title: Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions.
abstract: Following endoplasmic reticulum (ER) Ca(2+) depletion, STIM1 and Orai1 complexes assemble autonomously at ER-plasma membrane (PM) junctions to trigger store-operated Ca(2+) influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. 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.
authors: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.
authors: Minnie M
authors: MM
authors: Wu
authors: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.
authors: Elizabeth D
authors: ED
authors: Covington
authors: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.
authors: Richard S
authors: RS
authors: Lewis
mesh_terms: D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009010:Monte Carlo Method; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction; D000071737:Stromal Interaction Molecule 1
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium