Figure 1. Ca 2 tunneling in primary... | Rockefeller University Press

Figure 1.

Ca2+tunneling in primary salivary gland cells. (A) Cartoon depiction of Ca2+ tunneling. STIM1–Orai1 generates localized Ca2+ influx within ERPMCS defining the SOCE microdomain. Ca2+ taken into the ER by SERCA diffuses through the cortical ER and is released by IP3R to activate distal cortical targets. Generated using Biorender. (B) Comparison of SOCE and Ca2+ tunneling protocols. The ER Ca2+ stores are depleted by the reversible SERCA inhibitor CPA. This is followed by a washout phase to restore SERCA activity. SOCE develops upon the addition of extracellular Ca2+. Concurrent exposure to an agonist to produce IP3 or uncaging of IP3 activates Ca2+ tunneling. (C) Example traces of the cortical Ca2+ signals recorded during SOCE, SOCE induced by the irreversible blockade of the SERCA pump by thapsigargin (SOCE-Tg), and during tunneling. Traces are taken from Courjaret et al. (2018). (D) Cytosolic Ca2+ responses during SOCE and tunneling in isolated salivary gland cells. Tunneling is induced by the addition of CCh (10 µM) simultaneously to Ca2+ reperfusion. The bar chart quantifies the peak Ca2+ signal of tunneling versus SOCE (n = 26; unpaired t test, P < 0.0001). (E) Whole-cell Cl− currents in response to SOCE versus tunneling, showing similar profiles to the Ca2+ signals; and bar chart summarizing the data (n = 9; unpaired t test, P < 0.0001). (F) Localization at the whole cell level of mCh-STIM1 and GFP-Orai1 in HeLa cells. At rest (Ctr) the confocal plane is in the middle of the cell. After store depletion with thapsigargin (Tg) the image was acquired at the PM optical plane to visualize STIM1-Orai1 co-clusters. (G) Orthogonal sections from confocal z-stacks in control conditions and after store depletion. (H and I) Relative intensities of STIM1 and Orai1 across z-stack profiles as in G before (Ctr) and after store depletion (Tg). (J) Pearson correlation coefficient (PCC) between mCh-STIM and GFP-Orai1 measured in orthogonal slices before (Ctr) and after store depletion (Tg) (n = 13–16; unpaired t test, P < 0.0001).

Ca ²⁺ tunneling in primary salivary gland cells. (A) Cartoon depiction of Ca²⁺ tunneling. STIM1–Orai1 generates localized Ca²⁺ influx within ERPMCS defining the SOCE microdomain. Ca²⁺ taken into the ER by SERCA diffuses through the cortical ER and is released by IP₃R to activate distal cortical targets. Generated using Biorender. (B) Comparison of SOCE and Ca²⁺ tunneling protocols. The ER Ca²⁺ stores are depleted by the reversible SERCA inhibitor CPA. This is followed by a washout phase to restore SERCA activity. SOCE develops upon the addition of extracellular Ca²⁺. Concurrent exposure to an agonist to produce IP₃ or uncaging of IP₃ activates Ca²⁺ tunneling. (C) Example traces of the cortical Ca²⁺ signals recorded during SOCE, SOCE induced by the irreversible blockade of the SERCA pump by thapsigargin (SOCE-Tg), and during tunneling. Traces are taken from Courjaret et al. (2018). (D) Cytosolic Ca²⁺ responses during SOCE and tunneling in isolated salivary gland cells. Tunneling is induced by the addition of CCh (10 µM) simultaneously to Ca²⁺ reperfusion. The bar chart quantifies the peak Ca²⁺ signal of tunneling versus SOCE (n = 26; unpaired t test, P < 0.0001). (E) Whole-cell Cl⁻ currents in response to SOCE versus tunneling, showing similar profiles to the Ca²⁺ signals; and bar chart summarizing the data (n = 9; unpaired t test, P < 0.0001). (F) Localization at the whole cell level of mCh-STIM1 and GFP-Orai1 in HeLa cells. At rest (Ctr) the confocal plane is in the middle of the cell. After store depletion with thapsigargin (Tg) the image was acquired at the PM optical plane to visualize STIM1-Orai1 co-clusters. (G) Orthogonal sections from confocal z-stacks in control conditions and after store depletion. (H and I) Relative intensities of STIM1 and Orai1 across z-stack profiles as in G before (Ctr) and after store depletion (Tg). (J) Pearson correlation coefficient (PCC) between mCh-STIM and GFP-Orai1 measured in orthogonal slices before (Ctr) and after store depletion (Tg) (n = 13–16; unpaired t test, P < 0.0001).

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