Enforced tethering elongates the cortical endoplasmic reticulum and limits store-operated Ca2+ entry

Abstract

Recruitment of STIM proteins to cortical ER (cER) domains forming membrane contact sites (MCS) mediate the store-operated Ca2+ entry (SOCE) pathway essential for human immunity. The cER is dynamically regulated by STIM and tethering proteins during SOCE, but the ultrastructural rearrangement and functional consequences of cER remodelling are unknown. Here, we express natural (E-Syt1/2) and artificial (MAPPER-S/L) protein tethers in HEK-293T cells and correlate the changes in cER length and gap distance measured by electron microscopy with ionic fluxes. Native cER cisternae extended during store depletion and remained elongated at constant ER-PM gap distance during subsequent Ca2+ elevations. Tethering proteins enhanced store-dependent cER expansion, anchoring the enlarged cER at tether-specific gap distances of 12-15nm (E-Syts) and 5-9nm (MAPPERs). Cells with artificially extended cER had reduced SOCE and reduced agonist-induced Ca2+ release. SOCE remained modulated by calmodulin and exhibited enhanced Ca2+-dependent inhibition. We propose that cER expansion mediated by ER-PM tethering at a close distance negatively regulates SOCE by confining STIM-ORAI complexes to the periphery of enlarged cER sheets, a process that might participate in the termination of store-operated Ca2+ entry.

Architecture of Ca2+tunneling, a basic Ca2+signaling modality important for secretion

“…When the ERPMCS gap was artificially shorten using chemically induced linkers to below 6 nm, this prevented stabilization of the STIM1-Orai1 clusters within the junction and favored their enrichment at the junction periphery 32 . Furthermore, similar to our findings Henry et al showed that MAPPER localizes to a different subdomain than STIM1 within ERPMCS, whereas a longer tether Sec22 colocalized with STIM1 in ERPMCS 31 , similar to what we show herein for two endogenous tethers TMEM24 and E-Syt2 (Fig. 5C-E).…”

“…ERPMCS are close appositions between the ER and PM where the two membranes are <30 nm apart allowing the STIM1 cytoplasmic domain in its extended conformation to span the gap and activate Orai1 [26][27][28] . The gap distance between the ER and PM is controlled by different tethering proteins physiologically and can be modulated experimentally using artificial linkers [29][30][31][32] . ERPMCS are present at steady state (when Ca 2+ stores are full) and are stabilized by tethering proteins such as the extended synaptotagmins (E-Syt), TMEM24, ORPs, and GRAMDs 33 .…”

Extending the Contacts Breaks the Flow

“…Resolving MCS morphological changes is difficult with optical approaches, and numerous studies report alterations in MCS structures of little functional significance and functional defects occurring without changes in MCS structure. In a recent study, we attempted to fill this gap in knowledge by performing a quantitative and systematic evaluation of the ultrastructural changes occurring at MCS during the SOCE process using the gold standard of electron microscopy (Henry et al, 2022). SOCE is triggered physiologically following agonist stimulation by the generation of inositol trisphosphate that releases Ca 2+ from the ER into the cytoplasm.…”

Keep in contact: multiple roles of endoplasmic reticulum–membrane contact sites and the organelle interaction network in plants

“…At low levels of expression, MAPER‐GFP localises to the ER network and is preferably enriched at the ER–PM junction. However, when its expression is high, this reporter inevitably induces additional ER–PM interaction by enhancing ER–PM interaction and therefore affecting ER morphology (Henry et al ., 2022). So, when studying ER membrane interactions with any of the reporters that are currently available, it is essential that the impact of the level of reporter expression is considered.…”

“…Upon depletion of the ER calcium store, STIM1 oligomerises at the ER–PM junction (with the help of STIM2) and triggers the interaction and activation of Orai1 to allow Ca 2+ entry across the PM (Son et al ., 2020). This is a precisely regulated process, which requires a constant distance between ER–PM; cells with artificially enlarged cortical ER and enhanced ER–PM interaction are less sensitive to agonist‐induced Ca 2+ release (Henry et al ., 2022). Conversely, EPCS formation may also negatively regulate ion transport, for example potassium.…”