Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie05 - Supplemental Video 5: Real‐time trajectory of a tracked Orai1‐GFP particle in a store‐depleted HEK cell. A 16 s trajectory of an Orai1‐GFP particle depicted as a yellow dot moving in real time (20 frames per s). The trajectory is overlaid on the corresponding summed mCh‐STIM1 TIRF image (summed from the mCherry channel from the same 1000 frame movie). This HEK cell was expressing low levels of Orai1‐GFP and high/moderate levels of mCh‐STIM1 and treated with 1 μM TG in 0 Ca Ringer's for 5 min before imaging. Particle detection and tracking was done using u‐track software (see Methods). The entire particle trajectory is shown in Figure 3B. Bar, 1 μm.
- Movie07 - Supplemental Video 7: Real‐time trajectory of a tracked Orai1‐L273D‐GFP particle in a store‐depleted HEK cell. A 12 s trajectory of an Orai1‐L273D‐GFP particle depicted as a yellow dot moving in real time (20 frames per s). The trajectory is overlaid on the corresponding summed mCh‐STIM1 TIRF image (summed from the mCherry channel from the same 1000 frame movie). This HEK cell was expressing low levels of Orai1‐L273D‐GFP and high/moderate levels of mCh‐STIM1 and treated with 1 μM TG in 0 Ca Ringer's for 5 min before imaging. Particle detection and tracking was done using u‐track software (see Methods). The entire particle trajectory is shown in Figure 3D. Bar, 1 μm.
- Movie06 - Supplemental Video 6: Real‐time trajectory of a tracked GFP‐STIM1 particle in a store‐depleted HEK cell. A 14 s trajectory of a GFP‐STIM1 particle depicted as a yellow dot moving in real time (20 frames per s). The trajectory is overlaid on the corresponding summed mCh‐myc‐Orai1 TIRF image (summed from the mCherry channel from the same 1000 frame movie). This HEK cell was expressing low levels of GFP‐STIM1 and moderate levels of mCh‐myc‐Orai1 and treated with 1 μM TG in 0 Ca Ringer's for 3 min before imaging. Particle detection and tracking was done using u‐track software (see Methods). The entire particle trajectory is shown in Figure 3C. Bar, 1 μm.
- Movie03 - Supplemental Video 3: Diffusion and trapping of Orai1‐GFP in a store‐depleted HEK cell. A 50 s movie (acquired and displayed at 20 frames per s) of a HEK cell expressing low levels of Orai1‐GFP and moderate/high levels of mCherry‐STIM1 imaged with 488‐nm laser illumination in TIRF mode (488‐nm strongly excites GFP and weakly excites mCherry). The cell was treated with 1 μM TG in 0 Ca Ringer's for 6 min before imaging to deplete Ca2+ stores. The white box highlights a freely diffusing Orai1‐GFP particle becoming trapped by a mCh‐STIM1 punctum and then escaping (the same particle is shown in Figure 3A and Supp. Video 4). Bar, 5 μm.
- Movie01 - Supplemental Video 1: Single GFP‐STIM1 particles diffusing in the ER of a resting HEK cell. A 50 s movie (acquired and displayed at 20 frames per s) of a resting HEK cell (in 2 mM Ca Ringer's) expressing low levels of GFP‐STIM1, imaged with 488‐nm laser illumination in TIRF mode. This movie was acquired from the cell shown in Figure 1A. Bar, 5 μm.
- Movie02 - Supplemental Video 2: Single Orai1‐GFP particles diffusing in the PM of a resting HEK cell. A 50 s movie (acquired and displayed at 20 frames per s) of a resting HEK cell (in 2 mM Ca Ringer's) expressing low levels of Orail‐GFP, imaged with 488‐nm laser illumination in TIRF mode. This movie was acquired from the cell shown in Figure 1D. Bar, 5 μm.
- Movie04 - Supplemental Video 4: Real‐time trajectory of a tracked Orail‐GFP particle in a store‐depleted HEK cell. The 23 s trajectory of the same Orai1‐GFP particle highlighted in Supp. Video 3 is depicted as a yellow dot moving in real time (20 frames per s). The trajectory is overlaid on the mCh‐STIM1 image (summed from the mCherry channel from the same 1000 frame TIRF movie). Particle detection and tracking was done using u‐track software (see Methods). The entire particle trajectory is shown in Figure 3A. Bar, 1 μm.