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First published online 9 October 2007
doi: 10.1242/jcs.015735

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Role of the microtubule cytoskeleton in the function of the store-operated Ca²⁺ channel activator STIM1

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

View larger version (43K): [in this window] [in a new window]   Fig. 1. EYFP-STIM1 is organized in a microtubule-like array in HEK 293 cells. (A) Confocal image of HEK 293 cells overexpressing EYFP-STIM1. The arrowhead indicates what appears to be an `organizing center' of EYFP-STIM1 localization. (B) Confocal image of an HEK 293 cell that was fixed and immunostained for -tubulin. The arrowhead indicates the microtubule-organizing center near the nucleus, presumably the centrosome. (C) Confocal image of an HEK 293 cell overexpressing an ER-targeted CFP construct (ER-CFP). Bars, 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. EYFP-STIM1 colocalizes with -tubulin. (A) Upper panel: confocal images of an HEK 293 cell co-overexpressing EYFP-STIM1 (left panel; green) and mCherry--tubulin (middle panel; red) in the presence of 1.8 mM extracellular Ca2+. The arrowheads indicate a track of EYFP-STIM1 fluorescence that exactly matches a corresponding track of mCherry--tubulin fluorescence. Lower panel: EYFP-STIM1 and mCherry--tubulin localization 15 minutes following Ca2+ store depletion with thapsigargin (Tg; 2 µM) in a nominally Ca2+-free extracellular solution. (B) Upper panel: confocal images of an HEK 293 cell overexpressing EYFP-STIM1 that was fixed and immunostained with antibodies against EYFP (to enhance detection of EYFP-STIM1) (left panel; green) and -tubulin (middle panel; red). Lower panel: an HEK 293 cell that was store depleted with thapsigargin for 15 minutes before fixation and immunostaining. Arrowheads indicate a track of -tubulin along which several EYFP-STIM1 puncta appear to associate. Bars, 10 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Microtubule depolymerization causes dispersion of EYFP-STIM1 throughout the ER. (A) Confocal images of HEK 293 cells co-overexpressing EYFP-STIM1 (left panel; green) and an ER-targeted CFP (CFP-ER) (middle panel; red) in the presence of 1.8 mM extracellular Ca2+ (upper panel) and following a 20 minute treatment with nocodazole (NZL; 10 µM; lower panel). (B) Images of HEK 293 cells expressing EYFP-STIM1 that were treated with nocodazole for 20 minutes, followed by fixation and immunostaining with antibodies against GFP (left panel; green) and -tubulin (middle panel; red). Bars, 10 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Microtubule depolymerization inhibits SOCE. (A) Wild-type HEK 293 cells were treated with 100 µM colchicine for 20 minutes (red trace) or left untreated (control; black trace), and thapsigargin (Tg; 2 µM) was then added in the presence of 1.8 mM extracellular Ca2+. The intracellular Ca2+ concentration was monitored throughout the experiments, and SOCE became activated soon after Tg addition, as evidenced by the second peak in Ca2+ concentration, and remained active based on the sustained elevation of intracellular Ca2+ above baseline. Each trace represents the average response of all cells on a single coverslip (20-30 cells). (B) The average difference between the 340/380 value 15 minutes following Tg addition (sustained SOCE) and the 340/380 value just before Tg addition (baseline) was calculated for untreated control (n=150, three coverslips) and colchicine-treated (n=145, three coverslips) cells for experiments performed as described in (A). (C) SOCE was analyzed as described in (A) for cells treated for 20 minutes with 10 µM nocodazole (NZL; red trace) and for control cells treated with 0.1% DMSO (black trace). Because nocodazole treatment alone caused some store depletion and activation of SOCE (see Fig. 7), nocodazole and DMSO treatments were performed in a nominally Ca2+-free extracellular solution. (D) The average difference between the 340/380 value 15 minutes following Tg addition (sustained SOCE) and the 340/380 value just before Tg addition (baseline) was calculated for DMSO-treated (n=122, three coverslips) and nocodazole-treated (n=125, three coverslips) cells for experiments performed as described in (C). The data are reported as the mean±s.e.m.; the P values are based on t-tests.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Microtubule depolymerization inhibits ICRAC. (A) Whole-cell currents were measured in control (black trace) and nocodazole-treated (10 µM, 20 min; red trace) wild-type HEK 293 cells in the presence of 10 mM extracellular Ca2+; stores were depleted with a pipette solution containing 25 µM Ins(1,4,5)P3. Shown are the averaged responses (±s.e.m.) of seven control cells and eight cells treated with nocodazole (NZL). The means were not significantly different at any point along the traces. (B) Currents were measured as in D, except that the extracellular solution was switched from 10 mM Ca2+ to divalent-free at the time indicated. Averaged (±s.e.m.) traces from eight control (black trace) and six nocodazole-treated (red trace) cells are shown; the maximal Na+ current was significantly smaller in nocodazole-treated compared with control cells (t-test, P<0.01). (C) Averages of the peak Na+ currents from eight control and six nocodazole-treated cells. The data are reported as the mean±s.e.m.; the P value is based on a t-test.

