Journal: The Journal of Cell Biology

Article Title: Ca 2+ tunneling architecture and function are important for secretion

doi: 10.1083/jcb.202402107

Figure Lengend Snippet: CaTAr1 effects on tunneling and localization of ANO1–GFP and mCh-STIM1. (A) Dose-dependent inhibition of tunneling by CaTAr1. The amplitude of the tunneling signal is plotted as a function of the GFP signal normalized from minimum to maximum signal in each dish. The data is sorted in bins of 10%. (B) The relative localization of ANO1–GFP and mCh-STIM1 in NCL cells after store depletion is similar to the endogenous ANO1 channel and suggests the separation of ANO1 from the STIM1 cluster. (C) TIRF imaging of ANO1–GFP and mCh-STIM1 during store depletion with CPA confirmed the separation of the two proteins at the PM. Ctr and CPA images are from the same region of interest.

Article Snippet: The following primary antibodies were used at a 1:500 dilution: SERCA2 (NB300-581; Novus Biologicals), STIM1 (5668S; Cell Signaling and MAI-19451; Thermo Fisher Scientific), KRAP (14157-1-AP; Thermo Fisher Scientific), NFAT1 (5861S; Cell Signaling), and ANO1 (14476; Cell Signaling).

Techniques: Inhibition, Imaging

Journal: The Journal of Cell Biology

Article Title: Ca 2+ tunneling architecture and function are important for secretion

doi: 10.1083/jcb.202402107

Figure Lengend Snippet: Tunneling in NCL-SG3 cells. (A) Localization of ANO1 by immunocytochemistry and mCh-STIM1 before (Ctr) and after store depletion (Tg). An intensity plot performed along a line (arrow) crossing a STIM1 cluster illustrates the separation of STIM1 and ANO1 at the PM focal plane. (B–D) Comparative localization at the PM plane of mCh-STIM1 with ANO1–GFP and the endogenous ANO1 protein (ANO1–Ig) following store depletion. As indicated by the PCC and the peak-to-peak distances the two proteins do not colocalize laterally but are localized at the same optical plane (z distance) ( n = 8–18; one-way ANOVA, P < 0.0001 for B and C, P = 0.7 for D). (E) Intracellular Ca 2+ elevation induced by trypsin application in NCL-SG3 cells loaded with Fluo4-AM. The application of the SOCE inhibitor BTP-2 (10 μM) reduces the amplitude and the duration of Ca 2+ release ( n = 379/402; unpaired t test, P < 0.0001). (F) Confocal images of NCL-SG3 cells expressing the Cl − sensor mbYFPQS and mCh-STIM1 after store depletion. The orthogonal section through the cell indicates the PM localization of the chloride sensor. (G) Kymographs were measured during a tunneling event on cells expressing mbYFPQS and mCh-STIM1. The line passes through a SOCE cluster and an adjacent cell appendage labeled by mbYFPQS. The changes in Cl − concentration (upper) are distal from the STIM1 cluster, which indicates the Ca 2+ entry point. (H) Time course of the amplitude of the Cl − signal induced by Ca 2+ tunneling from NCL-SG3 cells in the same dish that either do not show any CaTAr2 expression (Ctr) and cells expressing CaTAr2. (I) Violin plots summarizing the amplitude of the Cl − signal 3 min after trypsin and Ca 2+ addition to stimulate tunneling ( n = 32–33; unpaired t test, P = 0.0004).

Article Snippet: The following primary antibodies were used at a 1:500 dilution: SERCA2 (NB300-581; Novus Biologicals), STIM1 (5668S; Cell Signaling and MAI-19451; Thermo Fisher Scientific), KRAP (14157-1-AP; Thermo Fisher Scientific), NFAT1 (5861S; Cell Signaling), and ANO1 (14476; Cell Signaling).

Techniques: Immunocytochemistry, Expressing, Labeling, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Ca 2+ tunneling architecture and function are important for secretion

doi: 10.1083/jcb.202402107

Figure Lengend Snippet: CaTAr2 and ANO1–GFP in NCL cells. (A) Airy scan images of the Co-expression of CaTAr2 and ANO1–GFP in NCL cells indicate the partial overlap between the two signals. (B) The colocalization is not influenced by store depletion. (C) Relative intensities along a virtual line scan (white arrows in A) reveal the colocalization of ANO1 and CaTAr2 but also enrichment in ANO1 at the edge of the CaTAr2-rich ER patch.

Article Snippet: The following primary antibodies were used at a 1:500 dilution: SERCA2 (NB300-581; Novus Biologicals), STIM1 (5668S; Cell Signaling and MAI-19451; Thermo Fisher Scientific), KRAP (14157-1-AP; Thermo Fisher Scientific), NFAT1 (5861S; Cell Signaling), and ANO1 (14476; Cell Signaling).

Techniques: Expressing