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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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Diminished STIM1 binding affinity allows Orai1-L273D to escape more readily from ER–PM junctions. ER Ca2+ stores in HEK cells were depleted with 1 μM TG in 0 Ca2+ Ringer's, and mCh-STIM1 was expressed at moderate levels in all experiments. (A) Histograms of diffusion coefficients for Orai1 (from Figures 1C and 2C) and Orai1-L273D (blue, 588 tracks, 4 cells) in store-depleted cells. (B) Cumulative histograms of the data shown in (A). (C) Average MSD vs. ∆t for Orai1 and Orai1-L273D (431 tracks, 4 cells) for particle trajectories that started in puncta (for depleted cells). Orai1 MSD plots from resting and depleted cells (black and red) are reproduced from Figure 2. (D) Average area of all puncta containing a tracked Orai particle, measured from thresholded mCh-STIM1 images (for Orai1, n = 439 puncta; for Orai1-L273D, n = 431 puncta).
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Figure 5: Diminished STIM1 binding affinity allows Orai1-L273D to escape more readily from ER–PM junctions. ER Ca2+ stores in HEK cells were depleted with 1 μM TG in 0 Ca2+ Ringer's, and mCh-STIM1 was expressed at moderate levels in all experiments. (A) Histograms of diffusion coefficients for Orai1 (from Figures 1C and 2C) and Orai1-L273D (blue, 588 tracks, 4 cells) in store-depleted cells. (B) Cumulative histograms of the data shown in (A). (C) Average MSD vs. ∆t for Orai1 and Orai1-L273D (431 tracks, 4 cells) for particle trajectories that started in puncta (for depleted cells). Orai1 MSD plots from resting and depleted cells (black and red) are reproduced from Figure 2. (D) Average area of all puncta containing a tracked Orai particle, measured from thresholded mCh-STIM1 images (for Orai1, n = 439 puncta; for Orai1-L273D, n = 431 puncta).

Mentions: Previous studies have suggested that Orai1 becomes trapped at ER–PM junctions through binding of the CAD domain of STIM1 to the C-terminus of Orai1 (Kawasaki et al., 2009; Park et al., 2009; Yuan et al., 2009). To test this model at the single-particle level, we examined the mobility of Orai1-L273D-GFP, an Orai1 mutant that does not bind STIM1 (Li et al., 2011). When expressed at very low levels, Orai1-L273D-GFP is expected to form multimers (presumably hexamers; Hou et al., 2012) with endogenous Orai1, consistent with the low fluorescence intensity and primarily one- to two-step photobleaching of Orai1-L273D-GFP particles (unpublished data). Thus these channels would be expected to have a reduced capacity to bind STIM1 compared with the wild-type (wt)-Orai1 channel. In depleted cells, diffusion of Orai1-L273D-GFP within junctions was nearly as slow as wt-Orai1-GFP (Figure 5, A and B). The slowed diffusion of Orai1-L273D in puncta could be due to STIM1 binding to endogenous wt-Orai1 subunits assembled with Orai1-L273D as well as to molecular crowding within the junction. The MSD versus ∆t graphs clearly show that in depleted cells the Orai1-L273D curves, while displaying anomalous diffusion, do not approach an asymptote as wt-Orai1 does (Figure 5C), even though L273D- and wt-Orai1 molecules start in puncta of equivalent size (Figure 5D). These data indicate that diffusion of Orai1-L273D is significantly less confined than diffusion of wt-Orai1, which is consistent with Orai1-L273D–containing channels having reduced binding to STIM1. Accordingly, we observed more frequent examples of escape of Orai1-L273D than wt-Orai1 from puncta (Figure 3D and Video S7).


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)

Diminished STIM1 binding affinity allows Orai1-L273D to escape more readily from ER–PM junctions. ER Ca2+ stores in HEK cells were depleted with 1 μM TG in 0 Ca2+ Ringer's, and mCh-STIM1 was expressed at moderate levels in all experiments. (A) Histograms of diffusion coefficients for Orai1 (from Figures 1C and 2C) and Orai1-L273D (blue, 588 tracks, 4 cells) in store-depleted cells. (B) Cumulative histograms of the data shown in (A). (C) Average MSD vs. ∆t for Orai1 and Orai1-L273D (431 tracks, 4 cells) for particle trajectories that started in puncta (for depleted cells). Orai1 MSD plots from resting and depleted cells (black and red) are reproduced from Figure 2. (D) Average area of all puncta containing a tracked Orai particle, measured from thresholded mCh-STIM1 images (for Orai1, n = 439 puncta; for Orai1-L273D, n = 431 puncta).
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Figure 5: Diminished STIM1 binding affinity allows Orai1-L273D to escape more readily from ER–PM junctions. ER Ca2+ stores in HEK cells were depleted with 1 μM TG in 0 Ca2+ Ringer's, and mCh-STIM1 was expressed at moderate levels in all experiments. (A) Histograms of diffusion coefficients for Orai1 (from Figures 1C and 2C) and Orai1-L273D (blue, 588 tracks, 4 cells) in store-depleted cells. (B) Cumulative histograms of the data shown in (A). (C) Average MSD vs. ∆t for Orai1 and Orai1-L273D (431 tracks, 4 cells) for particle trajectories that started in puncta (for depleted cells). Orai1 MSD plots from resting and depleted cells (black and red) are reproduced from Figure 2. (D) Average area of all puncta containing a tracked Orai particle, measured from thresholded mCh-STIM1 images (for Orai1, n = 439 puncta; for Orai1-L273D, n = 431 puncta).
Mentions: Previous studies have suggested that Orai1 becomes trapped at ER–PM junctions through binding of the CAD domain of STIM1 to the C-terminus of Orai1 (Kawasaki et al., 2009; Park et al., 2009; Yuan et al., 2009). To test this model at the single-particle level, we examined the mobility of Orai1-L273D-GFP, an Orai1 mutant that does not bind STIM1 (Li et al., 2011). When expressed at very low levels, Orai1-L273D-GFP is expected to form multimers (presumably hexamers; Hou et al., 2012) with endogenous Orai1, consistent with the low fluorescence intensity and primarily one- to two-step photobleaching of Orai1-L273D-GFP particles (unpublished data). Thus these channels would be expected to have a reduced capacity to bind STIM1 compared with the wild-type (wt)-Orai1 channel. In depleted cells, diffusion of Orai1-L273D-GFP within junctions was nearly as slow as wt-Orai1-GFP (Figure 5, A and B). The slowed diffusion of Orai1-L273D in puncta could be due to STIM1 binding to endogenous wt-Orai1 subunits assembled with Orai1-L273D as well as to molecular crowding within the junction. The MSD versus ∆t graphs clearly show that in depleted cells the Orai1-L273D curves, while displaying anomalous diffusion, do not approach an asymptote as wt-Orai1 does (Figure 5C), even though L273D- and wt-Orai1 molecules start in puncta of equivalent size (Figure 5D). These data indicate that diffusion of Orai1-L273D is significantly less confined than diffusion of wt-Orai1, which is consistent with Orai1-L273D–containing channels having reduced binding to STIM1. Accordingly, we observed more frequent examples of escape of Orai1-L273D than wt-Orai1 from puncta (Figure 3D and Video S7).

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