Inositol-1,4,5-trisphosphate (IP3)-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake

Abstract

Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER–plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains.

Mitochondrial Ca2+ uptake controls actin cytoskeleton dynamics during cell migration

“…These microdomains, also called Ca 2+ flickers are thought to be generated by Ca 2+ entry from the plasma membrane51 and by IP 3 -induced Ca 2+ release from the ER20. Here, we report that both cytosolic and ER Ca 2+ pools are decreased in mcu knocked down cells, confirming the requirement of an intact mitochondrial Ca 2+ uptake machinery to sustain SOCE1838. In mcu -silenced cells, altered SOCE may thus compromise the generation of Ca 2+ pulses, affecting in turn FAPs dynamics and cell migration.…”

“…This phenotype appears to be mediated by the decrease of RhoA and Rac1 activities as well as downregulation of total Calpain activity, two Ca 2+ dependent parameters. Actually, a direct lowering of intracellular Ca 2+ levels using BAPTA-AM or inhibition of the SOCE by silencing STIM118 mimicked mcu knock down phenotype. Importantly, treatment of the cells with the potent MCU inhibitor Ru360, resulted in a decreased capacity of the cells to close the gap, which illustrates the primordial role of mitochondrial Ca 2+ homeostasis in the cell migration process.…”

“…These organelles can rapidly uptake substantial amounts of Ca 2+ though the existence of Ca 2+ hot spots localized at the interface between the mitochondria and the endoplasmic reticulum (ER)14. The mitochondrial Ca 2+ uptake capacities have been also linked to an efficient Store-Operated Ca 2+ Entry (SOCE)15161718. Interestingly, the role of the SOCE process, which is regulated in part by the ER-resident Stromal Interacting Molecule 1 (STIM1) and Calcium release-activated calcium channel protein 1 (Orai1), has been highlighted in the actomyosin contractility1920 and breast tumor cell migration21.…”

Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

“…In contrast, the release of calcium from the ER in response to the SERCA inhibitor thapsigargin, which has been reported not to promote IP 3 production, was not inhibited by pyrrophenone [23, 37]. Thapsigargin does not generate the formation of high-concentration Ca 2+ micro-domains that are required for mitochondrial calcium uptake consistent with studies showing that it is not as effective in increasing mitochondrial calcium as the IP 3 -mediated pathway [31, 38, 39]. Steady state ER calcium levels are maintained by the continuous cycling of calcium in and out of the ER through SERCA-mediated uptake and passive leakage through basal ER calcium-leak channels [40].…”

The molecular era of the mitochondrial calcium uniporter

“…The uniporter seems to be important for two additional processes that are relevant to immune signalling: store-operated calcium entry (SOCE) and activation of the NOD-, LRR-and pyrin domaincontaining 3 (NLRP3) complex termed the NLRP3 inflammasome. Specifically, loss of MCU has been shown to reduce SOCE after inositol trisphosphate-mediated calcium release 54 and to blunt activation of the NLRP3 inflammasome induced by both the membrane attack complex in human lung epithelial cells 55 and by Pseudomonas aeruginosa in airway epithelial cells from patients with cystic fibrosis 56 . Taken together, these lines of evidence confirm a role for the uniporter in cellular calcium signallin g and point to its importance in the immune system.…”