S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse
- Department of Cell Physiology and Metabolism, Switzerland
- Faculty of Life Sciences, Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Switzerland
- Department of Physiology, Yonsei University Wonju College of Medicine, Republic of Korea
Figures
Figure 1 with 2 supplements
ORAI1 is S-acylated at cysteine C143.
(A) ORAI1 immunoblot of HeLa cells treated with PEG-5k to label S-acylation sites after exposure to NEM to block free thiols and then to hydroxylamine (HA) to break acyl-thioester bonds. (B) Western blot and corresponding autoradiogram of HeLa cells labelled for 2 hr with 3H-palmitic acid with or without HA and immunoprecipitated with anti-ORAI1. (C, D) Western blots and corresponding autoradiograms of HeLa (C) and RPE-1 (D) cells expressing the indicated YFP-tagged ORAI1 mutants labelled with 3H-palmitic acid and immunoprecipitated with anti-GFP. Graph bars correspond to the mean ± SEM 3H-palmitic acid incorporation normalised to WT construct in three to four independent experiments. (E) Normalised mean fura-2 responses evoked by Ca2+ readmission in HEK-TKO cells transiently transfected with the indicated ORAI1-YFP constructs and pre-exposed to Tg 1 µM for 8 min. (F) Area under the curve of the responses in E. Data are mean ± SEM of eight independent experiments. One-way ANOVA Dunnett’s multiple comparisons test.
Figure 1—figure supplement 1
Right, Orai1 protein sequences aligned with CLustalW for the indicated organisms.
Cysteines susceptible to be S-acylated are highlighted in yellow. Left, Schematic ORAI1 representation. Superimposed structures of the WT and H206A dOrai (PDB ID: 4HKR and 6BBF) conformations in ribbon representation highlighting cysteine residues at position 126, 143, and 195.
Figure 1—figure supplement 2
Top; fluorescence images of WT O1/S1 cells (left) and averaged mCh-STIM1 and ORAI1-YFP fluorescence of the different O1/S1 stable cell lines Data are mean ± SE of 53–132 cells.
Bottom; Averaged fura-2 responses (left) and integrated response (right) of O1/S1 cells bearing or not the indicated ORAI1 mutation(s). Data are mean ± SEM of 44–82 cells from two independent experiments. One-way ANOVA Dunnett’s multiple comparisons test.
Figure 2 with 2 supplements
Preventing ORAI1 S-acylation reduces ICRAC currents.
(A) Averaged fura-2 responses evoked by Ca2+ readmission to Tg-treated (1 µM for 8 min) HEK-293 cells stably expressing mCh-STIM1 and ORAI1-YFP (O1/S1) bearing or not the C143A mutation. (B) Fold peak amplitude (Peak divided by basal) of the responses in A. Data are mean ± SEM of 196 (WT) and 198 (C143A) cells from five independent experiments. (C) Averaged ICRAC recordings of WT and C143A O1/S1 cells, measured every 5 sec at –100 mV. ICRAC was activated by cell dialysis with 10 mM BAPTA and 10 µM Gd3+ added at the end of the recordings (see Figure 2—figure supplement 1). Data are mean ± SEM of 12 (WT) and 14 (C143A) cells. (D) Averaged current-voltage relationship of steady-state Gd3+-sensitive currents in the cells in C. (E) Peak current densities (Imax) of WT and C143A O1/S1 cells after subtraction of basal or Gd3+-insensitive currents (same cells as C). (F) Time-course of current activation. Left: Averaged recordings with basal and maximal values set to 0 and –1, respectively, aligned at t = 0 for break-in. Cells with pre-activated currents were not included. Right: Statistical evaluation of the activation time. Data are mean ± SEM, of 11 (WT and C143A) cells. Two-tailed unpaired Student’s t-test.
Figure 2—figure supplement 1
Representative ICRAC recordings of WT and C143A O1/S1 HEK-293 cells.
Bottom, time-course of current activation in cells without pre-activated currents. Basal and maximal values were set to 0 and –1, respectively, and aligned to the time of break-in. Dashed line indicates 90 % of current development.
