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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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Related in: MedlinePlus

Trapping and escape of single Orai1 and STIM1 particles at ER–PM junctions. All images are from HEK cells treated with 1 μM TG + 0 Ca2+ Ringer's for 3–6 min. (A and B) Orai1-GFP tracks (yellow) overlaid on the corresponding mCh-STIM1 TIRF image (red). (C) GFP-STIM1 track (yellow) overlaid on the mCh-myc-Orai1 TIRF image (red). (D) Orai1-L273D-GFP track (yellow) overlaid on the mCh-STIM1 TIRF image (red). Starting and ending points of tracks are indicated, and dashed yellow lines indicate tracking gaps identified and linked by u-track (see Materials and Methods).
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Figure 3: Trapping and escape of single Orai1 and STIM1 particles at ER–PM junctions. All images are from HEK cells treated with 1 μM TG + 0 Ca2+ Ringer's for 3–6 min. (A and B) Orai1-GFP tracks (yellow) overlaid on the corresponding mCh-STIM1 TIRF image (red). (C) GFP-STIM1 track (yellow) overlaid on the mCh-myc-Orai1 TIRF image (red). (D) Orai1-L273D-GFP track (yellow) overlaid on the mCh-STIM1 TIRF image (red). Starting and ending points of tracks are indicated, and dashed yellow lines indicate tracking gaps identified and linked by u-track (see Materials and Methods).

Mentions: Importantly, SPT offers the unique opportunity to observe the trapping event itself. Figure 3 and Videos S3–S6 show examples of single STIM1 and Orai1 particles undergoing free diffusion before being captured and escaping from a diffusion trap at the ER–PM junction, clearly illustrating the distinction between free diffusion outside junctions and confined diffusion within junctions. As indicated by the D histograms (Figure 2C), the most common behavior within puncta was very low mobility, and these particles bleached without escaping. Escaping particles generally showed a higher mobility within the punctum before escaping.


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)

Trapping and escape of single Orai1 and STIM1 particles at ER–PM junctions. All images are from HEK cells treated with 1 μM TG + 0 Ca2+ Ringer's for 3–6 min. (A and B) Orai1-GFP tracks (yellow) overlaid on the corresponding mCh-STIM1 TIRF image (red). (C) GFP-STIM1 track (yellow) overlaid on the mCh-myc-Orai1 TIRF image (red). (D) Orai1-L273D-GFP track (yellow) overlaid on the mCh-STIM1 TIRF image (red). Starting and ending points of tracks are indicated, and dashed yellow lines indicate tracking gaps identified and linked by u-track (see Materials and Methods).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230625&req=5

Figure 3: Trapping and escape of single Orai1 and STIM1 particles at ER–PM junctions. All images are from HEK cells treated with 1 μM TG + 0 Ca2+ Ringer's for 3–6 min. (A and B) Orai1-GFP tracks (yellow) overlaid on the corresponding mCh-STIM1 TIRF image (red). (C) GFP-STIM1 track (yellow) overlaid on the mCh-myc-Orai1 TIRF image (red). (D) Orai1-L273D-GFP track (yellow) overlaid on the mCh-STIM1 TIRF image (red). Starting and ending points of tracks are indicated, and dashed yellow lines indicate tracking gaps identified and linked by u-track (see Materials and Methods).
Mentions: Importantly, SPT offers the unique opportunity to observe the trapping event itself. Figure 3 and Videos S3–S6 show examples of single STIM1 and Orai1 particles undergoing free diffusion before being captured and escaping from a diffusion trap at the ER–PM junction, clearly illustrating the distinction between free diffusion outside junctions and confined diffusion within junctions. As indicated by the D histograms (Figure 2C), the most common behavior within puncta was very low mobility, and these particles bleached without escaping. Escaping particles generally showed a higher mobility within the punctum before escaping.

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