Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking
- Tata Institute of Fundamental Research, India
- National Centre for Biological Sciences, India
- Northwestern University, Feinberg School of Medicine, United States
- Institute for Immunodeficiency, University of Freiburg, Germany
- Institute for Transfusion Medicine and Gene Therapy, University of Freiburg, Germany
- Dr. Shahrooei Lab, Iran
Figures
Figure 1 with 3 supplements
STX11 is required for SOCE.
Measurement of SOCE in STX11-depleted Jurkat and FHLH4 patient T cells. (A–B) Representative Fura-2 calcium imaging assay (A) and quantification of independent repeats (B) measuring thapsigargin (TG) induced SOCE in Jurkat T cells treated with Scr (black), STX11#1 (red) or STX11#2 (blue) shRNAs. (n=30–40 cells/group, N=3). (C–D) Representative Fura-2 calcium imaging assay (C) and quantification of repeats (D) showing reconstitution of SOCE in STX11-depleted Jurkat T cells by ectopic expression of STX11. Scr shRNA with empty vector (EV) expression (black), STX11 shRNA with EV expression (red), STX11 shRNA with STX11 expression (green). (n=30–40 cells/group, N=3). (E) Sanger sequencing of FHLH4 patient DNA showing deletion of a single adenine at the 752nd position and the resulting frameshift. (F) Schematic showing the predicted domain distribution of the wildtype versus FHLH4 mutant STX11 protein. (G) Western blot on WCLs of healthy donor (HD) and FHLH4 patient PBMCs showing the relative molecular weight and abundance of the wildtype and mutant STX11 bands (red arrow). (H) Western blot on WCLs of HEK293 cells over-expressing wildtype or FHLH4 mutant STX11 (red arrow) and empty vector. (I) Fura-2 calcium imaging assay measuring anti-CD3 induced SOCE in HD (black) and FHLH4 (red) T cells. (n=30–40 cells/group). (J) Fura-2 calcium imaging assay measuring SOCE in HD expressing empty vector (EV) (black) and FHLH4 T cells expressing human STX11 (red), respectively. (n=30–40 cells/group). (K–L) Representative Fura-2 calcium imaging assay (K) and quantification of repeats (L) measuring SOCE in Jurkat T cells treated either with Scr shRNA expressing empty vector (EV) (black), STX11 shRNA expressing EV (red), STX11 shRNA expressing wildtype STX11 (green), or STX11 shRNA expressing FHLH4 mutant STX11 (blue).
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Figure 1—source data 1
Numerical data underlying the quantitative analyses represented in Figure 1A–D and I–L.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-data1-v1.xlsx
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Figure 1—source data 2
Folder containing the original sequencing result files, shown in Figure 1E.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-data2-v1.zip
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Figure 1—source data 3
Folder containing labeled PDF files of the original western blots for Figure 1G–H.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-data3-v1.zip
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Figure 1—source data 4
Folder containing the original images of western blots displayed in Figure 1G–H.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-data4-v1.zip
Figure 1—figure supplement 1
Targeted RNAi screen for genes involved in SOCE.
(A–H) Fura-2 calcium imaging assays measuring thapsigargin (TG) induced SOCE in HEK293 and/or Jurkat cell lines treated with Scr shRNA or different shRNA sequences against the gene of interest for 3–4 days. Shown here are the average traces from 30 to 40 cells per group.
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Figure 1—figure supplement 1—source data 1
Numerical data underlying the quantitative analyses represented in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Validation of STX11 knockdown in HEK293 and Jurkat T cells.
(A–B) Representative Fura-2 calcium imaging assay (A) and its quantification from repeats (B) measuring thapsigargin (TG) induced SOCE in HEK293 cells treated with Scr or STX11 shRNA. Bars show relative SOCE ± SE from three independent experiments in (A) where SOCE from Scr shRNA-treated group in one experiment was set at 100%. (C) Quantitative PCR to assess the efficiency of knockdown in shRNA-treated HEK293 (C) and Jurkat T cells (D) from three independent repeats. Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to beta-actin housekeeping control.
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Figure 1—figure supplement 2—source data 1
Numerical data underlying the quantitative analyses represented in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
RNAi screen for genes involved in primary HLH and/or vesicle fusion for their role in SOCE.
(A–F) Fura-2 calcium imaging assays measuring thapsigargin (TG) induced SOCE in HEK293 or Jurkat T cells treated with Scr shRNA or different shRNA sequences against the gene of interest for 3–4 days. Shown here are the average traces from 30 to 40 cells per group.
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Figure 1—figure supplement 3—source data 1
Numerical data underlying the quantitative analyses represented in Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig1-figsupp3-data1-v1.xlsx
Figure 2 with 3 supplements
STX11 depletion does not affect PM Orai1 levels and global membrane trafficking or secretion.
(A–C) Measurement of Bungarotoxin binding site (BBS) tagged Orai1-YFP in the PM of Scr (black) and STX11 shRNA-treated (red), resting (solid line), and store-depleted (broken line), HEK293 cells. Measurement of Orai1-YFP (A) and Alexa Fluor 647-conjugated Bungarotoxin binding (B) in a population of cells using flow-cytometry. (C) Quantification of Orai1-YFP and BTX-A647 in individual cells using confocal imaging. Line profile showing overlay of Orai1-YFP (green) and BTX-A647 (red) signal across the representative cells for individual groups. (D) Confocal images and quantification of Orai1-YFP and BTX-A647 in scramble and STX11 shRNA-treated, resting, and store-depleted Jurkat T cells. (n=30–40, N=3) (E–G) Quantification of receptor-mediated uptake and recycling in STX11 shRNA-treated cells. Scr or STX11 shRNA-treated HEK293 and Jurkat T cells were loaded with Alexa-647-Transferrin (30 min pulse), washed, chased for 0, 5, 10, and 20 min and imaged to quantify total Transferrin levels inside each cell. Representative images from HEK293 cells (E) and quantification from 50 to 60 HEK293 (F) and Jurkat T cells (G) is shown. N=3 (H) Quantification of non-receptor mediated endocytosis in STX11-depleted HEK293 cells. Representative images of FITC-conjugated Dextran uptake in Scr and STX11-shRNA-treated HEK293 cells (left panel) and quantification of the mean intensity per cell (right panel). (I) Quantification of biosynthesis and secretion in STX11-depleted cells. Scr or STX11 shRNA-treated HEK293 cells were transfected with secretory alkaline phosphatase (AP) expressing plasmid and levels of AP secreted into the culture supernatants were measured using p-nitrophenyl phosphate (pNPP) substrate at 48 hr post-transfection. (J–K) Immunolabeling of HEK293 cells to assess ER (J) and Golgi (K) health. Scr and STX11 shRNA-treated HEK293 cells were stained with anti-KDEL (J) and anti-GM130 antibodies (K), respectively, and imaged. (L) Measurement of ER calcium content in Scr and STX11 shRNA-treated HEK293 cells using transiently transfected ER-localized CEPIA. Cells were incubated in Ringer’s buffer containing 0 mM calcium followed by 1 mM EGTA, stimulated with 1 μM ionomycin and imaged. (N=3).
