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First published online 19 February 2008
doi: 10.1242/jcs.023903

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Ca²⁺-store-dependent and -independent reversal of Stim1 localization and function

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, PO Box 12233, Research Triangle Park, NC 27709, USA

View larger version (26K): [in this window] [in a new window]   Fig. 1. Ca2+ store refilling reverses the rearrangement of EYFP-Stim1. (A,B) TIRFM fluorescence intensity and relative intracellular Ca2+ concentrations were measured simultaneously in the same HEK293 cells overexpressing EYFP-Stim1 and the m5 muscarinic receptor. As indicated, cells were treated with 300 µM carbachol in nominally Ca2+-free extracellular medium to deplete intracellular Ca2+ stores. Carbachol signaling was then terminated by the addition of 50 µM atropine, after which extracellular Ca2+ was restored to 1.8 mM. Fifteen minutes later, the cells were treated with 2 µM thapsigargin to demonstrate that store refilling had occurred. This protocol was performed with cells treated in the absence (A) or presence (B) of 5 µM Gd3+ throughout. Note that in panel B SOCE did not occur upon restoration of extracellular Ca2+, and EYFP-Stim1 was not reversed. The upper traces show the TIRFM intensity profiles, and the bottom traces show the 360/380 fluorescence intensities representative of relative Ca2+ responses; each trace represents the average response of four cells measured in a single experiment. The bottom panels show TIRFM images taken at the times indicated (i-iv) in the intensity profiles. Bars, 10 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Rearrangements of both Stim1 and Orai1 are reversed by Ca2+ store refilling. (A) Confocal images of HEK293 cells co-overexpressing EYFP-Stim1 and the m5 muscarinic receptor in the presence of 1.8 mM extracellular Ca2+ before store depletion (left panel), 10 minutes following treatment with 300 µM carbachol in nominally Ca2+-free extracellular solution (center panel), and 10 minutes following restoration of 1.8 mM extracellular Ca2+ in the presence of 50 µM atropine (right panel). (B) The same protocol described in panel A was repeated with cells co-overexpressing EYFP-Stim1, CFP-Orai1 and the m5 muscarinic receptor. EYFP-Stim1 fluorescence is shown in the upper row, CFP-Orai1 is shown in the center row, and the bottom row shows merged images of EYFP-Stim1 (green) and CFP-Orai1 (red). Bars, 10 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 3. ML-9 dose-dependently inhibits SOCE and Icrac. (A) Relative intracellular Ca2+ concentrations were monitored in wild-type HEK293 cells treated with 100 µM ML-9 (red trace) or left untreated (black trace). Ca2+ stores were depleted with 2 µM thapsigargin in nominally Ca2+-free extracellular medium, and extracellular Ca2+ was restored to 1.8 mM 15 minutes later to reveal SOCE. ML-9 was removed at the end of the experiment to demonstrate the reversibility of ML-9 inhibition. Each trace represents the averaged response of all cells measured on a single coverslip. (B) The average peak SOCE responses above baseline from experiments performed as described in panel A were averaged for untreated control cells (n=150; five coverslips) or cells treated with 1 µM (n=78; three coverslips), 10 µM (n=76; three coverslips), 50 µM (n=84; three coverslips) or 100 µM (n=64; three coverslips) ML-9. Data are reported as the percentage of untreated control±s.e.m.; *, significant difference compared with control (P<0.001) based on one-way ANOVA. (C) Experiments were performed as described in panel A, but ML-9 was added 5 minutes following restoration of extracellular Ca2+ (red trace). (D) For experiments performed as described in panel C, the baseline-subtracted 340/380 ratio 5 minutes following ML-9 addition was divided by that just before addition. Data are reported as the percentage of untreated control±s.e.m. for untreated controls (n=151; five coverslips), and for 1 µM (n=90; four coverslips), 10 µM (n=80, three coverslips), 50 µM (n=61, three coverslips) and 100 µM (n=74, three coverslips) ML-9; *, significant difference compared with control (P<0.001) based on one-way ANOVA. (E) Whole-cell patch clamp analysis was performed with a pipette solution containing 20 mM BAPTA and 25 µM Ins(1,4,5)P3 to deplete intracellular Ca2+ stores. The cell was initially perfused with an extracellular solution containing 10 mM Ca2+. At the time indicated, perfusion was switched to a divalent-free (DVF) solution, which resulted in the development of a Na+ current. Perfusion was then returned to 10 mM Ca2+, and 100 µM ML-9 was added at the time indicated. Note the decrease in the Ca2+ current upon ML-9 addition. In the continued presence of ML-9, perfusion was again switched to DVF solution. At the end of the experiment, 10 mM extracellular Ca2+ was restored and ML-9 was removed to demonstrate reversal of inhibition of the Ca2+ current. (F) For experiments performed as described in panel E, the peak Na+ currents at the initial switch to DVF solution in the absence of ML-9 (control) and that at the second switch to DVF in the presence of 100 µM ML-9 were averaged and are expressed as mean±s.e.m. (n=6); *, significant difference compared with control (P<0.005) based on Student's t test.

