STIM1 and Endoplasmic Reticulum-Plasma Membrane Contact Sites Oscillate Independently of Calcium-Induced Calcium Release

Calcium (Ca²⁺) release from intracellular stores, Ca²⁺ entry across the plasma membrane, and their coordination via store-operated Ca²⁺ entry (SOCE) are 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 endoplasmic reticulum (ER)-localized Ca²⁺ sensor that activates the Orai1 channel on the plasma membrane for SOCE, in mast cells, we found that a significant proportion of cells exhibited STIM1 oscillations with the same periodicity as Ca²⁺ oscillations. These cortical oscillations, shared with ER-plasma membrane (ER-PM) contact sites proteins, were only detectable using total internal reflection fluorescence microscopy (TIRFM). Notably, STIM1 oscillations could occur independently of Ca²⁺ oscillations. Simultaneous imaging of cytoplasmic Ca²⁺ and ER Ca²⁺ with CEPIA1er revealed that receptor activation does not deplete ER Ca²⁺, whereas receptor activation without extracellular Ca²⁺ influx induces cyclic ER Ca²⁺ depletion. However, under such nonphysiological conditions, cyclic ER Ca²⁺ oscillations lead to sustained STIM1 recruitment, indicating that oscillatory Ca²⁺ release is neither necessary nor 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 oscillation frequency. Together, these findings suggest an active cortical mechanism governs the rapid dissociation of ER-PM contact sites, thereby control amplitude of oscillatory Ca²⁺ dynamics during receptor-induced Ca²⁺ oscillations.细胞内钙库释放钙离子（Ca²⁺）、钙离子跨质膜内流，以及二者通过钙池操纵性钙内流（store-operated Ca²⁺ entry, SOCE）实现的协同调控，对于受体激活的钙振荡至关重要。然而，钙振荡的确切机制，以及其调控环路定位于质膜还是细胞内区域，目前仍未明确。本研究以肥大细胞为模型，监测了定位于内质网（endoplasmic reticulum, ER）的钙离子感受器——基质相互作用分子1（stromal interaction molecule 1, STIM1）的动态变化，该蛋白可激活质膜上介导SOCE的Orai1通道。研究发现，相当比例的细胞呈现出与钙振荡周期一致的STIM1振荡。这类与内质网-质膜（ER-PM）接触位点蛋白共有的细胞皮层振荡，仅能通过总内反射荧光显微镜（total internal reflection fluorescence microscopy, TIRFM）观测到。值得注意的是，STIM1振荡可独立于钙振荡发生。利用CEPIA1er探针对细胞质钙离子与内质网钙离子进行同时成像后发现：受体激活并不会导致内质网钙离子耗竭；而在无细胞外钙离子内流的条件下，受体激活会诱导周期性内质网钙离子耗竭。但在这类非生理条件下，周期性内质网钙振荡会引发STIM1的持续募集，这表明振荡性钙释放既不是STIM1振荡发生的必要条件，也非充分条件。利用光遗传工具调控ER-PM接触位点的动态变化后，本研究发现持续存在的ER-PM接触位点会降低钙振荡的振幅，但不会改变振荡的频率。综上，上述研究结果提示，存在一种活跃的细胞皮层调控机制，可介导内质网-质膜接触位点的快速解离，从而在受体诱导的钙振荡过程中调控钙振荡动态的振幅。