Research ArticleCell Biology

STIM2 enhances receptor-stimulated Ca2+ signaling by promoting recruitment of STIM1 to the endoplasmic reticulum–plasma membrane junctions

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Science Signaling  13 Jan 2015:
Vol. 8, Issue 359, pp. ra3
DOI: 10.1126/scisignal.2005748
  • Fig. 1 Targeted knockout of STIM2 in mouse salivary glands.

    (A) Saliva secretion after stimulation with pilocarpine (0.25 mg/kg body weight) in wild-type (WT), STIM2fl/fl, and STIM2fl/fl + Cre mice. (B) Bar graph showing total saliva collected 10 min after treatment with the indicated amounts of pilocarpine [*P < 0.05; n = 9 or more mice per group; analysis of variance (ANOVA) was used to compare the saliva volume for STIM2fl/fl and STIM2fl/fl + Cre mice with the WT mice]. (C and D) Representative traces showing changes in fura2 fluorescence after CCh stimulation of dispersed salivary gland acinar cells. Data are representative of three or more independent cell preparations, with 15 to 25 acini imaged per experiment. Each cell preparation used salivary glands from one mouse. (D) Peak increase in fura2 fluorescence (FF0), calculated using data from three different cell preparations (at each [CCh]) (*P < 0.05; n = 72 to 77 cells from three different preparations; compared to the respective value for STIM2fl/fl using Student’s t test). (E) Nuclear translocation of GFP-NFAT in control and siSTIM2‐treated HEK293 cells after stimulation with CCh or Tg. The images shown are representative of results obtained with 30 to 40 cells per experiment from three independent experiments. (F) Proportion (%) of cells showing nuclear translocation of GFP-NFAT at 25 min after the addition of the indicated concentrations of CCh or 10 min after the addition of 1 μM Tg (n = 100 to 110 cells per group from three separate experiments; *P < 0.05; χ2 test was used to compare nuclear translocation of GFP-NFAT between control and siSTIM2 cells at each stimulation).

  • Fig. 2 Modulation of CCh-induced Ca2+ responses by STIM2.

    (A to D) Fura2 fluorescence was monitored in control and siSTIM2-treated HEK293 cells stimulated with the indicated concentrations of CCh. Traces are representative of the type of response exhibited by most cells at each [CCh] in control (left trace) and siSTIM2 (right trace) cells (n = at least 70 cells for each [CCh] in each of the three or more independent experiments). The bar graphs show the proportion (%) of the cell population displaying various patterns of [Ca2+]i changes at each [CCh]. Overall pattern of responses in siSTIM2 cells were significantly different from that in control cells at each [CCh] (n = 400 to 600 cells from four to six experiments; P < 0.05, χ2 test). (E) Dose-response curves showing the relative increases in [Ca2+]i over baseline for each [CCh] ranging from 50 nM to 1000 μM in control and siSTIM2 cells. (F) Proportion of the cells showing the elevated plateau response at each [CCh] in control and siSTIM2 cells. (G) Proportion of the cells showing baseline Ca2+ oscillations at each [CCh] in control and siSTIM2 cells. For (F) and (G), the n values are the same as in (A) to (D).

  • Fig. 3 Clustering of STIM1 and STIM2 in response to relatively low and high levels of stimulation.

    Fluorescent tagged proteins were expressed, individually or together, in HEK293 cells. Cells were stimulated with the indicated concentrations of CCh, and TIRF microscopy was used to image the fluorescence and visualize puncta in ER-PM junctions. The bar in this and subsequent figures indicates a scale of 10 μm. (A) Pattern of YFP-STIM1 (top) and YFP-STIM2 (bottom) fluorescence in cells individually expressing each construct sequentially stimulated with 1 and 100 μM CCh. (B) Bar graph showing representative changes in puncta fluorescence intensity for YFP-STIM1 and YFP-STIM2 at each [CCh], relative to the fluorescence at 100 μM CCh for each protein. A region of interest (ROI) was drawn around each punctum to quantify the changes in the fluorescence of individual punctum after each stimulation (n = 15 to 20 ROIs from each cell with four to six cells analyzed per experiment and with three or more separate experiments performed). (C) Pattern of CFP-STIM2 (top) and YFP-STIM1 (middle) fluorescence and the merged fluorescence (overlay in bottom) in a cell coexpressing both proteins sequentially stimulated with 1 and 100 μM CCh. The graphs below the images show line scan measurements with the line scan region indicated in the rightmost image. (D) Bar graph showing relative increase in puncta fluorescence intensity for YFP-STIM1 and CFP-STIM2 after each stimulation in cells coexpressing both proteins [data are from three cells (average of 15 to 20 ROIs per cell) and are representative of 10 to 15 cells from three or more separate experiments performed]. (E) The time-dependent increase in coexpressed YFP-STIM1 and CFP-STIM2 fluorescence after stimulation with 1 and 100 μM CCh [ROIs and number of cells and experiments as described in (D)]. (F) Coimmunoprecipitation of endogenous STIM2 with endogenous STIM1 after stimulation with 1 and 100 μM CCh. The bar graph below the blot shows the abundance of STIM2 relative to STIM1 in the immunoprecipitates at each [CCh] (n = 3 or more separate experiments; *P < 0.05, ANOVA with Sidak multiple comparisons test). IP, immunoprecipitation.

