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. 2021 Dec 16;10:e72051. doi: 10.7554/eLife.72051

Figure 3. 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 3—figure supplement 1. Fractional TIRF vs. total ORAI1-YFP fluorescence of WT and C143A O1/S1 cells.

Figure 3—figure supplement 1.

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).

Figure 3—figure supplement 2.

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.

Figure 3—figure supplement 3.

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.