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Figure 2—source data 1
Folder containing the original flow cytometry files and gating strategy for Figure 2A–B.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-data1-v1.zip
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Figure 2—source data 2
Numerical data underlying the quantitative analyses represented in Figure 2C–D, F–I and L.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Validation of STX11 knockdown in Orai1-BBS-YFP expressing HEK293 and Jurkat T cells.
(A) Schematic showing the design of recombinant Orai1-BBS-YFP fusion protein. Shown here is the placement of the bungarotoxin binding site (BBS) inside the second extracellular loop and YFP tag in the C-terminus of Orai1 as well as binding of BTX-A647 to BBS. Created with BioRender.com. (B) Representative quantitative PCR to assess the efficiency of knockdown in shRNA-treated HEK293 cells stably expressing Orai1-BBS-YFP. Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to RPL30 housekeeping control. (C) Representative Fura-2 calcium imaging assay measuring Thapsigargin (TG) induced SOCE in Orai1-BBS-YFP expressing HEK293 cells treated with Scr or STX11 shRNA. (D) Representative quantitative PCR to assess the efficiency of knockdown in shRNA-treated Jurkat T cells expressing Orai1-BBS-YFP. Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to RPL30 housekeeping control.
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Figure 2—figure supplement 1—source data 1
Numerical data underlying the quantitative analyses represented in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Measurement of total Orai1 levels in the plasma membrane of STX11-depleted U2OS cells.
(A–B) Measurement of total Orai1 levels in the plasma membrane of STX11-depleted cells by flow-cytometry. U2OS cells stably expressing Orai1-BBS-YFP were transduced with Scr (black) or STX11 (red) shRNA, stimulated with 1 µM TG (broken lines) or control (solid lines) and incubated with alpha-bungarotoxin alexa fluor 647 (BTX-AF647). Total Orai1-YFP levels (A) and BTX-AF647 binding to surface Orai1 (B) were measured where binding to wildtype U2OS cells was used as the background fluorescence. N=3 (C) Representative quantitative PCR to assess the efficiency of knockdown in shRNA-treated Orai1-BBS-YFP expressing U2OS cells from (A–B). Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to RPL30 housekeeping gene. (D–E) Measurement of thapsigargin (TG) induced SOCE in U2OS cells treated with STX11 RNAi in (A–B). (D) Representative averaged traces of Fura-2 calcium imaging assay performed on cells in panels (A–B). (E) Bar plot showing relative mean % SOCE ± SE from three independent experiments in (D).
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Figure 2—figure supplement 2—source data 1
Folder containing the original flow cytometry files and gating strategy for Figure 2—figure supplement 2A–B.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Numerical data underlying the quantitative analyses represented in Figure 2—figure supplement 2C–E.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-figsupp2-data2-v1.xlsx
Figure 2—figure supplement 3
SOCE in RNAi-treated Jurkat T cells to assess the role of SNAPs.
(A–C) Fura-2 calcium imaging assay measuring SOCE in cells treated with Scr shRNA or different shRNA sequences against SNAP23 (A) SNAP25 (B) SNAP29 (C). Average traces from 30 to 40 cells per group are shown. (D) Western blot images of co-IPs to test the association of Orai1 with SNAP23/SNAP25/SNAP29. HEK293 cells were co-transfected with Orai1-Myc and untagged SNAP23/SNAP25/SNAP29, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2) (E) Western blot images of co-IP to test the association of Stim1 with SNAP23/SNAP25/SNAP29. HEK293 cells were co-transfected with YFP-Stim1 and Myc-tagged SNAP23/SNAP25/SNAP29, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2).
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Figure 2—figure supplement 3—source data 1
Numerical data underlying the quantitative analyses represented in Figure 2—figure supplement 3A–C.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-figsupp3-data1-v1.xlsx
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Figure 2—figure supplement 3—source data 2
Folder containing labeled PDF files of the original western blots for Figure 2—figure supplement 3D–E.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-figsupp3-data2-v1.zip
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Figure 2—figure supplement 3—source data 3
Folder containing the original images of western blots shown in Figure 2—figure supplement 3D–E.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig2-figsupp3-data3-v1.zip
Figure 3
STX11 regulates ICRAC.
(A–D) Measurement of ICRAC in STX11-depleted Jurkat T cells in the whole-cell recording configuration in 20 mM extracellular Ca2+ Ringer’s solution. ICRAC was induced by passive depletion of intracellular Ca2+ stores by dialyzing 8 mM BAPTA into the cell via the patch-pipette. (A–B) Representative CRAC current (ICRAC) at –100 mV in Jurkat T cells treated with Scr shRNA (A) and STX11 shRNA (B) construct. The current is blocked by extracellular La3+ (10 µM) and replacing the 20 mM Ca2+ Ringer’s solution with a divalent-free solution (DVF) evokes a large Na+ current which depotentiates over tens of seconds. The current-voltage (I-V) relationship of the Ca2+ and DVF currents are shown on the right. Both Ca2+ and Na+ current amplitudes are reduced in the STX11 shRNA-treated group relative to Scr control cells. The I–V relationships (right plots) show no change in ion selectivity. (C–D) Summary of the current amplitudes of Ca2+ and Na+ currents (C) and current reversal potentials (D) in Scr and STX11 knockdown cells.
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Figure 3—source data 1
Numerical data underlying the quantitative analyses represented in Figure 3.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig3-data1-v1.xlsx
Figure 4
STX11 regulates NFAT activation, IL-2 gene expression, and degranulation in FHLH4 patient T cells.
