JCS023903 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. CFP-Orai1 does not rearrange upon store depletion when Stim1 is not overexpressed. Shown are confocal images of cells overexpressing CFP-Orai1 alone in the presence of 1.8 mM extracellular Ca²⁺ (left panel), following store depletion with thapsigargin (2 µM) in a nominally Ca²⁺-free extracellular solution (center panel) and following store refilling with atropine and 1.8 mM extracellular Ca²⁺ (right panel). Bar, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. ML-9 reverses EYFP-Stim1 punctae when Ca²⁺ stores are depleted with ionomycin. TIRFM was performed on EYFP-Stim1-expressing HEK293 cells that were treated at the times indicated with 400 nM ionomycin in nominal extracellular Ca²⁺, followed by restoration of 1.8 mM extracellular Ca²⁺ and finally addition of 100 µM ML-9. Shown is the fluorescence intensity profile averaged from seven cells measured on a single coverslip, representative of three identical experiments (n=12 cells total).

• Movie 1 -

  Movie 1. EYFP-Stim1 exhibits constitutive movements when the punctate localization is reversed by store refilling. A single EYFP-Stim1-overexpressing HEK293 cell was imaged by TIRFM beginning in an extracellular solution containing 1.8 mM Ca²⁺. At the times indicated in the upper-left area, the extracellular solution was changed to 300 µM carbachol (Carb) in nominal Ca²⁺, then to 50 µM atropine (Atn) with 1.8 mM Ca²⁺ and finally to 2 µM thapsigargin (Tg) in nominal Ca²⁺. Images were captured every 10 seconds, and the frame rate of the movie is ten frames per second. Bar, 10 µm.

• Movie 2 -

  Movie 2. Reversal with ML-9 re-establishes constitutive movements of EYFP-Stim1. A single EYFP-Stim1-overexpressing HEK293 cell was imaged by TIRFM beginning in an extracellular solution containing 1.8 mM Ca²⁺. At the times indicated in the upper-left area, the extracellular solution was changed to 2 µM thapsigargin (Tg) in nominal Ca²⁺ and then to 100 µM ML-9 in the continued presence of Tg and nominal Ca²⁺. Images were captured every 5 seconds, and the frame rate of the movie is ten frames per second. Bar, 10 µm.

• Movie 3 -

  Movie 3. ML-9 induces constitutive movements of EYFP-D76N/D78N-Stim1. A single HEK293 cell overexpressing EYFP-D76N/D78N-Stim1 was imaged by TIRFM beginning in an extracellular solution containing 1.8 mM Ca²⁺. At the time indicated in the upper-left area, the extracellular solution was changed to 100 µM ML-9 in the continued presence of 1.8 mM Ca²⁺. Images were captured every 5 seconds, and the frame rate of the movie is ten frames per second. Bar, 10 µm.

• Movie 4 -

  Movie 4. Pre- and post-reversal EYFP-Stim1 punctae exhibit a close spatial correlation over time. As described in Fig. 9C, images before reversal with ML-9 were pseudocolored red and merged with images taken following ML-9 washout (pseudocolored green). This movie shows a time-lapse series of merged images, zoomed into a region of the cell to demonstrate the spatial correlation between red and green punctae. The frame rate of the movie is five frames per second. The total experimental duration represented by this movie is 2 minutes.

• Movie 5 -

  Movie 5. EYFP-Stim1 punctae are similar before and after reversal and reformation. HEK293 cells overexpressing EYFP-Stim1 were imaged by TIRFM beginning in an extracellular solution containing 1.8 mM Ca²⁺. At the times indicated in the upper-left area, the extracellular solution was changed to 2 µM thapsigargin (Tg) in nominal Ca²⁺, then to 100 µM ML-9 in the continued presence of Tg and nominal Ca²⁺ and finally to Tg in nominal Ca²⁺ without ML-9 (Wash). Images were captured every 10 seconds, and the frame rate of the movie is ten frames per second. Bar, 10 µm. Images correspond to the experiment presented in Fig. 9.