Journal: Journal of Innate Immunity

Article Title: Mannan-Binding Lectin Reduces Epithelial-Mesenchymal Transition in Pulmonary Fibrosis via Inactivating the Store-Operated Calcium Entry Machinery

doi: 10.1159/000524693

Figure Lengend Snippet: Primers for the target genes

Article Snippet: The membranes were then stained with HRP-conjugated secondary antibody (S0001; Affinity Biosciences) at room temperature for another 1 h. Antibodies involved in this subsection are listed below: E-cadherin (14472, mouse anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), N-cadherin (22018-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), α-SMA (14395-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), vimentin (10366-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Orai1 (66223-1-Ig, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Stim1 (5668, rabbit anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), SGK1 (23394-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), PDK1 (17086-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), GAPDH (60004-1-Ig, mouse anti-human monoclonal antibody, 1:1,000 dilution; Proteintech), and Ub (sc-8017, mouse anti-human monoclonal antibody, 1:1,000 dilution; Santa Cruz Biotechnology).

Techniques:

Journal: Journal of Innate Immunity

Article Title: Mannan-Binding Lectin Reduces Epithelial-Mesenchymal Transition in Pulmonary Fibrosis via Inactivating the Store-Operated Calcium Entry Machinery

doi: 10.1159/000524693

Figure Lengend Snippet: MBL mediated Orai1 ubiquitination. a, b HBE cells were incubated with 10-ng/mL TGF-β in the presence or absence of 10-μg/mL MBL for 24 h. a mRNA levels of Orai1 and Stim1 were assessed by quantitative RT-PCR. b Orai1 and Stim1 expression were determined by Western blot. c Orai1 and Stim1 levels in mice lung tissues were detected by Western blot. d HBE cells were incubated with 10-ng/mL TGF-β in the presence or absence of 10-μg/mL MBL for 24 h. Cells were treated with 10-μM MG132 4 h before being harvested. The ubiquitination level of Orai1 was detected by immunoprecipitation assay. ns, not significant, * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results. Unpaired Student's t test was used in a .

Article Snippet: The membranes were then stained with HRP-conjugated secondary antibody (S0001; Affinity Biosciences) at room temperature for another 1 h. Antibodies involved in this subsection are listed below: E-cadherin (14472, mouse anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), N-cadherin (22018-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), α-SMA (14395-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), vimentin (10366-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Orai1 (66223-1-Ig, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Stim1 (5668, rabbit anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), SGK1 (23394-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), PDK1 (17086-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), GAPDH (60004-1-Ig, mouse anti-human monoclonal antibody, 1:1,000 dilution; Proteintech), and Ub (sc-8017, mouse anti-human monoclonal antibody, 1:1,000 dilution; Santa Cruz Biotechnology).

Techniques: Ubiquitin Proteomics, Incubation, Quantitative RT-PCR, Expressing, Western Blot, Immunoprecipitation

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Activity Assay, Incubation, Expressing, Fluorescence, Control, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Inhibition, Patch Clamp, Transferring, Control, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

doi: 10.1371/journal.pone.0151031

Figure Lengend Snippet: (A ) Ca 2+ flux induced by Bourgeonal, Oxa and DNFB in the presence of 2-APB (100 μM) and BTP2 (10 μM) in NK cells. The response by the odorant/hapten is shown in red, that in the presence of 2-APB is in blue, and that of BTP2 in yellow. The bar graph depicts the mean fold change (±SEM) in Indo-1 emission (i.e. the Indo-1V/Indo-1B ratio observed at the peak response relative to that of the baseline) induced by the stimulus alone (black bar) or the stimulus plus 2-APB (dark grey bar) or BTP2 (light grey) in NK cells from naive mice. n = 3 independent experiments, statistics as compared to stimulus alone using t-test ** p<0.01, * p<0.05, ns not significant (p>0.05). ( B ) Ca 2+ flux induced by Bourgeonal, Oxa and DNFB in the presence of 2-APB (100 μM) and BTP2 (10 μM) in Jurkat cells ( B ). Representative of 2–3 independent experiments. (C ) HEK293 cells were stably transfected with hSTIM1 without or with hORAI1, hORAI2 or hORAI3. Transfectants were kept in Ca 2+ -free medium and intracellular Ca 2+ stores were depleted using Thapsigargin (TG) (1 μM). Ca 2+ entry was measured following the addition of extracellular CaCl 2 (1 mM). Maximal Ca 2+ entry was detected when HEK293 cells co-expressed hSTIM1 plus hORAI1, hORAI2 or hORAI3. (F ) Oxa (0.4 mM) and DNFB (0.25 mM) fail to induce Ca 2+ flux in hSTIM1/hORAI transfected HEK293 cells.

