Supplementary information (Nature 2005-04-04198)
Supplementary Methods
Molecular cloning and mutagenesis
A cDNA clone, pAc5.1/D-STIM, encoding full-length DrosophilaStim was generated for transfection into S2 cells. Briefly, a 1.7 kb fragment was isolated from total mRNA of Drosophila S2 cells by RT-PCR, and subcloned between the XhoI and NotI sites of pAc5.1/V5-His B expression vector. Primers were designed based on the deposited sequence of Stim (accession number: AF328906). The pAc5.1/D-STIM-V5-His clone was made by deleting the original stop codon during RT-PCR, and subcloned into the XhoI and NotI sites of pAc5.1/V5-His A vector. Resulting clones were confirmed by sequencing. pAc5.1/EGFP was generated by cutting the EGFP coding region from pEGFP-N1(Clontech) and inserting into pAc5.1/V5-His A vector between EcoRI and NotI sites). Stim and Stim-V5-His EF hand mutants were made by exchanging the corresponding codons (EF1A3A: GAT to GCT and GAC to GCC; EF1N3N: GAT to AAT and GAC to AAC; EF12A: GAG to GCG; EF12Q: GAG to CAG; see supplementary figure 2) using the QuikChange site-directed mutagenesis kit (Stratagene). Cloning of full-length STIM1 (pcDNA5/FRT/TO/rSTIM1) was performed by subcloning a 2.1 kb cDNA fragment (from total mRNA of RBL-2H3 cells, accession number XM_341896) into a pcDNA5/FRT/TO-TOPO expression vector. Resulting clones were sequenced. The predicted sequence revealed 97% amino acid identity with human STIM1. QuikChange was used to make rat STIM1 EF hand mutants (EF1A3A: GAT to GCT and GAT to GCT; EF12Q: GAA to CAA; supplementary figure 2).
Solutions for Ca2+ imaging
Concentrations are in mM and osmolality in mOsm/kg. S2 Ringer solutions contained 2.5 mM probenecid. Ca2+ - free Ringer solutions contained 1 mM EGTA. All solutions contained 10 mM d-glucose.
External / Na+ / K+ / Ca2+ / Mg2+ / Cl- / HEPES / pH / OsmolalityS2 Ringer (Ca2) / 150 / 5 / 2 / 4 / 167 / 10 / 7.2 / 328
Jurkat Ringer (Ca2) / 155 / 4.5 / 2 / 1 / 165.5 / 5 / 7.4 / 310
Ca2+-free S2 Ringer (Ca0) / 150 / 5 / - / 6 / 167 / 10 / 7.2 / 332
Low-Ca2+ Jurkat Ringer (Ca0.4) / 155 / 4.5 / 0.4 / 2 / 164.3 / 5 / 7.4 / 310
Ca2+-free Jurkat Ringer (Ca0) / 153 / 4.5 / - / 3 / 163.5 / 5 / 7.4 / 310
Solutions and protocols for perforated patch recording
The internal solution contained (in mM): 45 CsCl, 70 Cs2SO4, 7 mM MgCl2, 1 CaCl2, 10 HEPES-Na+, pH = 7.3. The external solution contained: 6 CaCl2, 150 Na+ aspartate, 2 Cs+ methanesulfonate, 10 HEPES-Na+, 2 glucose, pH= 7.3. Voltage ramps 211 ms in duration from –110 to +85 mV were applied every 2.5 seconds, and inward current amplitude was measured at –100 mV.
Cell preparation for immunocytochemistry, light and electron microscopy
After PBS washing, cells were fixed in 4% paraformaldehyde for 20 minutes. Fixed cells were blocked and permeabilized in PBS with 10% NGS and 0.2% Triton X-100 for 20 minutes, then incubated with primary (anti-STIM1 rabbit polyclonal antibodies, used at a dilution of 1:500; anti-SERCA2 monoclonal antibodies from Sigma, used at a dilution of 1:500; and anti-PDI monoclonal antibodies from Molecular Probes, used at a dilution of 1:1000) and secondary antibodies for 1 hour at room temperature. All fluorescence dye-conjugated secondary antibodies from Molecular Probes (Alexa488 goat anti-rabbit; Alexa594 goat anti-rabbit) were used at a dilution of 1:1000. Texas-red goat anti-mouse secondary antibodies from Jackson Lab were used at a dilution of 1:300. Control staining with secondary antibody alone demonstrated the specificity of the polyclonal anti-STIM1 antibodies (data not shown). Qdot655 goat anti-rabbit secondary antibodies (Quantum Dot Corporation) were used at a dilution of 1:250.
Surface biotinylation
3×107 Jurkat cells were placed on ice for 30 min after treatment. The cells were biotinylated with 0.5 mg/ml sulfo-NHS-SS-biotin/PBS at 4°C for 45 min. Following quench of unreacted biotinylation reagent, the cells were washed and lysed. After insoluble materials were removed by centrifugation, biotinylated proteins were collected by incubation with immobilized NeutrAvidin Gel overnight at 4°C. Proteins were eluted by boiling in 5×SDS-PAGE denaturing sample buffer (Pierce) for electrophoresis and Western blotting with STIM1 Ab. Anti-CD3ε HRP-conjugated mAb (Santa Cruz) was used to verify and normalize for cell surface expression of T cell receptors. Anti-GAPDH mAb was used to confirm the absence of intracellular proteins.