(A–C) Estimation of nuclear translocation of NFAT. (A) Western blot showing nuclear translocation of NFAT in Jurkat T cells treated with Scr or STX11 shRNA and stimulated with PMA +TG. N=3. (B) Representative confocal images of control and STX11-depleted Jurkat T cells stimulated with anti-CD3, immunolabeled with anti-NFAT1 antibody, and counter-stained with DAPI. (N=3) (C) Box and whisker plot showing quantification of nuclear NFAT from cells populating 10 randomly chosen fields per group in (B). (D) Quantification of IL-2 mRNA in anti-CD3 stimulated Jurkat T cells treated with Scr or STX11 shRNA using qPCR. The bars show mean ± SE of relative IL-2 mRNA. N=3. (E) Quantification of IL-2 EIA on supernatants of Scr (black) or STX11 (red) shRNA-treated Jurkat T cells stimulated with anti-CD3. (F) qPCR analysis of IL-2 mRNA expression in various stimulated HD (black) and FHLH4 T cells (red). (G–H) Flow cytometry contour plot showing granule release assay performed on HD (black) and FHLH4 CD8 T (red) cells (G) and its quantification (H).
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Figure 4—source data 1
Folder containing labeled PDF files of original western blots for Figure 4A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig4-data1-v1.zip
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Figure 4—source data 2
Folder containing the original images of western blots shown in Figure 4A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig4-data2-v1.zip
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Figure 4—source data 3
Numerical data underlying the quantitative analyses represented in Figure 4C–F and H.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig4-data3-v1.xlsx
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Figure 4—source data 4
Folder containing the original flow cytometry files and gating strategy for Figure 4G.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig4-data4-v1.zip
Figure 5 with 12 supplements
STX11 directly binds resting Orai1 in the plasma membrane.
Intracellular localization of STX11 and its association with Orai1. (A) (Upper panel) Schematic showing epitopes for anti-STX11 and anti-HA antibodies used in the lower panel. (Lower panel) Representative confocal images of HEK293 cells transfected with either HA-tagged STX11 or untagged STX11 and stained using anti-HA antibody or anti-STX11 antibody, respectively. N=3 (B–D) Localization of HA-tagged STX11 with respect to Orai1-YFP in the basal as well as middle plane of resting (B) and store-depleted (C) HEK293 cells. (D) Pearson’s correlation coefficient showing Orai1 and STX11 colocalization from (B) and (C). N=3. (E) Representative confocal images of FHLH4 mutant STX11 expressing HEK293 cells. (F–G) Co-IP to assess STX11 binding to Orai1. Whole cell lysates of resting and store-depleted HEK293 cells co-expressing either Flag-Orai1 and STX11 (F) or Orai1-Myc and STX11 (G) were subjected to IP and western blot using anti-Myc, anti-Flag, or anti-STX11 antibodies, as indicated. (N=3) (H) Schematic showing key domains of STX11 and Orai1 used for in vitro pull-down assays. (I) Pull-down assay showing in vitro binding of His-tagged STX11 to Orai1. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. Input: 1/5th of the protein. N=4. (J) Pull-down assay showing in vitro binding of His-tagged Habc domain of STX11 to Orai1 fragments. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-His antibody. Input: 1/5th of the protein. N=3. (K) Binding free energy distribution of STX11-Habc and Orai1 C-terminus interactions. (L) Sphere and cartoon representation of the structure of STX11 Habc (green) and Orai1 C-terminus (cyan) complex after MD simulation. The N-termini are highlighted in blue, and C-termini in red. (M) Representative confocal images showing localization of STX11 with respect to Orai1:Stim1 puncta in store-depleted cells. HEK293 cells expressing Orai1-YFP, CFP-Stim1 were transfected with HA-tagged STX11, store-depleted, and stained using anti-HA antibody. (N=3) (N) Pearson’s correlation coefficient showing quantification of the localization of STX11 with respect to Stim1:Orai1 puncta in store-depleted cells from (M).
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Figure 5—source data 1
Numerical data underlying the quantitative analyses represented in Figure 5D, K and N.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-data1-v1.xlsx
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Figure 5—source data 2
Folder containing labeled PDF files of the original western blots for Figure 5F, G, I and J indicating the relevant bands.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-data2-v1.zip
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Figure 5—source data 3
Folder containing the original images of western blots shown in Figure 5F, G, I and J.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-data3-v1.zip
Figure 5—figure supplement 1
Degradation and mis-localization of N- and C-terminally tagged STX11 and interaction of STX11-SNARE domain with MBP-Orai1.
(A) Schematic showing a comparison of the key domains in STX11 and STX1A. (B) Western blot of whole cell lysates prepared from HEK293 cells expressing YFP-STX11 and STX11-YFP. Note the mismatch in molecular weight of the N- versus C-terminally tagged STX11 and the degradation products (arrows). N=3. (C) Confocal images showing localization of ectopically expressed mCherry-STX11 or STX11-mCherry in HEK293 cells. N=3. (D) Pull-down assay showing in vitro binding of His-tagged SNARE domain of STX11 to MBP-tagged Orai1 N- and C-termini. (Top panel) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 cytosolic tails. (Bottom panel) Western blot using anti-His antibody.
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Figure 5—figure supplement 1—source data 1
Folder containing labeled PDF files of the original western blots for Figure 5—figure supplement 1B, D.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Folder containing the original images of western blots shown in Figure 5—figure supplement 1B, D.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Contact frequency and RMSD of STX11-Habc and Orai1 C-terminus from AF3 predicted models.
(A) Frequency of interface residues of STX11-Habc and Orai1 C-terminus across AF3 predicted models. Residues with more than 50% frequency are shown. (B) RMSD of STX11 Habc and Orai1 C-terminus upon superposition on the 0th ns frame. The STX11-Habc RMSD indicates the backbone RMSD upon superposition of all frames on the STX11-Habc backbone of 0th ns. The Orai1 C-terminus RMSD suggests the stability of Orai1 with respect to STX11 and is calculated for the Orai1 C-terminus after superposition of all frames on the STX11-Habc backbone of 0th ns.
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Figure 5—figure supplement 2—source data 1
Numerical data underlying the quantitative analyses represented in Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp2-data1-v1.xlsx
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Figure 5—figure supplement 2—source data 2
Folder containing the custom script used in Figure 5—figure supplement 2A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp2-data2-v1.zip
Figure 5—figure supplement 3
Binding free energy, types of interactions and interacting residues of STX11-Habc and Orai1 C-terminus complex.
(A) Binding energy (ΔG) of STX11-Habc and Orai1 C-terminus complex across the simulation time for all three replicates. (B) Interactions between STX11-Habc and Orai1 C-terminus across the simulation time in three replicates. The interactions that persist for more than 30% of simulation time are shown and highlighted in different colors. H-bond has been categorized as sidechain-sidechain (ss), sidechain-backbone (sb), and backbone-sidechain (bs). (C) Residues predicted to be involved in interaction between STX11 Habc and the Orai1 C-terminus identified from all-atom MD simulations.