Article Snippet: Yasuo Mori, Kyoto University), pcDNA3.1-ORAI1 (Addgene #21638), ORAI2 (#16369), ORAI3 (#16370) and human STIM1-YFP (#19754) using calcium-phosphate transfection.

Techniques: Stable Transfection, Transfection

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Photostimulation was applied at 470 nm (4.0 mW/cm 2 ). Data were shown as mean ± sem. Scale bar, 5 µm. a Domain architecture of the human STIM1. SP, signal peptide; EF-SAM, EF-hand and sterile alpha-motif; TM, transmembrane domain; CC1, coiled-coil domain 1; SOAR, STIM-Orai activating region; P/S, proline/serine-rich region; TRIP, the S/TxIP microtubule-binding motif; PB, polybasic tail. b Schematic of STIM1–ORAI1 coupling at the ER–PM junction that mediates store-operated Ca 2+ entry. c – e Use of the iLID-sspB optical dimerizer to trigger STIM1ct activation and Ca 2+ influx through endogenous ORAI channels. c Schematic of the design. iLID or sspB was fused to the N-terminus of STIM1ct at residue 233. d Confocal images showing photoswitchable Ca 2+ influx in HeLa cells co-transfected with a red Ca 2+ sensor (R-GECO 1.2) and the iLID/sspB fused STIM1ct chimeras. Cells were exposed to two repeated dark-light cycles. e Quantitative analysis of Ca 2+ signals in response to repeated photostimulation ( n = 40 cells from three independent experiments). The half-lives ( t 1/2 ) of on and off kinetics were fitted with one phase exponential decay (“±” means 95% confidence interval). f – h Use of the CRY2-CIBN optical dimerizer to photo-activate STIM1ct and Ca 2+ influx. f Schematic of the design. CRY2 was used to photo-crosslink CIBN-STIM1ct and trigger STIM1ct activation to induce Ca 2+ entry. g Confocal images showing light-induced co-localization of mCherry (mCh)-tagged CIBN-STIM1ct with YFP-ORAI1 in HeLa cells. h Reversible Ca 2+ responses monitored by R-GECO 1.2 ( n = 30 cells). Blue bar, photostimulation at 470 nm with a power density of 4 mW/cm 2 . i – k ER-tethered CRY2-STIM1ct mimics STIM1 puncta formation at ER–PM junctions to evoke localized Ca 2+ influx. i Schematic of the design. j Confocal images illustrating light-induced clustering of ER-resident CRY2-STIM1ct at the footprint of HeLa cells. Enlarged views of the boxed regions were shown on the right. k Cytosolic Ca 2+ signals reported by R-GECO1.2 in HeLa cells subjected to two repeated dark-light cycles ( n = 30).

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Binding Assay, Activation Assay, Transfection