  • Fig. 4 Clustering of fluorescent protein-tagged STIM1, STIM2, and Orai1 in response to relatively low and high levels of stimulation.

    Fluorescent tagged proteins were expressed, individually or together, in HEK293 cells that were stimulated with the indicated concentrations of CCh. TIRF microscopy was used to image the fluorescence and visualize puncta in ER-PM junctions. (A) Cells coexpressing YFP-STIM1 and Orai1-CFP were sequentially stimulated with 1 and 100 μM CCh. The merged fluorescence (overlay of images) of both proteins is shown in the upper panels, with the line scans immediately below each image for each [CCh] (the line scan region is indicated in the rightmost panel). Individual images showing the clustering of YFP-STIM1 and Orai1-CFP after stimulation with 100 μM CCh are below the line scans. The bar graph shows puncta fluorescence intensity (arbitrary units) for YFP-STIM1 and Orai1-CFP in cells stimulated with CCh, relative to that at 100 μM CCh (set at 100%). Data are from four to six cells (15 to 20 ROIs per cell) and are representative of results obtained in three or more independent experiments. (B) Cells coexpressing Cherry-STIM1, YFP-STIM2, and Orai1-CFP were sequentially stimulated with 1 and 100 μM CCh. The merged fluorescence (overlay) of all proteins is shown in the upper panels, with the line scans immediately below each image for each [CCh] (the region of the scan is indicated in the rightmost panel). Individual images show the clustering of Cherry-STIM1, YFP-STIM2, and Orai1-CFP after stimulation with 100 μM CCh are shown below the line scans. The bar graph shows puncta fluorescence intensity (arbitrary units) for Cherry-STIM1, YFP-STIM2, and Orai1-CFP in cells stimulated with each [CCh] (fluorescence intensity at 100 μM CCh was set as 100% for each protein; n = 15 to 20 ROIs from each cell with two to four cells analyzed per experiment and with three or more separate experiments performed).

  • Fig. 5 Effect of the STIM2ΔK5 mutant on CCh-induced STIM1 and Orai1 clustering and SOCE.

    Fluorescent tagged proteins were expressed, individually or together, in HEK293 cells that were stimulated with the indicated concentrations of CCh. TIRF microscopy was used to image the fluorescence and visualize puncta in ER-PM junctions. (A) Fluorescence of cells expressing YFP-STIM2ΔK5 alone in the presence or absence of Tg (1 μM for 5 min) to deplete ER Ca2+ stores. (B) Fluorescence of cells coexpressing YFP-STIM2ΔK5 and Orai1-CFP in the presence or absence of Tg (1 μM for 5 min). (C) Fluorescence of cells coexpressing YFP-STIM2ΔK5 and CFP-STIM1 after sequential stimulation with the indicated concentrations of CCh. The line scans are shown immediately below each image for each [CCh] (the line scan region is indicated in the rightmost panel). (D) Fluorescence of HEK293 cells coexpressing YFP-STIM2ΔK5, Cherry-STIM1, and Orai1-CFP after sequential stimulation with the indicated concentrations of CCh. The line scans are shown immediately below each image for each [CCh] (the line scan region is indicated in the rightmost panel). Images are representative of results obtained in 15 to 20 cells from at least four independent experiments. (E and F) Fluorescence traces representing Ca2+ responses exhibited by most control cells (left) and HEK293 cells expressing STIM2ΔK5 (right) after stimulation with 1 (E) and 100 (F) μM CCh. Bar graphs show the percentage (%) of cells showing a given pattern of Ca2+ responses at each concentration of CCh. Overall pattern of responses in the two groups was significantly different (n = 170 to 240 cells per group from three to five separate experiments; *P < 0.05, χ2 test).