Figure 2—figure supplement 2
Families of currents evoked by 200 ms voltage steps to voltages ranging from –120 to –60 mV in steps of 20 mV in WT and C143A O1/S1 perfused with 10 mM EGTA to enable FCDI.
Left: representative traces. Middle: Fractional current at 197 ms vs. 3 ms recorded at the indicated voltages. Right: Inactivation kinetics (Tau fast) derived from exponential fits to the first 10 ms at the indicated voltages. Data are mean ± SEM, of 12 vs 12 cells. Two-way ANOVA.
Figure 3 with 3 supplements
Preventing ORAI1 S-acylation reduces channel clustering and affinity for lipid rafts.
(A) Cartoon of the YFP-HA-ORAI WT or C143A constructs with HA exposed extracellularly between TM3 and TM4. HEK-293 cells transiently expressing the indicated constructs were stained for HA in non-permeabilising conditions to decorate the ORAI1 PM fraction. Graph bars represent the mean ± SEM of 15 (WT), 17 (C143A) and 10 (WT+ NP-40) cells. Right, Representative confocal images of YFP (green) and HA (red) staining in the indicated conditions. Scale bar = 25 µm. (B) Representative TIRF images of WT and C143 O1/S1 cells exposed to 10 µM CPA for 10 min to induce mCh-STIM1 and ORAI1-YFP clustering. Bars = 5 µm. (C–E) Quantification of ORAI1-YFP clusters numbers (C), of the average ORAI1-YFP fluorescence inside STIM1 clusters (D), and of the Pearson’s co-localisation coefficient between mCh-STIM1 and ORAI1-YFP (E) 10 min after CPA exposure. Data are mean ± SEM of 13 (WT) and 12 (C143A) cells from three independent experiments. (C, E) Two-tailed unpaired Student’s t-test. (D) One tailed.
Figure 3—figure supplement 1
Fractional TIRF vs. total ORAI1-YFP fluorescence of WT and C143A O1/S1 cells.
Data are mean ± SEM of 65 (WT) and 61 (C143A) cells in three independent experiments.
Figure 3—figure supplement 2
Time-course of CPA-induced changes in ORAI1-YFP (top) and mCh-STIM1 (bottom) fluorescence (left), number of clusters (middle), and relative cluster area (right, with minimal value set to 0 and maximal value to 100).
Data are mean ± SEM of 13 (WT) and 12 (C143A) fluorescence-matched cells from three independent experiments. Bottom representative TIRF images ORAI1-YFP and mCh-STIM1 clusters formation in WT and C143A O1/S1 cells treated with CPA.
Figure 3—figure supplement 3
FRAP recordings of WT and C143 O1/S1 cells.
Left: original recordings and normalised fluorescence decay and recovery, Middle: representative images, bar = 5 µm. Right: diffusion coefficient (left) and plateau values (right) for the indicated cell lines. Data are mean ± SEM of 7 (WT) and 7 (C143A) fluorescence-matched cells from three independent experiments. Two-tailed unpaired Student’s t-test.
Figure 4 with 2 supplements
PAT20 S-acylates ORAI1 and modulates its activity.
(A) Western blot and matching autoradiogram of RPE-1 cells expressing ORAI1-YFP plus the indicated PAT isoform, labelled with 3H-palmitic acid and immunoprecipitated with anti-GFP. Graph bar (right) shows densitometry analysis of the tritiated bands relative to GFP in RPE1 (Blue, N = 2) and HeLa cells (yellow, N = 1). (B) Functional effect of PAT3, 7, and 20 expression. Representative western blot of HeLa cells expressing Myc-tagged PAT isoforms (left), averaged SOCE responses (middle), and peak amplitude (right). Data are mean ± SEM of 49–74 cells from three independent experiments. (C) Averaged SOCE responses of WT (left) or C143A (middle) S1/O1 cells expressing these PAT isoforms and their peak amplitude (right). Data are mean ± SEM of 31–129 cells from five independent experiments. (D) Lipid partitioning of ORAI1 in giant plasma membrane vesicles from HEK-293 cells transiently transfected with PiP2-Cherry and WT or C143 ORAI1-YFP together with PAT20 or PCDNA3. Left: representative fluorescence images of vesicles from cells expressing PAT20 and WT or C143 ORAI1-YFP (green) stained with cholera toxin subunit B (Magenta) as raft marker and PiP2-Cherry (Red) as non-raft marker (scale bar = 2.5 µm). Right: Graph bar representing the % of ORAI1 preference for raft domains. Data are mean ± SEM of 13 (WT empty), 12 (C143A empty), 23 (WT+ PAT20), and 16 (C143A + PAT20) vesicles from four independent experiments. (A–C) One-way ANOVA Dunnett’s multiple comparisons test. (D) One-tailed unpaired Student’s t-test.