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of a split STIM1 molecule (at residue 342) to monitor CC1–SOAR interaction in trans at real time. CC1–SOAR maintains STIM1ct in an inactive configuration at rest. As a result, Part II (mCh-STIM1 342–685 ) tightly docks to the ER-resident Part I (STIM1 1–342 -YFP) when the store remains full. Upon store depletion, structural changes propagate toward the CC1 region to weaken its association with SOAR, thereby leading to the cytosolic dispersion of Part II as shown in panel b . b Confocal images showing the distribution of split STIM1 molecules (green, STIM1 1–342 -YFP; red, mCh-STIM1 343–685 ) before and after thapsigargin (TG)-induced store depletion in HeLa cells. c Schematic illustrating the design of a LOV2-SOAR (STIM1 336–486 ) chimera to mimic the CC1–SOAR interaction that locks STIM1ct in an inactive state. CC1 is replaced by LOV2 (light-oxygen-voltage domain 2) to tightly cage SOAR in the dark. Upon blue light stimulation, the Jα helix becomes disordered to uncage SOAR, thereby restoring its activity to engage and gate ORAI channels. If the ER-resident Part I (STIM1 1–342 -YFP) and PM-embedded ORAI1 are co-expressed, LOV2-SOAR can be used to determine the relative binding strength of SOAR toward ER-anchored CC1 or PM-resident ORAI1 channels. d Light-inducible cytosol-to-ER translocation of mCh-LOV2-SOAR in HEK293 cells co-transfected with Part I as shown in panel c . e Quantification of cytosolic mCherry signals (images in panel d ) following three repeated light-dark cycles ( n = 18 cells). f – h Comparison of the relative strength of SOAR-CC1 and SOAR-ORAI1 interactions. f Top: Light-induced cytosol-to-PM translocation of mCh-LOV2-SOAR (gray) observed in HEK293 cells co-transfected with YFP-ORAI1 (green). Bottom: Confocal images of HEK293 cells co-expressing mCh-LOV2-SOAR (gray), YFP-ORAI (green) and STIM1 1–342 -CFP (cyan). In the dark, mCh-LOV2-SOAR was evenly distributed in the cytosol. Upon photostimulation, mCh-LOV2-SOAR preferred to translocate toward ER membrane but not to PM. g The fluorescence intensities (YFP, green; mCh, red; CFP, cyan) across the dashed line were plotted to evaluate the degree of colocalization. h Light-induced Ca 2+ response curves (quantified by GCaMP6s) in HEK293 cells transfected with LOV2-SOAR (red), LOV2-SOAR + ORAI1 (green) or LOV2-SOAR + Part I (STIM1 1–342 ; blue). n = 30 cells.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Activity Assay, Binding Assay, Translocation Assay, Transfection, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of an optogenetic clustering assay to examine real-time protein–protein interactions in living cells. b Summary of mCh-tagged baits and YFP-tagged preys used to map critical domains in STIM1ct that dictate STIM1 oligomerization. c Representative confocal images showing the intracellular distribution of the bait (mCh-CRY2-STIM1 233–448 ) and two different preys (P1—top panel, YFP-STIM1 233–658 ; P3—bottom panel, YFP-STIM1 233–342 ) before and after blue light stimulation in HeLa cells. d Time courses showing the kinetics of light-induced clustering (F cluster /F neighbor ) of the bait and its co-clustering with P1 (blue), but not with P3 (green), as seen in panel c . n = 18 cells. ( e ) Quantification of the degrees of light-inducible co-clustering for the five indicated preys in HeLa cells co-transfected with the bait. n = 18 cells from three independent experiments. f Summary of the optogenetic co-clustering assay results for the bait–prey combinations shown in panel b . “+” means co-clustering notably observed after photo-illumination; “−” means no appreciable cluster formation before and after blue light stimulation. g – i Light inducible co-clustering to dissect the STIM1 luminal EF-SAM domain. g Schematic showing the design of bait–prey constructs. h – i Representative confocal images of HeLa cells co-expressing ( h ) mCh-CRY2-EF (B4, STIM1 32–128, EF-hand ; red) with YFP-EF (P6, green), or ( i ) mCh-CRY2-SAM (B5, STIM1 128–200, SAM ; red) with YFP-SAM (P7; red) under dark (left) and blue light (right). The selected regions (dashed boxes) were enlarged to aid visualization. Scale bar 5 µm.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Transfection, Construct, Expressing

