title: Visualisation and identification of the interaction between STIM1s in resting cells.
abstract: Store-operated Ca(2+) channels are a major Ca(2+) entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca(2+) sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca(2+) stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.
authors.affiliation: Division of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. junhe@mails.tjmu.edu.cn
authors.forename: Jun
authors.initials: J
authors.lastname: He
authors.forename: Tao
authors.initials: T
authors.lastname: Yu
authors.forename: Jingying
authors.initials: J
authors.lastname: Pan
authors.forename: He
authors.initials: H
authors.lastname: Li
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001896:Boron Compounds / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D038761:Photobleaching; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D000071737:Stromal Interaction Molecule 1
chemical_list: D001426:Bacterial Proteins; D001896:Boron Compounds; D015220:Calcium Channels; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011993:Recombinant Fusion Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; C054562:yellow fluorescent protein, Bacteria; C109986:2-aminoethoxydiphenyl borate