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Figure 5—figure supplement 3—source data 1
Numerical data underlying the quantitative analyses represented in Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp3-data1-v1.xlsx
Figure 5—figure supplement 4
Co-IP of STX11 with Stim1 and measurement of Orai1 inside Stim1:Orai1 clusters.
(A) Co-immunoprecipitation (co-IP) to test the association of Stim1 with STX11. HEK293 cells were co-transfected with Stim1-Myc-His and STX11, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2) (B) Quantification of the fraction of Orai1 inside and outside Stim1:Orai1 clusters in store-depleted HEK293 cells expressing CFP-Orai1 and Stim1-YFP.
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Figure 5—figure supplement 4—source data 1
Folder containing labeled PDF files of the original western blots for Figure 5—figure supplement 4A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp4-data1-v1.zip
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Figure 5—figure supplement 4—source data 2
Folder containing the original images of western blots shown in Figure 5—figure supplement 4A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp4-data2-v1.zip
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Figure 5—figure supplement 4—source data 3
Numerical data underlying the quantitative analyses represented in Figure 5—figure supplement 4B.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp4-data3-v1.xlsx
Figure 5—figure supplement 5
Binding free energy (ΔG) of Orai1 C-termini and SOAR dimer complex.
(A) Schematic of human Stim1 and Orai1 showing the domains used for MD simulation. TM: Transmembrane, P/S: Proline/Serine rich, and CC: represent Coiled coil domains. (B) ΔG values of the three runs across all chains of SOAR dimer and Orai1 C termini. (C) ΔG values of chains AC and BD of SOAR dimer and Orai1 C-termini complex across the simulation time for all three runs.
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Figure 5—figure supplement 5—source data 1
Numerical data underlying the quantitative analyses represented in Figure 5—figure supplement 5B–C.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp5-data1-v1.xlsx
Figure 5—figure supplement 6
RMSD and cartoon representation of SOAR and Orai1 C-termini complex.
(A) RMSD of SOAR dimer and Orai1 C-termini complex. (B) Cartoon representation of the structure of Stim1-SOAR (344-444) dimer in complex with Orai1 C-termini.
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Figure 5—figure supplement 6—source data 1
Numerical data underlying the quantitative analyses represented in Figure 5—figure supplement 6A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig5-figsupp6-data1-v1.xlsx
Figure 5—video 1
Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_HABC domain, run 1.
Figure 5—video 2
Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_HABC domain, run 2.
Figure 5—video 3
Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_HABC domain, run 3.
Figure 5—video 4
Molecular dynamic simulation of interaction of Orai1 C-terminus with the Stim1 SOAR domain, run 1.
Figure 5—video 5
Molecular dynamic simulation of interaction of Orai1 C-terminus with the Stim1 SOAR domain, run 2.
Figure 5—video 6
Molecular dynamic simulation of interaction of Orai1 C-terminus with the Stim1 SOAR domain, run 3.
Figure 6
Residues involved in Orai1:STX11 complex formation are crucial for SOCE.
Analysis of STX11 and Orai1 mutants. (A) Schematic showing STX11 mutations (Top). STX11 N147A_E150A mutant expressing HEK293 co-labeled with Alexa Fluor-488 conjugated wheat germ agglutinin (WGA) or co-transfected with Orai1-YFP and immunolabeled with anti-STX11 to assess co-expression in PM of resting and store-depleted cells (Bottom). Scale bar 10 μm. (B) Western blot to compare total expression of mutant and wildtype STX11 using anti-STX11 antibody. (C–D) Representative Fura-2 calcium imaging assay (C) and quantification of repeats (D) measuring rescue of SOCE in Jurkat cells treated either with Scr shRNA (black) or STX11 shRNA expressing empty vector (red), wildtype STX11 (green), or N147A_E150A mutant STX11 (blue). (E) Coomassie blue stained SDS-PAGE showing expression and purification of His-tagged wildtype or N147A_E150A mutant STX11 (red arrow) from E. coli. (F–G) Pull-down assay measuring their binding to MBP-tagged Orai1 C-terminus. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4 (G) Quantification of STX11 band intensities in (F). (H) Schematic showing Orai1 mutations (Top). Confocal images of HEK293 cells expressing YFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 (Bottom). (I–J) Representative Fura-2 calcium imaging assay (I) and quantification of repeats (J) measuring rescue of SOCE in HEK293 cells treated either with Scr (black) or Orai1 shRNA expressing empty vector (red), wildtype Orai1 (green), or R289A_E272A_E275A_E278A mutant Orai1 (blue). (K–L) Pull-down assay measuring the binding of MBP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 C-terminus to wildtype or N147A_E150A mutant STX11. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4. (L) Quantification of STX11 band intensities in (K). (M–N) Schematic showing CAD domain in Stim1 (top panel) and representative confocal images (lower panel) (M) and quantification (N) of PM localized YFP-CAD in CFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 expressing HEK293 cells. (O–P) Representative Fura-2 calcium imaging assay (O) and quantification of repeats (P) of constitutive calcium flux from cells imaged in panels (M–N).
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Figure 6—source data 1
Folder containing labeled PDF files of the original gels and western blots for Figure 6B, E, F, K.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig6-data1-v1.zip
-
Figure 6—source data 2
Folder containing the original images of gels and western blots shown in Figure 6B, E, F and K.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig6-data2-v1.zip
-
Figure 6—source data 3
Numerical data underlying the quantitative analyses represented in Figure 6C–D, G, I–J, L and N–P.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig6-data3-v1.xlsx
Figure 7 with 1 supplement
Non-functional clustering of Orai1 in STX11-depleted cells.