  • Fig. 6 Effect of the STIM1ΔK mutant on CCh-induced STIM2 clustering.

    Fluorescent tagged proteins were expressed, individually or together, in HEK293 cells, and TIRF microscopy was used to image the fluorescence and visualize puncta in ER-PM junctions. (A) Fluorescence of cells expressing YFP-STIM1ΔK and CFP-STIM2 in the presence or absence of Tg (1 μM for 5 min) to deplete ER Ca2+ stores. (B) Fluorescence of cells coexpressing YFP-STIM1ΔK and CFP-STIM2 after sequential stimulation with the indicated concentrations of CCh. The merged fluorescence (overlay) of all proteins is shown in the upper panels, with the line scans immediately below each image for each [CCh] (the line scan region is indicated in the rightmost panel). (C) The bar graph shows the changes in puncta fluorescence intensity for YFP-STIM1ΔK and CFP-STIM2 after each stimulation in cells coexpressing both proteins, relative to the intensity at 100 μM CCh. ROI was drawn around each punctum to quantify the changes in the fluorescence of individual proteins within the punctum after each stimulation (n = 15 to 20 ROIs from each cell with two to four cells analyzed per experiment and with three or more separate experiments performed).

  • Fig. 7 Model depicting the role of STIM2 in the clustering of STIM1 and activation of SOCE after intracellular Ca2+ store depletion.

    In unstimulated cells, ER [Ca2+] is high, and the STIM proteins reside and are diffusively distributed in the ER membrane. Stimulation with relatively low concentration of agonist results in minimal depletion of the ER Ca2+ stores and mobilization of STIM2. These conditions promote the formation of STIM2-STIM1 heteromers, which translocate to the ER-PM junctions where STIM1 interacts with Orai1 and activates SOCE. Substantial depletion of the ER Ca2+ stores after stimulation with high concentrations of agonist drives the formation of STIM1 homomers and their recruitment to the ER-PM junctions where STIM1 activates Orai1. Because both STIM2-STIM1 heteromers and STIM1 homomers would form in response to high concentrations of agonist, we predict that Ca2+ influx would be greater than at the lower levels of stimulation.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/359/ra3/DC1

    Fig. S1. Effects and specificity of targeted knockout of STIM2 in mouse salivary glands.

    Fig. S2. Specificity of siRNA-mediated STIM2 knockdown and its effects on CCh-stimulated Ca2+ signaling.

    Fig. S3. Effect of STIM1 or STIM2 knockdown on CCh-stimulated [Ca2+]i responses.

    Fig. S4. Store depletion–induced clustering of STIM proteins in cells coexpressing YFP-STIM1 and CFP-STIM2 or expressing only YFP-STIM1EF→STIM2.

    Fig. S5. Effect of deleting the polybasic tail domain of STIM1 on store depletion–induced clustering.

  • Supplementary Materials for:

    STIM2 enhances receptor-stimulated Ca2+ signaling by promoting recruitment of STIM1 to the endoplasmic reticulum–plasma membrane junctions

    Hwei Ling Ong, Lorena Brito de Souza, Changyu Zheng, Kwong Tai Cheng, Xibao Liu, Corinne M. Goldsmith, Stefan Feske, Indu S. Ambudkar*

    *Corresponding author. E-mail: indu.ambudkar{at}nih.gov

    This PDF file includes:

    • Fig. S1. Effects and specificity of targeted knockout of STIM2 in mouse salivary glands.
    • Fig. S2. Specificity of siRNA-mediated STIM2 knockdown and its effects on CCh-stimulated Ca2+ signaling.
    • Fig. S3. Effect of STIM1 or STIM2 knockdown on CCh-stimulated [Ca2+]i responses.
    • Fig. S4. Store depletion–induced clustering of STIM proteins in cells coexpressing YFP-STIM1 and CFP-STIM2 or expressing only YFP-STIM1EF→STIM2.
    • Fig. S5. Effect of deleting the polybasic tail domain of STIM1 on store depletion–induced clustering.

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    Citation: H. L. Ong, L. B. de Souza, C. Zheng, K. T. Cheng, X. Liu, C. M. Goldsmith, S. Feske, I. S. Ambudkar, STIM2 enhances receptor-stimulated Ca2+ signaling by promoting recruitment of STIM1 to the endoplasmic reticulum–plasma membrane junctions. Sci. Signal. 8, ra3 (2015).

    © 2014 American Association for the Advancement of Science

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