Figure 4—figure supplement 1
Top graph bars represent the PAT3, 7, and 20 mRNA expression levels in cells transfected with the indicated siRNA (N = 6–12 biological replicates in two to four independent transfections).
Data are normalised to 18 s and to matching si-Scr control. Bottom;averaged SOCE responses (left), and peak amplitude (right) of WT and C143A S1/O1 cells transfected with the indicated siRNAs. Data are mean ± SEM of 61–126 (WT) and 180–250 (C143A) cells from two independent experiments. Student-t test (TOP) and One-way ANOVA Turkey multiple comparisons test (Bottom graph bars).
Figure 4—figure supplement 2
Averaged SOCE responses (left), and peak amplitude (right) of HEK-TKO cells transiently transfected with the indicated siRNAs plus mCh-STIM1 and either WT or C143A ORAI1-YFP.
Data are mean ± SEM of 36–42 cells from three independent experiments. or Sidak multiple comparisons tests.
Figure 5 with 7 supplements
ORAI1 S-acylation promotes Jurkat T cell activation.
(A) Averaged fura-2 responses, their peak amplitude (middle graph bar) and slope (right graph bar) evoked by Ca2+ re-addition in Tg treated (1 µM, 8 min) Jurkat cells lines generated by CRISPR with control or ORAI1-targeted guiding sequences and stably re-expressing either WT or C143A ORAI1-YFP. Data are mean ± SEM of 210 (Control), 242 (KO), 189 (WT), and 203 (C143A) cells from three independent experiments (B) Western blot showing the amount of biotinylated GFP immunoreactivity in the PM in Jurkat CRISPR ORAI1 cells reconstituted with WT or C143A ORAI1-YFP. Representative of 2 independent experiments. (C) Individual (thin lines) and averaged (thick line) fura-2 recordings of Jurkat CRISPR ORAI1 cells reconstituted with WT or C143A ORAI1-YFP, exposed to CD3/CD28-coated beads in Ca2+ containing solution (left). Averaged peak and integrated responses evoked by CD3/CD28 beads in individual cells during the recording period (right). Data are from 52 cells (WT) and 72 cells (C143A) from three independent experiments. (D) Relative changes in NFATC-Luciferase vs. housekeeping- Renilla luminescence evoked in 4 h by Tg (1 µM) and PMA (100 nM) in the indicated cell lines. Data are mean ± SEM of 8–12 biological replicates from three independent experiments. (E) Endogenous NFATC1 translocation evoked in 4 hr by Tg (1 µM) in the indicated cell lines, measured by immunofluorescence. Data are mean ± SEM of the nuclear to cytosol NFATC1 intensity ratio of 58–161 cells from four independent experiments. (F) Time-course of NFATC1 translocation evoked by plates coated with CD3 (OKT3 1 µg/ml). Data are mean ± SEM of 86–161 cells from five independent experiments. (G) IL-2 production evoked by untreated (NT) or surface coated CD3 (OKT3 1 µg/ml). Left, Representative density dot plots of Jurkat cells stained for IL-2. Graph bars represent the mean ± SEM of three independent experiments. One-way ANOVA Dunnett’s multiple comparisons test (A), Sidak multiple comparisons test (D and E), two-tailed unpaired Student’s t-test (C and G).
Figure 5—figure supplement 1
Sequences of genomic DNA used to generate the CRISPR ORAI1 Jurkat T cell lines (top) and FLAG or ORAI1 immunoblot of Jurkat T cells expressing FLAG-tagged Cas9 (bottom), WB are representative of two independent experiments.