(A–L) Quantification of Orai1 and Stim1 intensities in the ER-PM puncta of store-depleted cells. (A–B) Representative images of resting (A) and store-depleted (B) shRNA-treated HEK293 cells expressing N-terminal CFP-tagged Orai1 (CFP-Orai1) and C-terminal YFP-tagged Stim1 (Stim1-YFP) from confocal microscopy. (C–F) Mean Stim1-YFP intensity (C) and area (D) of Stim1 clusters. (E) Fraction of CFP-Orai1 intensities inside Stim1-YFP clusters of shRNA-treated cells, post store-depletion. (F) Pearson’s correlation coefficient of Stim1 and Orai1 co-localization inside Stim1 puncta. N=3. (G–L) Quantification of Orai1 and Stim1 intensities in the ER-PM puncta of C-terminal YFP-tagged Orai1 (Orai1-YFP) and N-terminal CFP-tagged Stim1 (CFP-Stim1) expressed in HEK293 cells and imaged using TIRF microscopy. (G) Representative TIRF images of resting and store-depleted shRNA-treated HEK293 cells. Scale bar 10 μM. (H–K) Mean CFP-Stim1 intensity (H) and area (I) of Stim1 clusters. Fraction of Orai1-YFP intensities (J) and area (K) of Orai1 clusters in control and STX11-depleted cells. (L) Pearson’s correlation coefficient of Stim1 and Orai1 co-localization inside Stim1 puncta. N=2. (M–N) Representative Fura-2 calcium imaging assay (M) and quantification of repeats (N) measuring TG-induced SOCE in Orai1-YFP and CFP-Stim1 expressing HEK293 cells treated with Scr (black) or STX11 (red) shRNA. (O–Q) Representative TIRF images of GFP-MAPPER in store-depleted Scr and STX11 shRNA-treated HEK293 cells (O). Quantification of area (P) and intensity (Q) of GFP-MAPPER puncta. n=15.
-
Figure 7—source data 1
Numerical data underlying the quantitative analyses represented in Figure 7C–F, H–N and P–Q.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Measurement of co-clustering of mutant Orai1 with Stim1.
(A–D) Representative confocal images (A) and quantification (B–D) of C-terminally YFP-tagged wild-type or R289A_E272A_E275A_E278A mutant Orai1 and CFP-Stim1 intensities and area of clusters in store-depleted HEK293 cells. n=20.
-
Figure 7—figure supplement 1—source data 1
Numerical data underlying the quantitative analyses represented in Figure 7—figure supplement 1B–D.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig7-figsupp1-data1-v1.xlsx
Figure 8 with 3 supplements
STX11 primes Orai1 for optimal on-site assembly.
Oligomerization of Orai1. (A) N-FRET analysis of Scr and STX11 shRNA-treated HEK293 cells co-expressing Orai1-CFP and Orai1-YFP before and after store-depletion. (B–C) Representative images (B) and quantification (C) of FRET analysis using acceptor photobleaching of Scr and STX11 shRNA-treated Orai1, Stim1 double knockdown resting HEK293 cells co-expressing Orai1-CFP (donor) and Orai1-YFP (acceptor). (D–E) Representative western blot (D) and quantification of band intensities from repeats (E) showing distribution of BS3 crosslinked Flag-Orai1 oligomeric bands B,C,D in control and STX11-depleted HEK293 cells. N=3. (F) Representative confocal images and quantification (right) of YFP-CAD localization in Scr and STX11 shRNA-treated HEK293 cells expressing Orai1-CFP. (G–H) Fura-2 calcium imaging assay (G) and quantification of repeats (H) to measure constitutive calcium influx in Orai1-CFP and YFP-CAD expressing, control (black) or STX11-depleted (red) HEK293 cells. n=80–90, N=3 (I–J) Fura-2 calcium imaging assay (I) and quantification of repeats (J) to measure constitutive calcium influx in Orai1-H134S mutant expressing control (black) and STX11-depleted (red) HEK293 cells. n=80–100, N=3 (K–L) Fura-2 calcium imaging assay (K) and quantification of repeats (L) to measure constitutive calcium influx in Orai1-ANSGA mutant expressing control (black) and STX11-depleted (red) HEK293 cells. n=50–60, N=3.
-
Figure 8—source data 1
Numerical data underlying the quantitative analyses represented in Figure 8A, C and E–L.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-data1-v1.xlsx
-
Figure 8—source data 2
Folder containing labeled PDF files of the original western blots for Figure 8D.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-data2-v1.zip
-
Figure 8—source data 3
Folder containing the original images of western blots shown in Figure 8D.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-data3-v1.zip
Figure 8—figure supplement 1
Lower half of the western blot of BS3 cross-linked Flag-Orai1 acquired at a higher exposure showing the monomeric Flag-Orai1 band (labeled as A).
-
Figure 8—figure supplement 1—source data 1
Folder containing labeled PDF files of the original western blots for Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-figsupp1-data1-v1.zip
-
Figure 8—figure supplement 1—source data 2
Folder containing the original images of western blots shown in Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-figsupp1-data2-v1.zip
Figure 8—figure supplement 2
Measurement of activation of constitutive calcium influx by Orai1-SOAR-SOAR in STX11 depleted cells.
(A) Schematic representation of SOAR domain in full-length Stim1. (B) Cartoon showing the design of the Orai1-SOAR-SOAR-EGFP (Orai1-S-S-GFP) construct. Created with BioRender.com. (C) Representative Fura-2 calcium imaging assay to measure constitutive calcium influx in Orai1-S-S-GFP expressing control (black) or STX11-depleted (red) HEK293 cells. Cells were imaged in Ringer’s buffer containing 0 mM followed by 2 mM extracellular Ca2+. (D) Quantification of constitutive calcium influx across experiments as shown in (C). n=90–100, N=3.
-
Figure 8—figure supplement 2—source data 1
Numerical data underlying the quantitative analyses represented in Figure 8—figure supplement 2.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-figsupp2-data1-v1.xlsx
Figure 8—figure supplement 3
Schematic representation and quantification of constitutively active Orai1 mutants in the PM of STX11 depleted cells.
(A) Schematic representation of H134S mutation in full-length Orai1. (B) Quantification showing percent Orai1-H134S-YFP intensity normalized to Orai1-WT-YFP in the PM of Scr and STX11 shRNA-treated HEK293 cells. n=30–40. (C) Schematic representation of ANSGA mutation in full-length Orai1. (D) Quantification showing percent Orai1-ANSGA-YFP intensity normalized to Orai1-WT-YFP in the PM of Scr and STX11 shRNA-treated HEK293 cells. n=30–40.
-
Figure 8—figure supplement 3—source data 1
Numerical data underlying the quantitative analyses represented in Figure 8—figure supplement 3.
- https://cdn.elifesciences.org/articles/111335/elife-111335-fig8-figsupp3-data1-v1.xlsx
Figure 9
Models depicting STX11-mediated priming of Orai1 and its role in T cell signaling and function.
