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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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STIM1 and Orai1 puncta fluorescence is stable over time. HEK cells expressing moderate levels of mCh-STIM1 (A and B) or mCh-myc-Orai1(C and D) together with PAGFP-labeled partner protein were store depleted with 1 μM TG in 0 Ca2+ Ringer's and imaged by confocal microscopy over time. Regions of interest marking selected puncta are superimposed on average images of 100-frame movies for mCh-STIM1 (A) and mCh-myc-Orai1 (C). Mean fluorescence vs. time (corrected for photobleaching) of the selected mCh-STIM1 (B) and mCh-myc-Orai1 (D) puncta shows stable puncta fluorescence over >5 min.
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Figure 6: STIM1 and Orai1 puncta fluorescence is stable over time. HEK cells expressing moderate levels of mCh-STIM1 (A and B) or mCh-myc-Orai1(C and D) together with PAGFP-labeled partner protein were store depleted with 1 μM TG in 0 Ca2+ Ringer's and imaged by confocal microscopy over time. Regions of interest marking selected puncta are superimposed on average images of 100-frame movies for mCh-STIM1 (A) and mCh-myc-Orai1 (C). Mean fluorescence vs. time (corrected for photobleaching) of the selected mCh-STIM1 (B) and mCh-myc-Orai1 (D) puncta shows stable puncta fluorescence over >5 min.

Mentions: Such a tight trap appears to be consistent with observations that STIM1 and Orai1 fluorescence in puncta is constant over minutes in cells with depleted stores (Figure 6). However, constant fluorescence can also result from particles entering and leaving the puncta at the same rate. Furthermore, escape of STIM1 and Orai1 particles may have been limited in the SPT experiments, because each particle was surrounded by a great excess of its binding partner (to facilitate identification of the junctions), whereas levels of endogenous STIM1 and Orai1 are probably more evenly matched. To examine the rate of escape under more physiological conditions, we examined puncta dynamics in cells expressing roughly equal amounts of PAGFP-STIM1 and mCh-Orai1 or PAGFP-Orai1 and mCh-STIM1. In its resting state, PAGFP is minimally fluorescent when excited at 488 nm, but its fluorescence increases ∼100-fold after activation by 405-nm light (Patterson and Lippincott-Schwartz, 2002). Transfected cells were treated with TG, and a 405-nm laser was focused on a mCh-labeled punctum (Figure 7A). A 5- to 20-ms flash activated PAGFP-STIM1 or PAGFP-Orai1 within the punctum, causing the fluorescence to reach a peak and subsequently decay over several minutes, revealing the loss of STIM1 or Orai1 from the punctum by diffusion (Figure 7, B and C). Because the membrane area into which the fluorescent protein diffuses is essentially infinite, the decay of fluorescence from photoactivation approximates the effective leaving rate for STIM1 and Orai1 from puncta. For both proteins, fluorescence decayed with a double-exponential time course with time constants of 22–25 and 357–376 s. The STIM1 exit rate was somewhat slower than that of Orai1 (t1/2 of fluorescence decay was ∼100 s for PAGFP-STIM1 and ∼50 s for PAGFP-myc-Orai1; Figure 7C). Given that the fluorescence intensities of mCh-labeled STIM1 and Orai1 puncta are constant over time (Figure 6), these results imply that when expressed at comparable levels, STIM1 and Orai1 in puncta are in diffusional equilibrium with their surrounding pools.


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)

STIM1 and Orai1 puncta fluorescence is stable over time. HEK cells expressing moderate levels of mCh-STIM1 (A and B) or mCh-myc-Orai1(C and D) together with PAGFP-labeled partner protein were store depleted with 1 μM TG in 0 Ca2+ Ringer's and imaged by confocal microscopy over time. Regions of interest marking selected puncta are superimposed on average images of 100-frame movies for mCh-STIM1 (A) and mCh-myc-Orai1 (C). Mean fluorescence vs. time (corrected for photobleaching) of the selected mCh-STIM1 (B) and mCh-myc-Orai1 (D) puncta shows stable puncta fluorescence over >5 min.
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Related In: Results  -  Collection

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Figure 6: STIM1 and Orai1 puncta fluorescence is stable over time. HEK cells expressing moderate levels of mCh-STIM1 (A and B) or mCh-myc-Orai1(C and D) together with PAGFP-labeled partner protein were store depleted with 1 μM TG in 0 Ca2+ Ringer's and imaged by confocal microscopy over time. Regions of interest marking selected puncta are superimposed on average images of 100-frame movies for mCh-STIM1 (A) and mCh-myc-Orai1 (C). Mean fluorescence vs. time (corrected for photobleaching) of the selected mCh-STIM1 (B) and mCh-myc-Orai1 (D) puncta shows stable puncta fluorescence over >5 min.
Mentions: Such a tight trap appears to be consistent with observations that STIM1 and Orai1 fluorescence in puncta is constant over minutes in cells with depleted stores (Figure 6). However, constant fluorescence can also result from particles entering and leaving the puncta at the same rate. Furthermore, escape of STIM1 and Orai1 particles may have been limited in the SPT experiments, because each particle was surrounded by a great excess of its binding partner (to facilitate identification of the junctions), whereas levels of endogenous STIM1 and Orai1 are probably more evenly matched. To examine the rate of escape under more physiological conditions, we examined puncta dynamics in cells expressing roughly equal amounts of PAGFP-STIM1 and mCh-Orai1 or PAGFP-Orai1 and mCh-STIM1. In its resting state, PAGFP is minimally fluorescent when excited at 488 nm, but its fluorescence increases ∼100-fold after activation by 405-nm light (Patterson and Lippincott-Schwartz, 2002). Transfected cells were treated with TG, and a 405-nm laser was focused on a mCh-labeled punctum (Figure 7A). A 5- to 20-ms flash activated PAGFP-STIM1 or PAGFP-Orai1 within the punctum, causing the fluorescence to reach a peak and subsequently decay over several minutes, revealing the loss of STIM1 or Orai1 from the punctum by diffusion (Figure 7, B and C). Because the membrane area into which the fluorescent protein diffuses is essentially infinite, the decay of fluorescence from photoactivation approximates the effective leaving rate for STIM1 and Orai1 from puncta. For both proteins, fluorescence decayed with a double-exponential time course with time constants of 22–25 and 357–376 s. The STIM1 exit rate was somewhat slower than that of Orai1 (t1/2 of fluorescence decay was ∼100 s for PAGFP-STIM1 and ∼50 s for PAGFP-myc-Orai1; Figure 7C). Given that the fluorescence intensities of mCh-labeled STIM1 and Orai1 puncta are constant over time (Figure 6), these results imply that when expressed at comparable levels, STIM1 and Orai1 in puncta are in diffusional equilibrium with their surrounding pools.

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