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of the high-throughput screening pipeline. The cytosol-to-PM translocation and Ca 2+ influx (GCaMP6s as readout) were used as two readouts. b Sequence alignment of human SOAR1 and SOAR2 domains and the 3D structure of SOAR1 (PDB entry: 3TEQ). Key residues at the interdimer interface or involved in ORAI1-binding were indicated by dots and triangles, respectively. Selected key residues were highlighted in the 3D structure. c Quantification of Ca 2+ responses (GCaMP6s) and PM translocation (mCherry signals) of selected CRY2-STIM1ct mutants before (dark dots) and after (blue dots) photostimulation. HeLa-GCaMP6s stable cells were co-transfected with each of the indicated mCh-CRY2-STIM1ct mutants and ORAI1-CFP. d Time courses showing the kinetics of light-induced Ca 2+ influx for WT and the indicated mCh-CRT2-STIM1ct variants. n = 60 cells. e – g Representative confocal images ( e ) and quantification of intracellular Ca 2+ signals, n = 60 cells. Box-whisker plots indicated the median, and the interquartile range with 5–95 percentile distribution. f , as well light-induced PM translocation, n = 8 cells ( g ), in HeLa cells expressing WT or the indicated mCh-CRY2-STIM1ct mutants. h Representative confocal images of HEK293 S1-KO cells expressing the GFP-tagged full-length STIM1-T393F mutant before and after TG-induced store depletion. i SOCE monitored by R-GECO1.2 in HEK293 S1-KO cells expressing GFP-STIM1 WT or the mutant T393F. n = 90 cells. j Summary of the degrees of Ca 2+ influx and PM translocation of cancer-associated mutations found in the SOAR domains of STIM1. HeLa cells were transfected with the indicated mCh-CRT2-STIM1ct mutants. Gain-of-function (H395Y and R424W; red) and loss-of-function (L402R, R426L/C, R429C; green) mutations were both identified. n = 60 cells.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: High Throughput Screening Assay, Translocation Assay, Sequencing, Binding Assay, Transfection, Whisker Assay, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Diagram of the STIM1 C-terminal fragment (residues 443–685) that contains both the S/TxIP EB1-binding motif and the positively charged polybasic tail (PB) that interacts with PM-resident PIPs. Mutations used in this study to perturb STIM1ct-target interactions were highlighted in red. b , c Schematic ( b ) and representative confocal images ( c ) showing light-inducible bimodal distribution (tracking of MT plus ends or cytosol-to-PM translocation) of mCh-CRY2-STIM1 443–685 in COS-7 cells. d Time courses of light-triggered MT plus-end tracking (green) and PM translocation (blue) of mCh-CRY2-STIM1 443–685 . Upon blue light illumination, cytosolic mCherry signals rapidly reduced ( t 1/2 = 18.2 ± 5.0 s), accompanied with the increase of MT tip tracking ( t 1/2 = 21.5 ± 7.3 s) or PM decoration ( t 1/2 = 27.6 ± 6.2 s). n = 12 cells. e , f Representative confocal images ( e ) and quantifications of MT plus-end tracking ( f ) of mCh-CRY2-STIM1ct variants (top, WT; bottom, P645N in the context of STIM1 443–670 ) in COS-7 cells co-transfected with EB1-GFP (green). The bar graph showed the averaged values of MT tip-to-cytosol intensity ratio under dark and lit conditions. n = 16 cells from three independent experiments. g , h Representative confocal images in HeLa cells ( g ) and quantification of the cytosol-to-PM translocation ( h ) of the indicated CRY2-PB variants before and after blue light illumination. n = 16 cells from three independent experiments. i – k Schematic showing light inducible ER-MT interactions in COS-7 cells cotransfected ER-anchored mCh-CRY2-STIM1 443–670 (red) with ( j ) EB1-GFP (green) or ( k ) GFP-tubulin (green). j Representative confocal images showing ER morphology change following blue light illumination. The mCherry and GFP fluorescent intensities across the dashed line were plotted next to the images to indicate the degree of signal overlaps. k Confocal images showing the clustering of ER-resident mCh-CRY2-STIM1 443–670 along with GFP-tubulin (green) upon blue light illumination. The surface profiles of a selected area (oval) were presented to aid the visualization of subcellular distribution. l , m Light-inducible assembly of ER-plasma membrane contact sites (MCSs) mediated by ER-resident mCh-CRY2-PB (STIM1 671–685 ). l Schematic of the design. m Representative confocal images of the footprint of HeLa cells transfected with mCh-CRY2-PB variants before and after blue light illumination.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Binding Assay, Translocation Assay, Transfection

