Figure 3.
Localization of PMCA4b and design of a tunneling inhibitor. (A) Distribution of GFP-PMCA4b and mCh-STIM1 in HeLa cells before (Ctr) and after (Tg) store depletion. The control confocal image is taken in the middle of the cell and images after store depletion at the PM plane. (B) Bar chart summarizing the PCC between mCh-STIM1, GFP-Orai1 (used as a maximum reference), and GFP-PMCA4b, measured in the orthogonal plane (n = 10–16; one-way ANOVA, P < 0.0001). (C) Quantification of the Z distance between mCh-STIM1 and GFP-PMCA4b peaks (n = 9–11; unpaired t test, P < 0.0001). (D and E) Intensity profiles along the x/y (D) and z (E) axis of mCh-STIM1 and GFP-PMCA4b across a STIM1 cluster (as indicated in A). (F) Cartoon summarizing the strategy to block cortical SERCA to specifically inhibit tunneling. (G) High magnification Airyscan images of GFP-MAPPER and mCh-STIM1 at rest (Ctr) and after store depletion (CPA). Line scans as indicated on the merged image show the colocalization of MAPPER (green) with the diffuse unclustered STIM1 (red) at rest, and the separation of clustered STIM1 from MAPPER. (H) Orthogonal sections of GFP-MAPPER and mCh-STIM1 before (Ctr) and after store depletion (CPA). The bar chart reports the distance in the z-axis between maximum intensities of GFP-MAPPER and mCh-STIM1 signals before (Ctr) and after store depletion (CPA) (n = 8–15; unpaired t test; P < 0.0001). (I) Cartoon depicting the structure of the Ca2+ tunneling attenuators (CaTAr1 and 2) compared to MAPPER. (J) Confocal images at the PM plane of CaTAr1 and mCh-STIM1 before (Ctr) and after (CPA) store depletion. The intensity plots using the white arrows in the high-magnification images report the colocalization of STIM1 and CaTAr1 at rest and their separation after store depletion. (K) Bar chart summarizing the PCC between CaTAr1 and mCh-STIM1 before (Ctr) and after store depletion (CPA) (n = 6; paired t test, P = 0.002). (L) Bar chart summarizing the distance between STIM1 clusters and CaTAr1 or MAPPER (MAP) (n = 6–7; unpaired t test, P = 0.91). Cartoons were generated using Biorender.

Localization of PMCA4b and design of a tunneling inhibitor. (A) Distribution of GFP-PMCA4b and mCh-STIM1 in HeLa cells before (Ctr) and after (Tg) store depletion. The control confocal image is taken in the middle of the cell and images after store depletion at the PM plane. (B) Bar chart summarizing the PCC between mCh-STIM1, GFP-Orai1 (used as a maximum reference), and GFP-PMCA4b, measured in the orthogonal plane (n = 10–16; one-way ANOVA, P < 0.0001). (C) Quantification of the Z distance between mCh-STIM1 and GFP-PMCA4b peaks (n = 9–11; unpaired t test, P < 0.0001). (D and E) Intensity profiles along the x/y (D) and z (E) axis of mCh-STIM1 and GFP-PMCA4b across a STIM1 cluster (as indicated in A). (F) Cartoon summarizing the strategy to block cortical SERCA to specifically inhibit tunneling. (G) High magnification Airyscan images of GFP-MAPPER and mCh-STIM1 at rest (Ctr) and after store depletion (CPA). Line scans as indicated on the merged image show the colocalization of MAPPER (green) with the diffuse unclustered STIM1 (red) at rest, and the separation of clustered STIM1 from MAPPER. (H) Orthogonal sections of GFP-MAPPER and mCh-STIM1 before (Ctr) and after store depletion (CPA). The bar chart reports the distance in the z-axis between maximum intensities of GFP-MAPPER and mCh-STIM1 signals before (Ctr) and after store depletion (CPA) (n = 8–15; unpaired t test; P < 0.0001). (I) Cartoon depicting the structure of the Ca2+ tunneling attenuators (CaTAr1 and 2) compared to MAPPER. (J) Confocal images at the PM plane of CaTAr1 and mCh-STIM1 before (Ctr) and after (CPA) store depletion. The intensity plots using the white arrows in the high-magnification images report the colocalization of STIM1 and CaTAr1 at rest and their separation after store depletion. (K) Bar chart summarizing the PCC between CaTAr1 and mCh-STIM1 before (Ctr) and after store depletion (CPA) (n = 6; paired t test, P = 0.002). (L) Bar chart summarizing the distance between STIM1 clusters and CaTAr1 or MAPPER (MAP) (n = 6–7; unpaired t test, P = 0.91). Cartoons were generated using Biorender.

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