Abstract
In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.
Highlights
Various cellular organelles communicate via membrane contact sites (MCSs) that mediate important cellular processes, such as intracellular signaling, metabolism, metabolite trafficking and organelle division[1]
Upon endoplasmic reticulum (ER) Ca2+ store depletion, STIM1 gradually aggregated to induce new ER-plasma membrane (PM) MCSs formation, as indicated by the increased number and size of the STIM1-EGFP and mCherry-Sec 61β and KDEL rings (Sec 61β), which remarkably co-clustered within 50 nm beneath the PM in the raw images, and the images rendered by volumetric imaging (Fig. 2a) and quantification analysis (Fig. 2c)
Based on our data and current knowledge, we propose the following model (Fig. 5): in non-excitable cells, the agonist- or antigen-mediated activation of G protein receptors on the PM increases phospholipid C activity, which hydrolyzes PIP2 to generate inositol trisphosphate to release ER Ca2+ stores
Summary
Various cellular organelles communicate via MCSs that mediate important cellular processes, such as intracellular signaling, metabolism, metabolite trafficking and organelle division[1]. Upon depletion of Ca2+ stores, STIM1 on the ER membrane changes configuration to form transient MCSs via its interaction with Orai[1] on the PM2, which opens Ca2+ release-activated Ca2+ (CRAC) channels to mediate the store-operated Ca2+ entry (SOCE)[3]. E-syt1-MCSs appear partially co-localized with STIM1 in live HeLa cells upon ER Ca2+ store depletion and replenishment[5], and catalyze phospholipid shuttling between ER and PM that mediates the sustained activation of the receptor-induced Ca2+ signaling pathway[6] and the slow Ca2+-dependent inactivation of the CRAC current[7]. Super-resolution imaging techniques were used to explore the structure of MCSs12, the prerequisite need for excessive photons has prevented the unequivocal visualization of the in vivo assembly and disassembly of ER-PM MCSs in live cells
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