Journal: eLife

Article Title: Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2+

doi: 10.7554/elife.80447

Figure Lengend Snippet: Figure 3. Regulation of SOCE by IP3R requires IP3 binding but not a functional pPore in SH-SY5Y cells. (A) SOCE is activated when loss of Ca2+ from the ER through IP3Rs activates STIM1 (i). Our results suggest an additional role for IP3Rs (ii). (B) SH-SY5Y cells expressing IP3R1-shRNA alone or with IP3R1 or IP3R1DA were stimulated with thapsigargin (Tg, 1 µM) in Ca2+-free HBSS before restoring extracellular Ca2+ (2 mM). Traces show mean ± s.e.m, for 100–150 cells from three experiments. (C) Cells expressing IP3R1-shRNA and IP3R1DA were treated with NS-siRNA or Orai1-siRNA before measuring Tg-evoked Ca2+ entry. Traces show mean ± s.e.m. for 85–100 cells from three experiments. (D) Summary results (mean ± s.e.m.) show peak increases in [Ca2+]c (Δ[Ca2+]c) evoked by Ca2+ restoration. (E) Tg-evoked Ca2+ entry in cells expressing IP3R1-shRNA with IP3R1, IP3R1RQ or IP3R1RQ/KQ. Traces show mean ± s.e.m, for 90–150 cells from three experiments. (F) Summary results (mean ± s.e.m.) show peak increases in [Ca2+]c (Δ[Ca2+]c) evoked by Ca2+ restoration. Different letter codes (panels D, F) indicate significantly different values, p<0.001, for multiple comparison one-way ANOVA and pair-wise Tukey’s test and for two genotype comparison Mann Whitney U-test. See also Figure 3—figure supplement 1—source data 1. Source data in Figure 3—source data 1.

Article Snippet: The siRNAs used were to human Orai1 (100 nM, Dharmacon, Cat# L- 014998- 00- 0005) or non- silencing (NS, Dharmacon, Cat# D- 495 001810- 10- 05), to human STIM1 (Santa Cruz Biotechnology, Cat# sc- 76589) or NS (Santa Cruz Biotechnology, Cat# sc- 37007).

Techniques: Binding Assay, Functional Assay, Expressing, shRNA, Comparison, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2+

doi: 10.7554/elife.80447

Figure Lengend Snippet: Figure 5. IP3Rs promote interaction of STIM1 with Orai1. (A–E) PLA analyses of interactions between STIM1 and Orai1 in SH-SY5Y cells expressing NS-shRNA (A) or IP3R1-shRNA alone (B) or with IP3R1 (C), IP3R1DA (D) or IP3R1RQ/KQ (E). Confocal images are shown for control cells or after treatment with thapsigargin (Tg, 1 µM) in Ca2+-free HBSS. PLA reaction product is red, and nuclei are stained with DAPI (blue). Scale bars, 5 µm. Summary results show

Article Snippet: The siRNAs used were to human Orai1 (100 nM, Dharmacon, Cat# L- 014998- 00- 0005) or non- silencing (NS, Dharmacon, Cat# D- 495 001810- 10- 05), to human STIM1 (Santa Cruz Biotechnology, Cat# sc- 76589) or NS (Santa Cruz Biotechnology, Cat# sc- 37007).

Techniques: Expressing, shRNA, Control, Staining

Journal: eLife

Article Title: Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2+

doi: 10.7554/elife.80447

Figure Lengend Snippet: Figure 6. Ligand-bound IP3R1 supports SOCE-dependent STIM1 movement to ER-PM contact sites. (A–B) Representative TIRF images of mVenus STIM1 co-transfected with either wild type mcherry-rat IP3R1 (A) or IP3R1RQ/KQ (ligand binding mutant), (B) in wild type SH-SY5Y cells before (Basal) and after CPA induced store depletion (CPA treated) at 4 min and 7 min. On the right are shown RGB profile plots of STIM1 (green) and IP3R1, wild type or mutant (magenta) corresponding to the rectangular selections (Cell 1 and Cell 2). Scale bar is 10 µm.(C–D) Changes in number of IP3R1 (C) and STIM1

Article Snippet: The siRNAs used were to human Orai1 (100 nM, Dharmacon, Cat# L- 014998- 00- 0005) or non- silencing (NS, Dharmacon, Cat# D- 495 001810- 10- 05), to human STIM1 (Santa Cruz Biotechnology, Cat# sc- 76589) or NS (Santa Cruz Biotechnology, Cat# sc- 37007).

