Bcl-2 suppresses ER-mitochondrial Ca2+ transfer by directly limiting ER-mitochondrial contact sites

<title>Abstract</title> <p> Anti-apoptotic B cell lymphoma (Bcl-2) tightly regulates apoptosis by controlling mitochondrial outer membrane permeabilization and intracellular Ca ²⁺ dynamics. At the endoplasmic reticulum (ER), Bcl-2 directly inhibits inositol 1,4,5-trisphosphate receptors (IP ₃ Rs), intracellular Ca ²⁺ -release channels, thereby limiting mitochondrial Ca ²⁺ overload. Yet, Bcl-2’s non-canonical function in cells not exposed to apoptotic insults remains poorly understood. Here, we set out to study this function by generating CRISPR-Cas9-mediated Bcl-2 knockout (Bcl-2 KO) in HeLa cells, which are not dependent on Bcl-2 for their survival. Bcl-2 KO did not evoke spontaneous cell death, but enhanced mitochondrial respiration. Mechanistically, Bcl-2 KO increased mitochondrial Ca ²⁺ uptake, which was restored by Bcl-2 re-expression. Moreover, Bcl-2-deficient cells displayed increased mitochondria-ER contact sites (MERCS), including short contacts (10 nm) and contacts at an optimal distance (20 nm) for efficient ER-mitochondrial Ca ²⁺ transfer, without altering the abundance of major MERCS-tethering proteins. Enforcing a 20 nm ER-mitochondrial distance using 20 nm molecular rulers increased mitochondrial Ca ²⁺ influx in control cells to similar levels as those in Bcl-2 KO cells. <italic>Vice versa</italic> , Bcl-2 overexpression limited the number of 10 and 20 nm MERCS, an effect of Bcl-2 that was largely dependent on the presence of IP ₃ Rs. Therefore, we identified a novel role of Bcl-2 at MERCS where it controls ER-mitochondrial distance, thereby providing a yet unappreciated action of Bcl-2 that helps preventing mitochondrial Ca ²⁺ overload. These new insights into Bcl-2’s non-canonical role may contribute to a better understanding of altered ER-mitochondrial Ca ²⁺ signalling in disease conditions associated with Bcl-2 upregulation such as haematological cancers. </p>