(A) STX11 deficient (unprimed) and sufficient (primed) Orai1 in resting and store-depleted cells. For simplicity, STX11 interaction with only two subunits of Orai1 has been shown. Created with BioRender.com. (B) Regulation of T cell SOCE, NFAT activation, and degranulation by STX11 in healthy donor and FHLH4 patient T cell. Created with BioRender.com.
Author response image 1
Representative Fura-2 traces of Scr (black) and STX11 (red) shRNA-treated HEK293 (A) and Jurkat (B) cells, where the cells were incubated with 2 mM Ca2+ followed by addition of 10 mM EGTA to quench the 2 mM Ca2+.
Since we do not have access to a perfusion system, we could not test Fura2 response after re-addition of 2mM calcium to the existing EGTA and Ca2+ mixture. However, the transition from 0mM to 2mM is already shown in Figure 8.
Author response image 2
Box plots representing quantification of individual repeats of constitutive calcium influx in Scr and STX11 shRNA-treated HEK293 cells expressing YFP-CAD and Orai1-CFP without (A) and with baseline subtraction (B).
(C-D) Quantification of individual repeats of Scr and STX11 shRNA-treated HEK293 cells expressing Orai1-H134S (C) and Orai1-ANSGA (D) mutants.
Tables
Table 1
AF3 predictions for STX11:Orai1 complex formation.
| Proteins/domains | ipTM | pTM |
|---|---|---|
| Full-length Orai1 - Full-length STX11 | 0.11 | 0.35 |
| STX11-Habc_41–167 and Orai1_N-term_1–87 | 0.27 | 0.59 |
| STX11-Habc_41–167 and Orai1_C-term_256–301 | 0.53 | 0.79 |
| STX11-SNARE_183–267 and Orai1_N-term_1–87 | 0.08 | 0.34 |
| STX11-SNARE_183–267 and Orai1_C-term_256–301 | 0.18 | 0.45 |
| STX11-Habc_41–167 and full-length Orai1 | 0.24 | 0.38 |
| STX11-SNARE_183–267 and full-length Orai1 | 0.08 | 0.37 |
| Orai1_N-term_1–87 and full-length STX11 | 0.15 | 0.5 |
| Orai1_C-term_256–301 and full-length STX11 | 0.16 | 0.54 |
Table 2
AF3 predictions for STX11-Habc and Orai1 C-terminus with different seeds.
| Seed values for STX11-Habc and Orai1 C-terminus | ipTM | pTM |
|---|---|---|
| 111 | 0.69 | 0.85 |
| 236 | 0.51 | 0.78 |
| 174 | 0.5 | 0.77 |
| 802 | 0.49 | 0.76 |
| 455 | 0.56 | 0.78 |
Table 3
AF3 predictions for SOAR and Orai1 in a 2:2 ratio.
| Two copies of SOAR and 2 copies of Orai1_C-term_256–301 | ipTM | pTM |
|---|---|---|
| Seed_101 | 0.73 | 0.78 |
| Seed_102 | 0.73 | 0.78 |
| Seed_103 | 0.73 | 0.78 |
| Seed_104 | 0.73 | 0.78 |
| Seed_105 | 0.74 | 0.79 |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal anti-myc (9E10) | Abcam | ab32 (RRID:AB_303599) | WB ( 1:1000) |
| Antibody | Rabbit polyclonal anti-SNAP23 | SYSY | 111203 | WB (1:1500) |
| Antibody | Mouse monoclonal anti-myc (9E10) | Culture supernatant, in house (this paper) | - | WB (0.25 μg/ml) |
| Antibody | Mouse monoclonal anti-SNAP25 | SYSY | 111011 | WB (1:1500) |
| Antibody | Rabbit polyclonal anti-SNAP29 | SYSY | 111303 | WB (1:1500) |
| Antibody | Rabbit polyclonal anti-GFP | Molecular Probes, Invitrogen | A11122 (RRID:AB_221569) | WB (1:2500) |
| Antibody | Rabbit polyclonal anti-flag | Sigma-Aldrich | F7425 (RRID:AB_439687) | Co-IP (1:1600) |
| Antibody | Mouse monoclonal anti-flag (M2) | Sigma-Aldrich | F3165 (RRID:AB_259529) | WB (1:1500) |
| Antibody | Mouse monoclonal anti-6X His | Thermo Fisher Scientific | MA1-135 (RRID:AB_2536841) | WB (1:2000) |
| Antibody | Rabbit polyclonal anti-STX11 | Sigma-Aldrich | SAB4301593 | IF, WB (1:1000) |
| Antibody | Rabbit polyclonal anti-STX11 | SYSY | 110113 | IF (1.67 μg/ml) |
| Antibody | Rabbit polyclonal anti-STX11 | Thermo Fisher Scientific | PA5-50800 (RRID:AB_2636252) | WB (1:2000) |
| Antibody | Rabbit polyclonal anti-STX11 | Proteintech | 13301–1-AP (RRID:AB_2198214) | IF (0.3 μg/ml) |
| Antibody | Mouse monoclonal anti-NFATc2 (4G6-G5) | Santa Cruz Biotechnology | sc-7296 (RRID:AB_628012) | WB (1:500) |
| Antibody | Rabbit polyclonal anti-NFAT1 | Cell Signaling Technology | 4389 (RRID:AB_1950418) | IF (1:200) |
| Antibody | Mouse monoclonal anti-Clathrin | BD Biosciences | 610499 (RRID:AB_397865) | WB (1:3000) |
| Antibody | Goat polyclonal anti-Lamin B (M-20) | Santa Cruz Biotechnology | sc-6217 (RRID:AB_648158) | WB (1:500) |
| Antibody | Rabbit monoclonal mAb HA-tag | Cell Signaling Technology | 3724 S (RRID:AB_1549585) | IF (1:1000) |
| Antibody | Alpaca polyclonal VHH anti-rabbit AF488 | Jackson | 611-544-152 | IF (1.375 μg/ml) |
| Antibody | A647 conjugated Goat polyclonal anti-rabbit | Molecular Probes | A-21245 (RRID:AB_2535813) | IF (1:800) |
| Other | DAPI | Thermo Fisher Scientific | D1306 (RRID:AB_2629482) | Nuclear staining in Figures 2 and 4 (2 μg/ml) |
| Antibody | Donkey polyclonal anti-rabbit A647 | Jackson | 711-605-152 | IF (1:800) |
| Antibody | HRP conjugated Mouse monoclonal anti-GAPDH | Sigma-Aldrich | G9295 (RRID:AB_1078992) | WB (1:40,000) |
| Antibody | HRP conjugated Donkey Polyclonal anti-Rabbit | Jackson | 711-035-152 | WB (1:50,000) |
| Antibody | HRP conjugated Donkey polyclonal anti-Mouse | Jackson | 715-035-151 | WB (1:50,000) |
| Antibody | Alexa fluor 647 conjugated Donkey polyclonal anti-mouse | Jackson | 715-705-150 | IF (1:500) |
