Mitochondria-ER contacts restrain store-operated Ca{superscript 2}⁺ entry via Ca{superscript 2}⁺ flickering and STIM1 trapping

Ca{superscript 2} homeostasis requires coordinated regulation of ER stores and mitochondrial buffering. While the ER replenishes Ca{superscript 2} via STIM1-mediated store-operated Ca{superscript 2} entry (SOCE), how mitochondria-ER contact sites (MERCs) influence this process remains unclear. Here, using fluorescent live imaging, we capture spontaneous mitochondrial Ca{superscript 2} flickering at MERCs, which is driven by the IPR-VDAC1-mediated Ca{superscript 2} transfer and exerts an inhibitory effect on SOCE. Mechanistically, MERCs establish local ER Ca{superscript 2} depletion microdomains through this Ca{superscript 2} 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, Ca{superscript 2}-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 Ca{superscript 2} overload.