Supplementary material from "STIM1 and Endoplasmic Reticulum-Plasma Membrane Contact Sites Oscillate Independently of Calcium-Induced Calcium Release"

Calcium (Ca²⁺) release from endoplasmic reticulum (ER) and Ca²⁺ entry across the plasma membrane are both critical for receptor-activated Ca²⁺ oscillations. However, the precise mechanism of Ca²⁺ oscillations and whether their control loop resides at the plasma membrane or intracellularly remain unresolved. By examining the dynamics of stromal interaction molecule 1 (STIM1), an ER-localized Ca²⁺ sensor that activates the Orai1 channel on the plasma membrane for store-operated Ca²⁺ entry (SOCE), in mast cells, we found that STIM1 could oscillate with the same periodicity as Ca²⁺ oscillations. These STIM1 oscillations, shared with other ER-PM contact sites proteins, could occur independently of Ca²⁺ oscillations. Simultaneous imaging of cytoplasmic and ER Ca²⁺ revealed that receptor activation does not deplete ER Ca²⁺, whereas receptor activation without extracellular Ca²⁺ influx induces cyclic ER Ca²⁺ depletion. Cyclic ER Ca²⁺ oscillations, under nonphysiological conditions, lead to sustained STIM1 recruitment but not oscillations, indicating that oscillatory Ca²⁺ release is not sufficient for STIM1 oscillations. Using optogenetic tools to manipulate ER-PM contact site dynamics, we found that persistent ER-PM contact sites reduced the amplitude of Ca²⁺ oscillations without alteration of their frequency. Together, these findings suggest a cortical mechanism governs the ER-PM contact sites and regulates receptor-induced Ca²⁺ oscillations.