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

Summary 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 [Ca 2+ ] changes ([Ca 2+ ] 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 Ca 2+ transfer. Pericam-tagged linkers revealed IP 3 -induced [Ca 2+ ] ER-mt signals that exceeded 9 μM and endured buffering bulk cytoplasmic [Ca 2+ ] increases. Altering linker length to modify the space available for the Ca 2+  transfer machinery had a biphasic effect on [Ca 2+ ] ER-mt signals. These studies provide direct evidence for the existence of high-Ca 2+ microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca 2+ transfer.