Bottom; representative flow cytometry Fluo eight responses evoked by the Tg/Ca2+protocol in the indicated cells (left) and their averaged response and peak amplitude (right). Data are mean ± SD of two independent experiments.
Figure 5—figure supplement 2
Fluorescence intensity profiles of CRISPR ORAI1 cells reconstituted with WT and C143 ORAI1-YFP measured by flow cytometry (N = 4).
Student-t test.
Figure 5—figure supplement 3
Representative Fura2 recordings of the responses evoked by CD3/CD28-coated beads in Jurkat CRISPR ORAI1 cells reconstituted with WT or C143A ORAI1-YFP (from Fig.Figure 5C).
Figure 5—figure supplement 4
Top left blot indicates the3H-palmitate incorporation in ORAI1-deficient Jurkat T cells reconstituted with WT ORAI1-YFP transiently expressing the indicated PAT isoforms.
Top right panel shows 3H-palmitate incorporation relative to total GFP IP for the indicated conditions in two independent experiments. Bottom left blot presents the 3H-palmitate incorporation in ORAI1-deficient Jurkat T cells reconstituted with C143A ORAI1-YFP transiently expressing PAT3, 7, and 20. Bottom left blot presents the3H-palmitate incorporation in ORAI1-deficient Jurkat T cells reconstituted with WT ORAI1-YFP transiently expressing each of the 23 PAT isoforms.
Figure 5—figure supplement 5
Averaged SOCE responses (left) and peak SOCE amplitude (middle) measured with YC3.6 in WT or ORAI1-deficient Jurkat T cells expressing PAT20 or a control plasmid.
Data are mean ± SEM of 10–40 cells from two independent experiments. Right,Fraction of IL-2-positive WT or ORAI1-deficient Jurkat T cells expressing PAT20 or a control plasmid treated with Tg for 4 hr or adhered 24 hr on plates coated with CD3 (1 µg/ml) with or without Ionomycin (1 µM) and PMA (20 nM added 2 hr before fixation). N = 1. One-way ANOVA Dunnett’s multiple comparisons test (H).
Figure 5—figure supplement 6
NFATC1 immunoreactivity of ORAI1-deficient Jurkat T cells reconstituted with WT and C143 ORAI1-YFP treated with Tg 1 µM for 4 hr to induce nuclear translocation of NFATC1.
Line scan: 30 µm. Line scan normalised intensities for Hoesch and NFATC1 are plotted to illustrate NFATC1 nuclear localisation.
Figure 5—figure supplement 7
Fraction of IL-2 positive ORAI1-deficient Jurkat T cells reconstituted with WT or C143A ORAI1-YFP and transfected with si-Scr and si-PAT20 adhered 24 hr on plates coated with CD3 (1 µg/ml).
Data are mean ± SEM of 6–9 samples from three independent experiments. Student-t test one way.
Figure 6 with 5 supplements
ORAI1 S-acylation regulates TCR enrichment and signaling at the immune synapse.