View larger version (23K): [in this window] [in a new window]   Fig. 4. ML-9 inhibits EYFP-Stim1 rearrangement. (A) Time-lapse TIRFM was performed on EYFP-Stim1-overexpressing HEK293 cells treated with 100 µM ML-9 or left untreated (control). The left panel shows TIRFM fluorescence intensity profiles for two control (black traces) and two ML-9-treated cells (red traces). Thapsigargin (Tg; 2 µM) was added in nominally Ca2+-free extracellular solution at the time indicated to deplete Ca2+ stores, and ML-9 was removed at the end of the experiment to demonstrate reversal of the ML-9 inhibition. The right-hand panel shows representative TIRFM images taken at the times indicated (i-iii) in the intensity profile. (B) TIRFM imaging was performed on three cells as described in panel A, but ML-9 (100 µM) was added after store depletion with thapsigargin. (C) The average baseline-subtracted TIRFM fluorescence intensity 5 minutes following ML-9 addition was divided by that just before addition for experiments performed as described in panel B for untreated controls (n=6; two coverslips), or cells treated with 1 µM (n=11; three coverslips), 10 µM (n=11, three coverslips), 50 µM (n=14, three coverslips) and 100 µM (n=10, three coverslips) ML-9. Data are expressed as the percentage of untreated control±s.e.m.; *, significant difference compared with control (P<0.001) based on one-way ANOVA. Bars, 10 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Reversal of Stim1 localization by ML-9 is complete. Shown are confocal images of cells in the presence of 1.8 mM extracellular Ca2+ (left panel), 15 minutes following store depletion with thapsigargin (Tg; 2 µM) in nominally Ca2+-free extracellular solution (center panel) and 5 minutes following addition of 100 µM ML-9 in the continued presence of Tg and Ca2+-free extracellular solution. Bars, 10 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Inhibition of SOCE and Icrac by ML-9 is due to inhibition of Stim1 rearrangement. (A) SOCE experiments were performed in which ML-9 was added following restoration of extracellular Ca2+ as described in Fig. 3C. As described in Fig. 3D, SOCE following ML-9 addition as a percentage of the untreated control was calculated and is plotted as a function of ML-9 concentration for HEK293 cells overexpressing unconjugated EYFP (black squares) and EYFP-Stim1 (blue triangles). (B) TIRFM fluorescence intensity (black trace) and relative intracellular Ca2+ concentration (360/380 ratio; blue trace) were measured simultaneously in the same cell. Ca2+ stores were depleted with thapsigargin (Tg; 2 µM) in the presence of 1.8 mM extracellular Ca2+, and 100 µM ML-9 was added 15 minutes later. (C) For the data shown in panel B, the TIRFM (black trace) and 360/380 (blue trace) values beginning just before ML-9 addition were normalized to the same minimum and maximum values.

View larger version (39K): [in this window] [in a new window]   Fig. 7. ML-9 reverses constitutive EYFP-D76N/D78N-Stim1 localization and SOCE activity. (A) Intracellular Ca2+ concentration was monitored in HEK293 cells overexpressing EYFP-D76N/D78N-Stim1 beginning in the presence of 1.8 mM extracellular Ca2+, followed at the time indicated by switch to a nominally Ca2+-free extracellular solution. Extracellular Ca2+ was then restored, and 100 µM ML-9 was added. In the continuous presence of ML-9, extracellular Ca2+ was again removed and restored. The trace represents the average response of all the cells measured on a single coverslip; representative of three independent experiments. (B) Time-lapse TIRFM was performed on a cell overexpressing EYFP-D76N/D78N-Stim1; 100 µM ML-9 was added at the time indicated in the fluorescence intensity profile (left panel). Right-hand panel: representative TIRFM images taken at the times indicated (i and ii) in the intensity profile. Representative of three independent experiments. (C) Confocal images of EYFP-D76N/D78N-Stim1-expressing cells in the absence (left panel) and presence (right panel) of 100 µM ML-9 in the presence of 1.8 mM extracellular Ca2+ throughout. Bars, 10 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Knockdown of MLCK protein expression by siRNA does not inhibit rearrangement of EYFP-Stim1. (A) HEK293 cells left untransfected (WT; lane 1), or transfected with control siRNA (lane 2) or one of three different MLCK siRNA constructs (lanes 3-5) were subjected to western blotting with an antibody against MLCK. (B) Three independent experiments were performed as described in panel A, and the average band intensities for each condition are expressed as a percentage of wild-type±s.e.m.; *, significant difference compared with wild-type cells (P<0.01) based on one-way ANOVA. (C) Cells were transfected with control siRNA or (D) with MLCK #3, and TIRFM experiments were performed in which stores were depleted with 2 µM thapsigargin in nominally Ca2+-free extracellular solution. Each trace represents a single cell, and the responses of all the cells measured from a single coverslip are shown.

View larger version (39K): [in this window] [in a new window]   Fig. 9. Single EYFP-Stim1 punctae form in similar locations upon multiple rearrangement stimuli. (A) Two EYFP-Stim1-expressing HEK293 cells were imaged by time-lapse TIRFM, during which Ca2+ stores were depleted with 2 µM thapsigargin in the presence of nominal extracellular Ca2+, 100 µM ML-9 was added to reverse punctae formation and ML-9 was removed to re-stimulate punctae formation. Shown is the fluorescence intensity profile, with each trace representing a single cell. (B) Representative TIRFM images taken at the times indicated (i-iv) in the intensity profile in panel A. The complete image series of this experiment is shown in supplementary material Movie 4. (C) The TIRFM image taken at 380 seconds (store-depleted with thapsigargin before ML-9 treatment) was pseudocolored red. This image was then merged with the image taken at 710 seconds (after ML-9 washout), which was pseudocolored green. Image `b' on the right is a close-up of the region denoted by the white rectangle in the full-size image on the left. Image `a' was generated from the merge of the images 60 seconds before the images used to generate the full-size image on the left, and image `c' was generated from the merge of the images 60 seconds after the images used to generate the full-size image on the left. The arrows in image `b' point to pairs of red and green punctae that remain consistent throughout the series of merged images. Scale bars: 10 µm in B and C; 1 µm in Ca-c.

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