Heterogeneity of the endoplasmic reticulum Ca²⁺ store determines colocalization with mitochondria

Contact sites between the endoplasmic reticulum (ER) and mitochondria play a pivotal role in cell signaling, and the interaction between these organelles is dynamic and finely regulated. We have studied the role of ER Ca²⁺ concentration ([Ca²⁺]_ER) in modulating this association in HeLa and HEK293 cells and human fibroblasts. According to Manders' coefficient, ER-mitochondria colocalization varied depending on the ER marker; it was the highest with ER-Tracker and the lowest with ER Ca²⁺ indicators (Mag-Fluo-4, erGAP3, and G-CEPIA1er) in both HeLa cells and human fibroblasts. Only GEM-CEPIA1er displayed a high colocalization with elongated mitochondria in HeLa cells, this ER Ca²⁺ indicator reveals low Ca²⁺ regions because this ion quenches its fluorescence. On the contrary, the typical rounded and fragmented mitochondria of HEK293 cells colocalized with Mag-Fluo-4 and, to a lesser extent, with GEM-CEPIA1er. The ablation of the three IP₃R isoforms in HEK293 cells increased mitochondria-GEM-CEPIA1er colocalization. This pattern of colocalization was inversely correlated with the rate of ER Ca²⁺ leak evoked by thapsigargin (Tg). Moreover, Tg and Histamine in the absence of external Ca²⁺ increased mitochondria-ER colocalization. On the contrary, in the presence of external Ca²⁺, both Bafilomycin A1 and Tg reduced the mitochondria-ER interaction. Notably, knocking down MCU decreased mitochondria-ER colocalization. Overall, our data suggest that the [Ca²⁺] is not homogenous within the ER lumen and that mitochondria-ER interaction is modulated by the ER Ca²⁺ leak and the [Ca²⁺]_i.