scholarly journals Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE

2022 ◽  
Vol 119 (3) ◽  
pp. e2114928118
Author(s):  
Moaz Ahmad ◽  
Hwei Ling Ong ◽  
Hassan Saadi ◽  
Ga-Yeon Son ◽  
Zahra Shokatian ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Gene Editing ◽  
Functional Communication ◽  
Store Depletion ◽  
Agonist Stimulation ◽  
First Response ◽  
N Terminus

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER–plasma membrane (ER–PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER–PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER–PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER–PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

scholarly journals Functional Communication between IP3R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

2021 ◽  
Author(s):  
Moaz Ahmad ◽  
Hwei Ling Ong ◽  
Hassan Saadi ◽  
Ga-Yeon Son ◽  
Zahra Shokatian ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Functional Communication ◽  
Functional Link ◽  
N Terminus

STIM proteins sense decreases in [Ca2+]ER and cluster in endoplasmic reticulum (ER)-plasma membrane (PM) junctions where they recruit and activate Orai1. While STIM1 clustering requires substantial [Ca2+]ER decrease, STIM2 displays pre-clustering under resting conditions and regulates basal Ca2+ entry. The mechanism(s) underlying constitutive clustering of STIM2 is not known. We show herein that endogenous STIM2 assembles as mobile and immobile clusters and that Orai1 is recruited to the latter. Anchoring of STIM2 clusters is triggered by decreases in local [Ca2+]ER that is mediated by ambient activity of IP3R and sensed by the STIM2 N-terminus. This functional link between IP3R and STIM2 governs constitutive STIM2 clustering and ensures coupling of [Ca2+]ER decrease at sub-threshold stimuli with activation of Ca2+ entry.

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+entry in mouse pancreatic acinar cells

2005 ◽  
Vol 288 (1) ◽  
pp. C214-C221 ◽  
Author(s):  
Juan A. Rosado ◽  
Pedro C. Redondo ◽  
Ginés M. Salido ◽  
Stewart O. Sage ◽  
Jose A. Pariente
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Cell ◽  
Botulinum Toxin A ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Amylase Secretion ◽  
Cell Type ◽  
Store Depletion

We recently reported that store-operated Ca2+entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

scholarly journals The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

2021 ◽  
Vol 118 (10) ◽  
pp. e2010789118
Author(s):  
Yihan Shen ◽  
Nagendra Babu Thillaiappan ◽  
Colin W. Taylor
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fluorescence Intensity ◽  
Small Cluster ◽  
Store Depletion ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cytosolic Domains ◽  
Intracellular Ca ◽  
The Many ◽  
Orai Channels

Increases in cytosolic Ca2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca2+ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca2+ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

scholarly journals Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates Ca2+ dynamics in T cells

2016 ◽  
Vol 113 (10) ◽  
pp. 2762-2767 ◽  
Author(s):  
Jin Seok Woo ◽  
Sonal Srikanth ◽  
Miyuki Nishi ◽  
Peipei Ping ◽  
Hiroshi Takeshima ◽  
...  
Keyword(s):  
T Cells ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Genetic Manipulation ◽  
Erk Signaling ◽  
Excitable Cells ◽  
Activated T Cells ◽  
Activation Markers ◽  
Extracellular Signaling ◽  
Store Depletion

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca2+ entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca2+ sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER–PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER–PM junctions to regulate Ca2+ signaling. Silencing or genetic manipulation of JP4 decreased ER Ca2+ content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca2+-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate–JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca2+ homeostasis and mediate SOCE in T cells.

scholarly journals State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

2014 ◽  
Vol 143 (5) ◽  
pp. 621-631 ◽  
Author(s):  
Anna Amcheslavsky ◽  
Olga Safrina ◽  
Michael D. Cahalan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cadmium Ions ◽  
Store Depletion ◽  
State Dependent ◽  
Conducting Pathway ◽  
Cysteine Scanning ◽  
Crac Channel ◽  
High Concentrations ◽  
Cysteine Scanning Mutagenesis

After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd2+). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd2+-induced block that is state dependent: Cd2+ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.

scholarly journals Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

2022 ◽  
Vol 79 (1) ◽  
Author(s):  
Jose Sanchez-Collado ◽  
Jose J. Lopez ◽  
Isaac Jardin ◽  
Alejandro Berna-Erro ◽  
Pedro J. Camello ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Store Depletion ◽  
Agonist Stimulation ◽  
Dependent Inactivation ◽  
Crac Channel ◽  
Membrane Location

AbstractThe identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

scholarly journals S-acylation of Orai1 regulates store-operated Ca2+ entry

2021 ◽  
Vol 135 (5) ◽  
Author(s):  
Savannah J. West ◽  
Goutham Kodakandla ◽  
Qioachu Wang ◽  
Ritika Tewari ◽  
Michael X. Zhu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Types ◽  
Central Component ◽  
Channel Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Crac Channel ◽  
Underlying Mechanisms ◽  
Different Cell Types

ABSTRACT Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER–PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER–PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER–PM junctions, subsequent binding to STIM1 and channel activation.

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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