| Other | Alexa fluor 647 conjugated Alpha Bungarotoxin | Thermo Fisher Scientific | B35450 | Labelling Orai1-BBS-YFP in Figure 2, Figure 2—figure supplements 1 and 2 (1 μg/ml) |
| Antibody | HRP conjugated rabbit polyclonal anti-GFP | Thermo Fisher Scientific | A10260 | WB (1:1000) |
| Antibody | HRP conjugated Goat polyclonal anti-rabbit Fc | Jackson | 111-035-008 | WB (1:50,000) |
| Other | HRP conjugated Streptavidin | Jackson | 016-030-084 | EIA in Figure 4 (0.1 μg/ml) |
| Antibody | Purified Mouse monoclonal anti-human IL-2 | BD Biosciences | BD #555051 (RRID:AB_395672) | EIA (1 μg/ml) |
| Antibody | Biotin conjugated Purified mouse monoclonal anti-human IL-2 | BD Biosciences | 555040 (RRID:AB_395666) | EIA (1 μg/ml) |
| Antibody | Ultra-LEAF Purified mouse monoclonal anti-human CD3 | BioLegend | 317326 (RRID:AB_11150592) | Stimulation for Degranulation assay (5–10 μg/ml) |
| Antibody | Ultra-LEAF Purified mouse monoclonal anti-human CD28 | BioLegend | 302934 (RRID:AB_11148949) | Stimulation for Degranulation assay (2 μg/ml) |
| Antibody | Ultra-LEAF Purified mouse monoclonal anti-human CD49d | BioLegend | 304340 (RRID:AB_2810444) | Stimulation for Degranulation assay (2 μg/ml) |
| Antibody | PE mouse monoclonal anti-human CD107a (LAMP-1) | BioLegend | 328608 (RRID:AB_1186040) | FACS (1:50) |
| Antibody | APC mouse monoclonal anti-human CD8 antibody | BioLegend | 344722 (RRID:AB_2075388) | FACS (0.5 μg/ml) |
| Antibody | FluoTag-X4 conjugated camelid monoclonal Anti-Rabbit IgG sdAb | SYSY | N2404-At488-S | IF (1:400) |
| Antibody | Mouse monoclonal Anti-rat GM130 | BD Biosciences | 610823 (RRID:AB_398142) | IF (0.5 μg/ml) |
| Antibody | Mouse monoclonal Anti-rat KDEL | Santa Cruz Biotechnology | sc-58774 (RRID:AB_784161) | IF (1:100) |
| Other | Fura-2-AM | Thermo Fisher Scientific | F1221 | Single cell calcium imaging in Figures 1, 6—8 (1 μM) |
| Other | Thapsigargin (TG) | Sigma-Aldrich | T9033-1MG | Store depletion in Figures 1, 2, 4—8 (1 μM) |
| Other | Polybrene (Hexadimethrine-bromide) | Sigma-Aldrich | 107689–10 G | Viral transduction in Figures 1—8 (8 μg/ml) |
| Other | Alexa Fluor 647 ChromPure Human Transferrin | Jackson | 009-600-050 | Transferrin recycling assay in Figure 2 (20 μg/ml) |
| Other | Disodium 4-Nitrophenyl Phosphate Hexahydrate | TCI | N0241 | Substrate for AP assay in Figure 2 (1 mg/ml) |
| Other | Dextran-FITC | TCI | F0918 | Dextran-FITC uptake assay in Figure 2 (1 mg/ml) |
| Other | NE-PER kit | Thermo Fisher Scientific | 78833 | Nuclear and cytosolic extracts in Figure 4 |
| Other | Protein A/G Mag Sepharose xtra | Cytiva | 28967056/66 | Co-IPs in Figure 5, Figure 2—figure supplement 3 and Figure 5—figure supplement 4 |
| Other | Protein A/G 4 Fast Flow Sepharose | Cytiva | 17528001 | Co-IPs in Figure 5—figure supplement 4, Figure 2—figure supplement 3 |
| Other | Mammalian Protease Inhibitor Cocktail | Sigma-Aldrich | 539134 | Cell lysis buffer in Figure 5, Figure 2—figure supplement 3 and Figure 5—figure supplement 4, Figures 1, 6 and 8 (1:200) |
| Other | Protease Inhibitor Cocktail (Bacterial) | Calbiochem | 539132 | In vitro binding assay in Figures 5 and 6 and associated figure supplements (1:200) |
| Other | Talon Beads | Takara | 635502 | In vitro binding assay in Figures 5 and 6 and associated figure supplements |
| Other | Dextrin Sepharose Beads | Cytiva | 28935597 | In vitro binding assay in Figures 5 and 6 and associated figure supplements |
| Other | HEPES | Sigma-Aldrich | H3375-100G | Ringer’s buffer, and HEK293FT culture media |
| Other | Lipofectamine 2000 | Thermo Fisher Scientific | 11668019 | Transfection reagent in Figures 1, 2, 5—8 |
| Other | Lipofectamine 3000 | Thermo Fisher Scientific | L3000001 | Transfection reagent in Figures 1, 2, 5—8 |
| Strain, strain background (Escherichia coli) | Lemo21(DE3) | New England Biolabs | C2528J | Chemically competent cells |
| Strain, strain background (Escherichia coli) | DH5α | New England Biolabs | C2987H | Chemically competent cells |
| Other | IPTG | Sigma-Aldrich | 16758–10 G | Induction of His-STX11 expression in Figures 5 and 6 and associated figure supplements |
| Other | Sarkosyl (N-lauroylsarcosine sodium salt) | Sigma-Aldrich | L9150-50G | Bacterial cell lysis in Figures 5 and 6 and associated figure supplements |
| Other | Lysozyme | Sigma-Aldrich | 62970–5 G-F | Bacterial cell lysis in Figures 5 and 6 and associated figure supplements |
| Other | DNase I Recombinant RNase-free | Roche | 04716728001 | Bacterial cell lysis in Figures 5 and 6 and associated figure supplements |
| Other | PMA (Phorbol Myristate Acetate) | Sigma-Aldrich | P8139 | Cell stimulation in Figure 4 (20–50 ng/ml) |
| Other | Ionomycin | Sigma-Aldrich | 407952 | Cell stimulation in Figures 2 and 4 (1 μM) |
| Other | Human IL-2 protein | Acro Biosystems | H5215 | Primary T cell culture in Figures 1 and 4 (50 ng/ml) |
| Other | Lectin from Phaseolus vulgaris (PHA) | Sigma-Aldrich | L8754 | Primary T cell culture in Figures 1 and 4 (2 μg/ml) |
