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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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Binding to the PM and Orai1 slows and confines STIM1 diffusion in store-depleted cells. All HEK cells were store depleted by treatment with 1 μM TG + 0 Ca2+ Ringer's. (A) Histograms of diffusion coefficients for STIM1 alone (green, 4077 tracks, 7 cells), STIM1 particles with Orai1 (black, 1288 tracks, 7 cells), STIM1-∆K alone (blue, 3305 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 729 tracks, 6 cells). (B) Cumulative histograms of data shown in A. (C) Average MSD vs. ∆t for STIM1-∆K alone (blue, 3305 tracks, 6 cells), STIM1 alone (green, 3574 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 753 tracks, 6 cells). STIM1 with Orai1 resting (gray) and depleted (black) D histograms and MSD plots are reproduced from Figures 1 and 2. (D) Average area of all puncta containing tracked Orai1 or STIM1 particles, measured from thresholded mCh-STIM1 images (for Orai1+STIM1, n = 439 puncta; for STIM1 only, n = 3574 puncta).
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Figure 4: Binding to the PM and Orai1 slows and confines STIM1 diffusion in store-depleted cells. All HEK cells were store depleted by treatment with 1 μM TG + 0 Ca2+ Ringer's. (A) Histograms of diffusion coefficients for STIM1 alone (green, 4077 tracks, 7 cells), STIM1 particles with Orai1 (black, 1288 tracks, 7 cells), STIM1-∆K alone (blue, 3305 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 729 tracks, 6 cells). (B) Cumulative histograms of data shown in A. (C) Average MSD vs. ∆t for STIM1-∆K alone (blue, 3305 tracks, 6 cells), STIM1 alone (green, 3574 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 753 tracks, 6 cells). STIM1 with Orai1 resting (gray) and depleted (black) D histograms and MSD plots are reproduced from Figures 1 and 2. (D) Average area of all puncta containing tracked Orai1 or STIM1 particles, measured from thresholded mCh-STIM1 images (for Orai1+STIM1, n = 439 puncta; for STIM1 only, n = 3574 puncta).

Mentions: On the other hand, accumulation is also expected from a diffusion trap mechanism in which the escape of STIM1 and Orai1 from junctions is restricted by a one-way barrier and/or by tethering. A general hallmark of particles diffusing within a restricted space (“corral”) is that the MSD versus ∆t is sublinear and approaches a plateau (Kusumi et al., 1993). Consistent with such a confinement mechanism, the MSD versus ∆t curves for STIM1 and Orai1 were highly nonlinear in depleted cells, in both cases reaching a similar asymptote within several seconds (Figure 2E). These results suggest that STIM1 and Orai1 encounter a common barrier that confines their movement within junctions. The dimensions of the barrier were estimated from the plateau MSD value (Saxton and Jacobson, 1997). For Orai1, the MSD plateau was ∼0.115 μm2 (Figure 2E), which can be approximated by a circular corral with a diameter of 0.68 μm (Saxton and Jacobson, 1997). For STIM1, the estimated corral diameter was 0.76 μm (estimated from an MSD plateau of ∼0.14 μm2). We also used local thresholding to measure the areas of all fluorescent mCh-STIM1 puncta that contained tracked Orai1 particles (see Materials and Methods). In depleted cells, Orai1 particles were located in mCh-STIM1 puncta with mean area of 0.95 μm2 (Figure 4D), equivalent to a circle with a diameter of 1.1 μm. Considering the varying size and shape of puncta, the 0.7- to 0.8-μm corral diameter approximated from the MSD plateaus corresponds reasonably well to the average puncta diameter of ∼1.1 μm, suggesting the boundaries of the diffusion trap correspond to the edges of ER–PM junctions.


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)

Binding to the PM and Orai1 slows and confines STIM1 diffusion in store-depleted cells. All HEK cells were store depleted by treatment with 1 μM TG + 0 Ca2+ Ringer's. (A) Histograms of diffusion coefficients for STIM1 alone (green, 4077 tracks, 7 cells), STIM1 particles with Orai1 (black, 1288 tracks, 7 cells), STIM1-∆K alone (blue, 3305 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 729 tracks, 6 cells). (B) Cumulative histograms of data shown in A. (C) Average MSD vs. ∆t for STIM1-∆K alone (blue, 3305 tracks, 6 cells), STIM1 alone (green, 3574 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 753 tracks, 6 cells). STIM1 with Orai1 resting (gray) and depleted (black) D histograms and MSD plots are reproduced from Figures 1 and 2. (D) Average area of all puncta containing tracked Orai1 or STIM1 particles, measured from thresholded mCh-STIM1 images (for Orai1+STIM1, n = 439 puncta; for STIM1 only, n = 3574 puncta).
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Figure 4: Binding to the PM and Orai1 slows and confines STIM1 diffusion in store-depleted cells. All HEK cells were store depleted by treatment with 1 μM TG + 0 Ca2+ Ringer's. (A) Histograms of diffusion coefficients for STIM1 alone (green, 4077 tracks, 7 cells), STIM1 particles with Orai1 (black, 1288 tracks, 7 cells), STIM1-∆K alone (blue, 3305 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 729 tracks, 6 cells). (B) Cumulative histograms of data shown in A. (C) Average MSD vs. ∆t for STIM1-∆K alone (blue, 3305 tracks, 6 cells), STIM1 alone (green, 3574 tracks, 6 cells), and STIM1-∆K with Orai1 (red, 753 tracks, 6 cells). STIM1 with Orai1 resting (gray) and depleted (black) D histograms and MSD plots are reproduced from Figures 1 and 2. (D) Average area of all puncta containing tracked Orai1 or STIM1 particles, measured from thresholded mCh-STIM1 images (for Orai1+STIM1, n = 439 puncta; for STIM1 only, n = 3574 puncta).
Mentions: On the other hand, accumulation is also expected from a diffusion trap mechanism in which the escape of STIM1 and Orai1 from junctions is restricted by a one-way barrier and/or by tethering. A general hallmark of particles diffusing within a restricted space (“corral”) is that the MSD versus ∆t is sublinear and approaches a plateau (Kusumi et al., 1993). Consistent with such a confinement mechanism, the MSD versus ∆t curves for STIM1 and Orai1 were highly nonlinear in depleted cells, in both cases reaching a similar asymptote within several seconds (Figure 2E). These results suggest that STIM1 and Orai1 encounter a common barrier that confines their movement within junctions. The dimensions of the barrier were estimated from the plateau MSD value (Saxton and Jacobson, 1997). For Orai1, the MSD plateau was ∼0.115 μm2 (Figure 2E), which can be approximated by a circular corral with a diameter of 0.68 μm (Saxton and Jacobson, 1997). For STIM1, the estimated corral diameter was 0.76 μm (estimated from an MSD plateau of ∼0.14 μm2). We also used local thresholding to measure the areas of all fluorescent mCh-STIM1 puncta that contained tracked Orai1 particles (see Materials and Methods). In depleted cells, Orai1 particles were located in mCh-STIM1 puncta with mean area of 0.95 μm2 (Figure 4D), equivalent to a circle with a diameter of 1.1 μm. Considering the varying size and shape of puncta, the 0.7- to 0.8-μm corral diameter approximated from the MSD plateaus corresponds reasonably well to the average puncta diameter of ∼1.1 μm, suggesting the boundaries of the diffusion trap correspond to the edges of ER–PM junctions.

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