Mitochondria-ER contacts restrain store-operated Ca²⁺ entry via Ca²⁺ flickering and STIM1 trapping

Ca²⁺ homeostasis requires coordinated regulation of ER stores and mitochondrial buffering. While the ER replenishes Ca²⁺ via STIM1-mediated store-operated Ca²⁺ entry (SOCE), how mitochondria–ER contact sites (MERCs) influence this process remains unclear. Here, using fluorescent live imaging, we capture spontaneous mitochondrial Ca²⁺ flickering at MERCs, which is driven by the IP₃R–VDAC1-mediated Ca²⁺ transfer and exerts an inhibitory effect on SOCE. Mechanistically, MERCs establish local ER Ca²⁺ depletion microdomains through this Ca²⁺ transfer. These microdomains act as molecular traps, triggering STIM1 accumulation at MERCs via its polybasic K-domain and sequestering it away from the plasma membrane (PM) to suppress SOCE. Furthermore, acute MERC induction redistributes constitutively active, Ca²⁺-insensitive STIM1 away from ER–PM junctions, demonstrating that MERCs directly outcompete the PM for STIM1 recruitment. Finally, the microtubule (EB1)–ER contacts serve as a finite-capacity reservoir that buffers excess STIM1, with its disruption unmasking the dynamic competition between MERCs and PM. Together, our findings establish a tripartite system—PM, mitochondria, and microtubules—that dictates SOCE by controlling STIM1 topography, thereby protecting cells against Ca²⁺ overload.