| Other | Retronectin | Takara | T100B | Primary T cell transduction in Figure 1 (20 μg/ml) |
| Other | RNeasy Plus Mini kit | Qiagen | 74134 | RNA isolation for all qPCR assays in Figure 4 and figure supplements associated with Figures 1 and 2 |
| Other | QIAshredder | Qiagen | 79654 | Cell disintegration before RNA isolation in Figure 4 and figure supplements associated with Figures 1 and 2 |
| Other | Superscript IV | Thermo Fisher Scientific | 18091300 | cDNA synthesis for qPCR assays in Figure 4 and figure supplements associated with Figures 1 and 2 |
| Other | Qubit ssDNA Assay kit | Thermo Fisher Scientific | Q10212 | cDNA quantification for qPCRs in Figure 4 and figure supplements associated with Figures 1 and 2 |
| Other | Human STX11 TaqMan probe (FAM-MGB) | Thermo Fisher Scientific | Hs00186823_m1 | qPCR in figure supplements associated with Figures 1 and 2 |
| Other | Human Beta Actin TaqMan probe (FAM-MGB) | Thermo Fisher Scientific | Hs01060665_g1 | qPCR in Figure 4 and figure supplements associated with Figure 1 |
| Other | Human IL2 Taqman probe (FAM-MGB) | Thermo Fisher Scientific | Hs00174114_m1 | qPCR in Figure 4 |
| Other | Human RPL30 Taqman probe (FAM-MGB) | Thermo Fisher Scientific | Hs00265497_m1 | qPCR in figure supplements associated with Figures 1 and 2 |
| Other | Paraformaldehyde | Electron Microscopy Sciences | 157–8 | Cell fixation for IF in Figures 2, 4—7 |
| Other | Bovine Serum Albumin | Jackson | 001000173 | Blocking agent for IF in Figures 2, 4—7 |
| Other | Luria Bertini Broth | Himedia | M1245 | Bacterial cell culture in Figures 5 and 6 |
| Other | HBSS | Sigma-Aldrich | H8264 | Cell washing and incubation in Figures 1, 2, 4—8 |
| Other | Terrific Broth | Sigma-Aldrich | T9179 | Lemo21 cell culture in Figures 5 and 6 |
| Other | Ampicillin | Sigma-Aldrich | A0166-5G | Antibiotic for bacterial selection (100 μg/ml) |
| Other | Kanamycin | Sigma-Aldrich | K1876-1G | Antibiotic for bacterial selection (50 μg/ml) |
| Other | Chloramphenicol | Sigma-Aldrich | C0378-5G | Antibiotic for bacterial selection (100 μg/ml) |
| Other | 30% Acrylamide:Bis Solution (29:1) | Biorad | 1610156 | SDS-PAGE gels in Figures 1, 4—6 and 8 and their associated figure supplements |
| Other | DTT (Dithiothreitol) | Biorad | 1610611 | 1 X Laemmli buffer for SDS-PAGE in Figures 1, 4—6 and 8 and associated figure supplements |
| Other | 0.05% Trypsin-EDTA | Gibco | 25300054 | Trypsinization of HEK293, and HEK293FT cells |
| Other | 0.25% Trypsin-EDTA | Gibco | 25200072 | Trypsinization of U2OS cells |
| Other | 100 X Penicillin Streptomycin | Sigma-Aldrich | P4333-100ML | Supplement in cell culture medium in Figures 1—8 |
| Other | Puromycin | Gibco | A11138-03 | Antibiotic for selection of shRNA treated cells in Figures 1—4, 6—8 and their associated figure supplements |
| Cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573 (RRID:CVCL_0045) | Figures 1, 2, 5—8 and associated figure supplements |
| Cell line (Homo sapiens) | Jurkat T cell line [Clone E6-1] | ATCC | TIB-152 (RRID:CVCL_0367) | Figures 1—4 and 6 and associated figure supplements |
| Cell line (Homo sapiens) | U2OS cell line | ATCC | HTB-96 (RRID:CVCL_0042) | Figure 2—figure supplement 2 |
| Cell line (Homo sapiens) | HEK293FT | Thermo Fisher Scientific | R70007 | Virus generation in Figures 1—8 |
| Other | Universal Mycoplasma Detection Kit | ATCC | 30–1012 K | Mycoplasma testing of cell lines |
| Biological sample (Homo sapiens) | Human PBMCs | Dr. Shahrooei Lab | - | Figures 1 and 4 |
| Software, algorithm | Schrödinger Suite (Protein Preparation Wizard, Desmond, Simulation Interaction Diagram, thermal_mmgbsa.py, trj_merge.py, trajectory clustering, analyze_trajectory_ppi.py) | Schrödinger, LLC | - | System preparation, MD simulation, MM/GBSA, and trajectory analysis in Figure 5 and associated figure supplements |
| Software, algorithm | PyMOL | Schrödinger, LLC | - | Figure generation in Figure 5 and associated figure supplements |
| Software, algorithm | PDBSum | EMBL-EBI | - | Interaction analysis in Figure 5 and associated figure supplements |
Additional files
-
Supplementary file 1
List of primers and shRNAs used in this paper.
- https://cdn.elifesciences.org/articles/111335/elife-111335-supp1-v1.xlsx
-
Supplementary file 2
List of plasmids, including the newly generated constructs used in this paper.
- https://cdn.elifesciences.org/articles/111335/elife-111335-supp2-v1.xlsx
-
Supplementary file 3
Folder containing the PDB file for Figure 5L.
- https://cdn.elifesciences.org/articles/111335/elife-111335-supp3-v1.zip
-
Supplementary file 4
Folder containing the PDB file for Figure 5—figure supplement 6B.
- https://cdn.elifesciences.org/articles/111335/elife-111335-supp4-v1.zip
-
MDAR checklist
- https://cdn.elifesciences.org/articles/111335/elife-111335-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
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Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking
eLife 15:RP111335.
https://doi.org/10.7554/eLife.111335.3