(A) Conditions used to study synapse formation between Raji and Jurkat T cells. Raji are pulsed with SEE prior to co-culture with Jurkat cells, IS formation evaluated in living (25 min) and fixed cells (1 H) and Jurkat activation at 24 hr. (B) Relative changes in NFAT-Luciferase vs. housekeeping- Renilla luminescence evoked by coculture of the indicated cell lines for 24 hr with naïve or SEE (1 µg/ml) pulsed Raji. Data are mean ± SEM of 15–20 biological replicates in cells from four independent experiments. (C) Representative images of ORAI1-deficient Jurkat T cells reconstituted with WT or mutant ORAI1-YFP co-cultured with pulsed RAJI cells stained with CellMask deep red. (Scale bar = 10 µm). (D) ORAI distribution in IS vs. opposite pole in IS forming between SEE-pulsed Raji and ORAI1-deficient Jurkat T cells reconstituted with WT (29 IS) or mutant (26 IS) ORAI1. Chi-square p value: 0.0014, two-sided Fisher’s exact test. (E) Quantification of ORAI1 enrichment in time-lapse images from C. IS accumulation was measured using kymographs of 20 pixels wide lines spanning the IS-distal cap axis, drawn on stable (not moving) IS forming in WT (12) and C143A (8) cells from three independent experiments. Graph bar shows mean ± SEM area under the curve of the kinetic enrichment graphs. (F) Representative confocal images of IS forming between SEE-pulsed Raji labeled with CellMask (Magenta, APC white labelling) and ORAI1-deficient Jurkat T cells reconstituted with WT or mutant ORAI1-YFP (Green), labelled with Phalloidin (Blue) and anti-TCR-PE (Red). Scale bar = 10 µm. Arrows indicate IS formation and asterisks accumulation of ORAI1 in the opposite pole. (G) IS enrichment for ORAI1-YFP, Phalloidin and TCR fluorescence in Jurkat T cells reconstituted with WT or C143A ORAI1-YFP. Fluorescence levels at the IS and opposite pole were extracted from 10 pixel wide line profiles. Graph bars shows mean ± SEM in WT (n = 25 IS) and C143A (n = 20 IS). Two-tailed unpaired Student’s t-test (B, E and G); Chi square (D). (H) Proposed model: S-acylation by PAT20 targets ORAI1 to lipid rafts, enabling the coordinated recruitment of ORAI1 and TCR to the immune synapse for efficient signalling.
Figure 6—figure supplement 1
Top; Representative images of IS formation in ORAI1-deficient Jurkat T cells reconstituted with WT or C143A ORAI1-YFP, related to Figure 6C.
Bottom; Procedure to study ORAI IS enrichment in kymographs from line-scan recordings (related to Figure 6E).
Figure 6—figure supplement 2
Surface TCR-PE expression in the indicated cell lines determined by flow-cytometry.
Data are mean ± SEM of three experiments.
Figure 6—figure supplement 3
Phosphorylated vs.total ERK1/2 immunoreactivity of ORAI1-deficient Jurkat T cells reconstituted with WT or C143A ORAI1-YFP adhered or not for 15 min on plates coated with CD3 (1 µg/ml).
N = 4 independent experiments. One sample T test (to hypothetical value of 1) (D).
Figure 6—video 1
Related to Figure 6C–E.
ORAI1-deficient Jurkat T cells reconstituted with WT ORAI1-YFP co-cultured with pulsed RAJI cells stained with CellMask deep red. Images were obtained every 30 s. As seen ORAI1 fluorescence is accumulated in the IS interphase.
Figure 6—video 2
Related to Figure 6C–E.
ORAI1-deficient Jurkat T cells reconstituted with C143A ORAI1-YFP co-cultured with pulsed RAJI cells stained with CellMask deep red. Images were obtained every 30 s. As seen ORAI1 fluorescence is accumulated in the opposite pole of the IS.
Author response image 1
CRISPR ORAI1 KO + ORAI1 WT Jurkat cells were treated with ML349 (10µM 24h) or untreated (left) or transfected with PAT3, PAT7 or PAT20 (Right).
Cells were pulsed for 2 H with palmitate and chased for the indicated times. Autoradiograms show IP of ORAI1-YFP with GFP beads.
Author response image 2
CRISPR ORAI1 KO + ORAI1-WT Jurkat cells transiently expressing each of 23 PAT isoforms were labeled with tritiated palmitate.
Autoradiograms show IP of ORAI1-YFP with GFP beads. Only PAT3, 7 and 20 promoted ORAI1 S-acylation.
Author response image 3
ORAI fluorescence in HEKs1/O1 WT or C143A in our previous submission and in the new one, with fluorescent matched basal levels.
Author response image 4
Author response image 5
Lipid raft preference of WT (left) or C143A (right) ORAI1-YFP co-expressed with an empty plasmid or PAT20.
Related to Figure 4D. Data was aggregated by experimental day and paired to demonstrate that despite the inter-experimental variability, the raft preference of ORAI1 WT consistently increased when PAT20 is coexpressed (n = 4 independent days).
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S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse
eLife 10:e72051.
https://doi.org/10.7554/eLife.72051
