER-mt) signals. These studies provide direct evidence for the existence of high-Ca2+ microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca2+ transfer.