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. (H) Western blot on WCLs of HEK293 cells over-expressing wildtype or FHLH4 mutant STX11 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), STX11 shRNA, expressing FHLH4 mutant STX11 (blue).

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 scramble and STX11 shRNA treated, resting and store-depleted, HEK293 cells. Measurement of Orai1-YFP (A) and Alexa Fluor 647-conjugated Bungarotoxin binding (B) in population of cells using flow-cytometry. Quantification of Orai1-YFP and BTX-A647 in individual cells using confocal imaging (C). (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) Scr or STX11 shRNA treated HEK293 and Jurkat T cells were loaded with Alexa-647-Transferrin (30 minute 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 50-60 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) 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 substrate at 48h post-transfection. (J-K) Immunolabelling 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 and stimulated with 1 μM ionomycin. (N=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) Representative current at −100 mV in Jurkat T cells treated with Scr shRNA 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. (B) ICRAC from a Jurkat T cell treated with STX11 shRNA. Both Ca2+ and Na+ current amplitudes are reduced relative to 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 and current reversal potentials in Scr and STX11 knockdown cells.

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, immunolabelled 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) Granule release assay performed on HD (black) and FHLH4 CD8 T (red) cells (G) and its quantification (H).

STX11 directly binds resting Orai1 in plasma membrane.

Intracellular localization of STX11 and its association with Orai1. (A) 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 nuclear 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. Input:1/5th of the protein. (Bottom) Western blot using anti-His antibody. 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) Quantification of the localization of STX11 with respect to Stim1:Orai1 puncta in store-depleted cells.

AF3 predictions for STX11:Orai1 complex formation

AF3 predictions for STX11-Habc and Orai1 C-terminus with different seeds

AF3 predictions for SOAR and Orai1 in 2:2 ratio

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-labelled with Alexa Fluor-488 conjugated wheat germ agglutinin (WGA) or co-transfected with Orai1-YFP and immunolabelled with anti-STX11 to assess co-expression in PM of resting and store-depleted cells (Bottom). Scale bar 10μm. (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 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) Representative confocal images (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.

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. (G-J) 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. (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 (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 scramble and STX11 shRNA treated HEK293 cells (O) and quantification of intensity (P) and area of GFP-MAPPER puncta (Q). n=15.

STX11 primes Orai1 for optimal on-site assembly.

Oligomerization of Orai1. (A) N-FRET analysis of scramble 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 in control and STX11 depleted HEK293 cells. N=3. (F) Representative confocal image and quantification (right) of YFP-CAD localization in Scr and STX11 shRNA treated HEK293 cells expressing Orai1-CFP. (G) 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=60-70, 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

Models depicting STX11 mediated priming of Orai1 and its role in T cell signaling and function.

(A) STX11 unbound (unprimed) and bound (primed) Orai1 in resting and store-depleted cells. For simplicity, STX11 interaction with only two subunits of Orai1 has been shown. (B) Regulation of T cell SOCE, NFAT activation and effector function by STX11.

(A-H) Targeted RNAi screen for SNAREs involved in SOCE. Fura-2 calcium imaging assays measuring thapsigargin (TG) induced SOCE in HEK293 and/or Jurkat cell lines treated with scramble (scr) shRNA or different shRNA sequences against the gene of interest for 3-4 days. Shown here are the average traces from 30-40 cells per group.

(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 scramble 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 3 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.

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 cells treated with scramble (scr) shRNA or different shRNA sequences against the gene of interest for 3-4 days. Shown here are the average traces from 30-40 cells per group.

(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. (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 house-keeping 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.

(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 1uM 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).

SOCE in RNAi treated Jurkat cells to assess the role of SNAPs.

(A-C) Fura-2 calcium imaging assay measuring SOCE in cells treated with scramble (scr) shRNA or different shRNA sequences against SNAP23 (A) SNAP25 (B) SNAP29 (C). Average traces from 30-40 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).

(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 STX-11-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.

(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.

(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.

(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 fraction of Orai1 inside and outside Stim1:Orai1 clusters in store-depleted HEK293 cells expressing CFP-Orai1 and Stim1-YFP.

(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) The binding free energy distribution of the three runs for the interactions between the SOAR dimer and Orai1 C termini. (C) Binding free energy (ΔG) of SOAR dimer and Orai1 C-termini complex across the simulation time for all three runs.

(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.

(A-D) Representative confocal images (A) and q uantification (B-D) of 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.

Lower half of the Western blot of BS3 cross-linked Flag-Orai1 acquired at a higher exposure showing the monomeric Flag-Orai1 band (labelled as A).

(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. (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= 80-90, N=3.

(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. (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.