Supplementary Figures (provided as pdf files)
Supplementary Figure 1 STIM1 and Stim sequence and domains. Protein sequence comparison shows an overall 31% identity and 60% similarity of DrosophilaStim and STIM1 (signal peptides were not included). Predicted functional domains are indicated: EF hand motif (yellow), potential N-glycosylation site (red), SAM domain (grey), transmembrane domain (turquoise), coiled-coil domain (bold italics). Green shading indicates conserved cysteine residues. Functional domains are highly conserved between Stim and STIM1: 92% similarity of the EF hand motif, 79% similarity of the SAM domain, 82% similarity of the TM domain, and 79% similarity of the coiled-coil domain.
Supplementary Figure 2 Expression of wild-type and EF hand mutant Stim and STIM1. a, Stim domains and EF hand mutation sites. b, Anti-V5 western blot of protein from untransfected S2 cells (lane 1), overexpression of Stim (lane 2) and Stim EF hand mutant 1A3A (lane 3). Equal protein amounts were loaded for each lane. c, STIM1 domains and EF hand mutation sites. d, Anti-STIM1 western blot of protein from untransfected Jurkat cells (lane 1), overexpression of WT STIM1 (lane 2), and STIM1 EF hand mutants 1A3A and 12Q (lanes 3 and 4, respectively). GAPDH protein levels showed equal loading of lanes.
Supplementary Figure 3 Effects of Stim and EF hand Stim overexpression on [Ca2+]i signals in S2 cells. a, Average intracellular [Ca2+]i responses in blank-transfected S2 cells, S2 cells overexpressing WT Stim, and S2 cells overexpressing Stim mutant EF1A3A, respectively. b, Effects of CRAC channel blockers 2-APB (50 M), SKF96365 (20 M), and Gd3+ (1 M) on TG-evoked Ca2+ influx in S2 cells. c, Reduction of the elevated resting [Ca2+]i in S2 cells transfected with mutant Stim EF1A3A by 2-APB (50 M), SKF96365 (20 M), and Gd3+ (1 M). Error bars: SEM.
Supplementary Figure 4 Single-cell analysis of resting [Ca2+]i and TG-independent Ca2+ influx. a, Histogram of resting [Ca2+]i in control Jurkat cells (114 untransfected cells), cells overexpressing WT STIM1 (171 cells) and cells overexpressing STIM1 EF hand mutants (126 cells; two cells >800 nM are not shown); respectively. B, TG-independent Ca2+ influx histogram of control Jurkat cells (114 untransfected cells); cells overexpressing WT STIM1 (136 cells); and cells overexpressing STIM1 EF hand mutants (103 cells; eight cells >800 nM are not shown). C, Resting Ca2+ histogram of control S2 cells (392 untransfected cells); cells overexpressing WT Stim (568 cells); and cells overexpressing Stim EF1A3A (425 cells; 18 cells >600 nM are not shown).
Supplementary Figure 5 Ca2+ release transients in control cells and cells transfected with EF hand mutants. a, Averaged traces of Ca2+ release evoked by TG in zero-Ca2+ external solution. b, cells overexpressing STIM1 EF12Q (from a) were arrayed by their resting [Ca2+]i level and put into 6 subgroups. Averaged traces of each group show there is no relation between resting [Ca2+]i level and store content. c, Comparison of the peak [Ca2+]i of Ca2+-release transients in control S2 cells and S2 cells overexpressing Stim mutant EF12Q. Error bars: SEM.
Supplementary Figure 6 Expression of Stim EF hand mutants inhibits S2 cell growth and induces apoptosis. a, Stim EF hand mutants (EF1A3A, EF1N3N, EF12A and EF12Q) inhibit S2 cell proliferation. Controls (blank transfection) and transfected cells were cultured with a beginning density of 1.25*106cells per ml. Viable cells were counted daily. b,Annexin-V staining and brightfield images of EF1A3A Stim(top) and WT Stim(bottom). Error bars: SEM.
Supplementary Figure 7 STIM1 translocation in RBL cells, human T cells, and PC12 cells. All cells bathed in 2 mM Ca2+ Ringer for 5 min (left) or treated with TG + zero-Ca2+ for 5 min (right).Scale bar is 5 m. a, RBL cells. b, Human T cells. c, PC12 cells.
Supplementary Figure 8 Colocalization of STIM1 with ER markers in RBL cells. a, STIM1 (green) and PDI (red) immunofluorescence staining of RBL cells in 2 mM Ca2+ Ringer solution or 1 M TG in zero-Ca2+ solution for 5 min. Bottom panels are colocalization images (yellow) that depict pixels that contained both STIM1 and ER fluorescence (pixels containing only STIM1 or ER staining were erased). Note the reduced colocalization caused by STIM1 translocation in TG-treated cells. b, Time course of CRAC channel activation in response to CPA (denoted by application bar) in RBL cells.
Supplementary Figure 9 Translocation of STIM1 occurs in Jurkat T cells when Ca2+ stores are depleted by TG or CPA treatment, but not under control conditions. Scale bar is 5 m. a, CPA (10 M) in zero-Ca2+ solution: note STIM1 hotspots. b, 2 mM Ca2+ + TG: note STIM1 hotspots. c, Cells treated with zero-Ca2+. d, Cells treated with 2 mM Ca2+ + DMSO.
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