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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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Diffusion of STIM1 and Orai1 in resting cells. (A) A resting HEK cell in 2 mM Ca2+ Ringer's expressing a low level of GFP-STIM1 and a moderate level of mCherry (mCh)-myc-Orai1. Colored GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A resting 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 far right. (C) Diffusion coefficient histograms for STIM1 (black) and Orai1 (red) in resting cells. STIM1 mean D = 0.116 μm2/s (1744 tracks, 3 cells); Orai1 mean D = 0.090 μm2/s (4994 tracks, 6 cells). (D) Cumulative histograms of the diffusion coefficients shown in C. (E) Average MSD vs. ∆t for all STIM1 and Orai1 tracks in resting cells (weighted average ± weighted SD; see Materials and Methods). The dashed lines indicate linear least-squares fits to points 2–5 (∆t = 50–200 ms) of the MSD vs. ∆t curves for STIM1 and Orai1.
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Figure 1: Diffusion of STIM1 and Orai1 in resting cells. (A) A resting HEK cell in 2 mM Ca2+ Ringer's expressing a low level of GFP-STIM1 and a moderate level of mCherry (mCh)-myc-Orai1. Colored GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A resting 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 far right. (C) Diffusion coefficient histograms for STIM1 (black) and Orai1 (red) in resting cells. STIM1 mean D = 0.116 μm2/s (1744 tracks, 3 cells); Orai1 mean D = 0.090 μm2/s (4994 tracks, 6 cells). (D) Cumulative histograms of the diffusion coefficients shown in C. (E) Average MSD vs. ∆t for all STIM1 and Orai1 tracks in resting cells (weighted average ± weighted SD; see Materials and Methods). The dashed lines indicate linear least-squares fits to points 2–5 (∆t = 50–200 ms) of the MSD vs. ∆t curves for STIM1 and Orai1.

Mentions: To enable single-particle tracking, we imaged fluorescently labeled STIM1 and Orai1 proteins under conditions in which the separation between adjacent proteins exceeded the diffraction limit (∼250 nm). We tested several labeling methods, including PAGFP, mEos2, and GFP. While all three methods gave qualitatively similar results, we found that GFP-labeled proteins produced the longest tracks before bleaching. Trajectories of single GFP-STIM1 and Orai1-GFP particles in resting HEK cells are shown in Figure 1 and Supplemental Videos S1 and S2. Several observations indicate that these particles represent single molecules of STIM1 in the ER and Orai1 in the PM. The fluorescence intensities of the particles were narrowly distributed and single- or double-step photobleaching events were observed, indicating that most particles were labeled with a single GFP (Supplemental Figure S1). Furthermore, after bleaching by prolonged total internal reflection fluorescence (TIRF) illumination, new Orai1-GFP particles entered the cell footprint from the edges of the cell (unpublished data), confirming that Orai1 molecules being tracked were in the PM, not in intracellular vesicles, which would be expected to insert throughout the cell footprint.


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)

Diffusion of STIM1 and Orai1 in resting cells. (A) A resting HEK cell in 2 mM Ca2+ Ringer's expressing a low level of GFP-STIM1 and a moderate level of mCherry (mCh)-myc-Orai1. Colored GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A resting 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 far right. (C) Diffusion coefficient histograms for STIM1 (black) and Orai1 (red) in resting cells. STIM1 mean D = 0.116 μm2/s (1744 tracks, 3 cells); Orai1 mean D = 0.090 μm2/s (4994 tracks, 6 cells). (D) Cumulative histograms of the diffusion coefficients shown in C. (E) Average MSD vs. ∆t for all STIM1 and Orai1 tracks in resting cells (weighted average ± weighted SD; see Materials and Methods). The dashed lines indicate linear least-squares fits to points 2–5 (∆t = 50–200 ms) of the MSD vs. ∆t curves for STIM1 and Orai1.
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Related In: Results  -  Collection

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Figure 1: Diffusion of STIM1 and Orai1 in resting cells. (A) A resting HEK cell in 2 mM Ca2+ Ringer's expressing a low level of GFP-STIM1 and a moderate level of mCherry (mCh)-myc-Orai1. Colored GFP-STIM1 tracks are overlaid on the mCh-myc-Orai1 TIRF image. (B) A resting 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 far right. (C) Diffusion coefficient histograms for STIM1 (black) and Orai1 (red) in resting cells. STIM1 mean D = 0.116 μm2/s (1744 tracks, 3 cells); Orai1 mean D = 0.090 μm2/s (4994 tracks, 6 cells). (D) Cumulative histograms of the diffusion coefficients shown in C. (E) Average MSD vs. ∆t for all STIM1 and Orai1 tracks in resting cells (weighted average ± weighted SD; see Materials and Methods). The dashed lines indicate linear least-squares fits to points 2–5 (∆t = 50–200 ms) of the MSD vs. ∆t curves for STIM1 and Orai1.
Mentions: To enable single-particle tracking, we imaged fluorescently labeled STIM1 and Orai1 proteins under conditions in which the separation between adjacent proteins exceeded the diffraction limit (∼250 nm). We tested several labeling methods, including PAGFP, mEos2, and GFP. While all three methods gave qualitatively similar results, we found that GFP-labeled proteins produced the longest tracks before bleaching. Trajectories of single GFP-STIM1 and Orai1-GFP particles in resting HEK cells are shown in Figure 1 and Supplemental Videos S1 and S2. Several observations indicate that these particles represent single molecules of STIM1 in the ER and Orai1 in the PM. The fluorescence intensities of the particles were narrowly distributed and single- or double-step photobleaching events were observed, indicating that most particles were labeled with a single GFP (Supplemental Figure S1). Furthermore, after bleaching by prolonged total internal reflection fluorescence (TIRF) illumination, new Orai1-GFP particles entered the cell footprint from the edges of the cell (unpublished data), confirming that Orai1 molecules being tracked were in the PM, not in intracellular vesicles, which would be expected to insert throughout the cell footprint.

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