Techniques: Transfection, Ligand Binding Assay, Mutagenesis

Journal: eLife

Article Title: Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2+

doi: 10.7554/elife.80447

Figure Lengend Snippet: Figure 7. Extended synaptotagmins rescue SOCE in cells lacking IP3R1. (A) SH-SY5Y cells expressing IP3R1-shRNA alone or with E-Syt1 were stimulated with Tg (1 µM) in Ca2+-free HBSS before restoring extracellular Ca2+ (2 mM). Traces show mean ± s.e.m, for 20–80 cells from three experiments. (B) Summary results show Δ[Ca2+]c evoked by restoring Ca2+ (SOCE). Mean ± s.e.m, ***p < 0.001, Mann-Whitney U- test. (C) Summary results (mean ± s.e.m, n=20–80 cells) show resting [Ca2+]c (left) and the peak Ca2+ signals (Δ[Ca2+]c) evoked by thapsigargin (Tg, 1 µM) in Ca2+-free HBSS for SH-SY5Y cells expressing IP3R1-shRNA alone or with human E-Syt1. (D) Cells over-expressing E-Syt1 and treated with IP3R1-shRNA in combination with either NS or STIM1 siRNA were stimulated with Tg (1 µM) in Ca2+-free HBSS before restoration of extracellular Ca2+ (2 mM). Mean ± s.e.m. from three experiments with 30–40 cells. (E, F) Summary results (mean ± s.e.m, n=30–40 cells) show SOCE evoked by Tg (E), resting [Ca2+]c and the Tg-evoked Ca2+ release from intracellular stores (F). ***p< 0.001, Mann-Whitney U- test. (G) Similar analyses of cells expressing NS shRNA alone or with human E-Syt1 and then treated

Article Snippet: The siRNAs used were to human Orai1 (100 nM, Dharmacon, Cat# L- 014998- 00- 0005) or non- silencing (NS, Dharmacon, Cat# D- 495 001810- 10- 05), to human STIM1 (Santa Cruz Biotechnology, Cat# sc- 76589) or NS (Santa Cruz Biotechnology, Cat# sc- 37007).

Techniques: Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2+

doi: 10.7554/elife.80447

Figure Lengend Snippet: Figure 8. Dual regulation of SOCE by IP3Rs. (A) SOCE is activated when loss of Ca2+ from the ER, usually mediated by opening of IP3Rs when they bind IP3, causes STIM to unfurl cytosolic domains (2). The exposed cytosolic domains of STIM1 reach across a narrow gap between the ER and PM at a MCS to interact with PIP2 and Orai1 in the PM. Binding of STIM1 to Orai1 causes pore opening, and SOCE then occurs through the open Orai1 channel. We show that IP3Rs when they bind IP3 also facilitate interactions between Orai1 and STIM, perhaps by stabilizing the MCS (1). Receptors that stimulate IP3 formation thereby promote both activation of STIM (by emptying Ca2+ stores) and independently promote interaction of active STIM1 with Orai1. (B) Other mechanisms, including ryanodine receptors (RyR), can also release Ca2+ from the ER. We suggest that convergent regulation of SOCE by IP3R with bound IP3 allows receptors that stimulate IP3 formation to selectively control SOCE.

Article Snippet: The siRNAs used were to human Orai1 (100 nM, Dharmacon, Cat# L- 014998- 00- 0005) or non- silencing (NS, Dharmacon, Cat# D- 495 001810- 10- 05), to human STIM1 (Santa Cruz Biotechnology, Cat# sc- 76589) or NS (Santa Cruz Biotechnology, Cat# sc- 37007).

Techniques: Binding Assay, Activation Assay, Control