View larger version (12K): [in this window] [in a new window]   Fig. 6. Overexpression of STIM1 rescues inhibition by nocodazole. To evaluate SOCE, changes in the intracellular Ca2+ concentration were monitored in HEK 293 cells in which Ca2+ stores were depleted with thapsigargin (Tg; 2 µM) in the presence of a nominally Ca2+-free extracellular solution, followed by addition of 1.8 mM Ca2+. In wild-type HEK 293 cells overexpressing unconjugated EYFP (A) and cells overexpressing EYFP-STIM1 (B), SOCE was evaluated following a 20 minute incubation in 10 µM nocodazole (NZL; red traces) or 0.1% DMSO (black traces). Each trace represents the average response of all cells on a single coverslip (20-30 cells). (C) The average difference between the peak 340/380 value following Ca2+ addition and the 340/380 value just before Ca2+ addition was calculated for EYFP-expressing cells treated with DMSO (n=89, three coverslips) or 10 µM nocodazole (n=110, four coverslips) and in EYFP-STIM1-expressing cells treated with DMSO (n=77, three coverslips) or with 10 µM nocodazole (n=84, 3 coverslips) for experiments performed as described in (A) and (B). The data are reported as the mean±s.e.m.; the P values are based on t-tests.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Nocodazole treatment activates SOCE and ICRAC in cells overexpressing EYFP-STIM1. (A) Cells overexpressing EYFP-STIM1 were treated with 10 µM nocodazole (NZL; red trace) or 0.1% DMSO (black trace) for 20 minutes in a nominally Ca2+-free extracellular solution, followed by addition of 1.8 mM extracellular Ca2+ in the absence of store depletion. Also shown is nocodazole-treated EYFP-STIM1-overexpressing cells in which Orai1 expression was reduced by RNAi (blue trace). Each trace represents the average response of all cells on a single coverslip (20-30 cells). (B) Cells expressing EYFP were treated with 10 µM nocodazole (red trace) or 0.1% DMSO (black trace), and Ca2+ entry was evaluated in the absence of store depletion, as described in (A). (C) The average difference between the peak 340/380 value following Ca2+ addition and the 340/380 value just before Ca2+ addition was calculated for cells expressing EYFP-STIM1 treated with 0.1% DMSO (n=85, three coverslips), 10 µM nocodazole (n=167, six coverslips) and in EYFP-STIM1-expressing, Orai1-knockdown cells treated with nocodazole (n=62, three coverslips) for experiments performed as described in (A). The data are represented as the mean±s.e.m.; P values are based on one-way ANOVA. (D-F) The responses of all the cells measured on a single coverslip are shown for the averaged traces shown in (A); (D) EYFP-STIM1 cells treated with DMSO; (E) EYFP-STIM1 cells treated with 10 µM nocodazole; (F) EYFP-STIM1-overexpressing, Orai1-knockdown cells treated with nocodazole. (G) Cells overexpressing EYFP-STIM1 were patched with an intracellular solution clamped at 0 mM Ca2+. Break-in was performed in 10 mM extracellular Ca2+, and the extracellular solution was switched to divalent-free at intervals of 1 minute, with intervals of 30 seconds of 10 mM Ca2+ in between. (H) The same protocol as in C was performed using a 0 mM Ca2+ intracellular solution on a nocodazole-treated cell overexpressing EYFP-STIM1.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Microtubule depolymerization does not inhibit the punctate localization of EYFP-STIM1. (A) Confocal images of HEK 293 cells in the presence of 1.8 mM extracellular Ca2+ (left panel), following a 20 minute incubation in 10 µM nocodazole (NZL; middle panel), and 15 minutes following store depletion with thapsigargin (Tg; 2 µM) in the continued presence of nocodazole (right panel). (B) TIRFM images of cells expressing EYFP-STIM1 in 1.8 mM extracellular Ca2+ (left panel; i), following a 20 minute treatment in 10 µM nocodazole (middle panel; ii) and 15 minutes following store depletion with thapsigargin in the continued presence of nocodazole (right panel; iii). The same experiment is shown in C, except that cells were treated with 0.1% DMSO instead of nocodazole. (D) Fluorescence intensity profiles over time for the cells shown in B and C; each trace represents the average fluorescence intensity measured in a region of interest encompassing a single cell. Red traces: nocodazole-treated cells; black traces: DMSO-treated cells. The labels i, ii and iii in the graph represent the times at which the still images in B and C were taken. (E) Average maximal TIRFM fluorescence intensity measured in EYFP-STIM1 cells following the 20 minute treatment in nocodazole (n=6 cells over three coverslips) or DMSO (n=5 cells over three coverslips) and in the same cells following store depletion with thapsigargin (Tg). The data are reported as the mean±s.e.m.; the P values are based on a t-test. Bars, 10 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 9. Colchicine potentiates but does not activate SOCE in cells overexpressing STIM1. (A) HEK 293 cells expressing EYFP-STIM1 were treated with 1.0 µM CPA alone (Control; black trace) or with CPA and 100 µM colchicine (red trace) for 20 minutes in a nominally Ca2+-free extracellular solution, followed by addition of 1.8 mM extracellular Ca2+ to reveal SOCE. Also shown are data from cells treated with colchicine alone (blue trace). Each trace represents the average response of all cells on a single coverslip (20-30 cells). (B) The average difference between the peak 340/380 value following Ca2+ addition and the 340/380 value just before Ca2+ addition was calculated for CPA-treated control (n=64, three coverslips) and colchicine-treated (n=97, three coverslips) cells for experiments performed as described in (A). The data are reported as the mean±s.e.m.; the P value is based on t-test.