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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.

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