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Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions.

Wu MM, Covington ED, Lewis RS - Mol. Biol. Cell (2014)

Bottom Line: After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER-PM junctions.While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels.Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.

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Restricted mobility of STIM1 and Orai1 after store depletion. (A) A store-depleted HEK cell (in 0 Ca2+ Ringer's + 1 μM TG) expressing a low level of GFP-STIM1 and a moderate level of mCh-myc-Orai1. GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A TG-treated HEK cell expressing a low level of Orai1-GFP and a moderate level of mCh-STIM1; Orai1-GFP tracks are overlaid on the mCh-STIM1 TIRF image. Tracks in A and B are color-coded by their average diffusion coefficient according to the color scale at right. (C) Diffusion coefficient histograms for STIM1 (1288 tracks, 7 cells) and Orai1 (832 tracks, 6 cells) in store-depleted cells. D values were calculated from junctional sojourns (see Materials and Methods). Histograms from resting cells in Figure 1C are overlaid for comparison. (D) Cumulative histograms of data shown in C. (E) Average MSD vs. ∆t for STIM1 (1225 tracks, 6 cells) and Orai1 (439 tracks, 5 cells) particle trajectories that started in puncta. MSD vs. ∆t graphs from resting cells in Figure 1E are overlaid for comparison.
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Figure 2: Restricted mobility of STIM1 and Orai1 after store depletion. (A) A store-depleted HEK cell (in 0 Ca2+ Ringer's + 1 μM TG) expressing a low level of GFP-STIM1 and a moderate level of mCh-myc-Orai1. GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A TG-treated HEK cell expressing a low level of Orai1-GFP and a moderate level of mCh-STIM1; Orai1-GFP tracks are overlaid on the mCh-STIM1 TIRF image. Tracks in A and B are color-coded by their average diffusion coefficient according to the color scale at right. (C) Diffusion coefficient histograms for STIM1 (1288 tracks, 7 cells) and Orai1 (832 tracks, 6 cells) in store-depleted cells. D values were calculated from junctional sojourns (see Materials and Methods). Histograms from resting cells in Figure 1C are overlaid for comparison. (D) Cumulative histograms of data shown in C. (E) Average MSD vs. ∆t for STIM1 (1225 tracks, 6 cells) and Orai1 (439 tracks, 5 cells) particle trajectories that started in puncta. MSD vs. ∆t graphs from resting cells in Figure 1E are overlaid for comparison.

Mentions: In store-depleted cells, trajectories of STIM1 and Orai1 were found mostly in puncta and were much more spatially restricted than in resting cells (compare Figure 2, A and B, with Figure 1, A and B). Diffusion coefficients of STIM1 and Orai1 within puncta of TG-treated cells were significantly reduced compared with D values in resting cells (STIM1 mean D = 0.031 ± 0.001 μm2/s, n = 1288 tracks; Orai1 mean D = 0.030 ± 0.001 μm2/s, n = 832 tracks). The mean D value for Orai1 in depleted cells agrees well with the average D measured by FRAP (0.036 ± 0.006 μm2/s; Park et al., 2009). Interestingly, the distributions of D values for STIM1 and Orai1 in store-depleted cells were virtually identical, unlike in resting cells (Figure 2, C and D), consistent with STIM1 and Orai1 moving together as a complex within the ER–PM junction. The size of the immobile fraction also increased significantly, from 5 to 24% for STIM1 particles and from 4 to 25% for Orai1. Possible contributors to the slowing of STIM1 and Orai1 in puncta include complex formation between the two proteins, interactions of STIM1 with the PM, molecular crowding, and diffusion within compartments whose sizes are comparable to the distance traveled within the sampling interval (see Discussion).


Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions.

Wu MM, Covington ED, Lewis RS - Mol. Biol. Cell (2014)

Restricted mobility of STIM1 and Orai1 after store depletion. (A) A store-depleted HEK cell (in 0 Ca2+ Ringer's + 1 μM TG) expressing a low level of GFP-STIM1 and a moderate level of mCh-myc-Orai1. GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A TG-treated HEK cell expressing a low level of Orai1-GFP and a moderate level of mCh-STIM1; Orai1-GFP tracks are overlaid on the mCh-STIM1 TIRF image. Tracks in A and B are color-coded by their average diffusion coefficient according to the color scale at right. (C) Diffusion coefficient histograms for STIM1 (1288 tracks, 7 cells) and Orai1 (832 tracks, 6 cells) in store-depleted cells. D values were calculated from junctional sojourns (see Materials and Methods). Histograms from resting cells in Figure 1C are overlaid for comparison. (D) Cumulative histograms of data shown in C. (E) Average MSD vs. ∆t for STIM1 (1225 tracks, 6 cells) and Orai1 (439 tracks, 5 cells) particle trajectories that started in puncta. MSD vs. ∆t graphs from resting cells in Figure 1E are overlaid for comparison.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Restricted mobility of STIM1 and Orai1 after store depletion. (A) A store-depleted HEK cell (in 0 Ca2+ Ringer's + 1 μM TG) expressing a low level of GFP-STIM1 and a moderate level of mCh-myc-Orai1. GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A TG-treated HEK cell expressing a low level of Orai1-GFP and a moderate level of mCh-STIM1; Orai1-GFP tracks are overlaid on the mCh-STIM1 TIRF image. Tracks in A and B are color-coded by their average diffusion coefficient according to the color scale at right. (C) Diffusion coefficient histograms for STIM1 (1288 tracks, 7 cells) and Orai1 (832 tracks, 6 cells) in store-depleted cells. D values were calculated from junctional sojourns (see Materials and Methods). Histograms from resting cells in Figure 1C are overlaid for comparison. (D) Cumulative histograms of data shown in C. (E) Average MSD vs. ∆t for STIM1 (1225 tracks, 6 cells) and Orai1 (439 tracks, 5 cells) particle trajectories that started in puncta. MSD vs. ∆t graphs from resting cells in Figure 1E are overlaid for comparison.
Mentions: In store-depleted cells, trajectories of STIM1 and Orai1 were found mostly in puncta and were much more spatially restricted than in resting cells (compare Figure 2, A and B, with Figure 1, A and B). Diffusion coefficients of STIM1 and Orai1 within puncta of TG-treated cells were significantly reduced compared with D values in resting cells (STIM1 mean D = 0.031 ± 0.001 μm2/s, n = 1288 tracks; Orai1 mean D = 0.030 ± 0.001 μm2/s, n = 832 tracks). The mean D value for Orai1 in depleted cells agrees well with the average D measured by FRAP (0.036 ± 0.006 μm2/s; Park et al., 2009). Interestingly, the distributions of D values for STIM1 and Orai1 in store-depleted cells were virtually identical, unlike in resting cells (Figure 2, C and D), consistent with STIM1 and Orai1 moving together as a complex within the ER–PM junction. The size of the immobile fraction also increased significantly, from 5 to 24% for STIM1 particles and from 4 to 25% for Orai1. Possible contributors to the slowing of STIM1 and Orai1 in puncta include complex formation between the two proteins, interactions of STIM1 with the PM, molecular crowding, and diffusion within compartments whose sizes are comparable to the distance traveled within the sampling interval (see Discussion).

Bottom Line: After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER-PM junctions.While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels.Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus