visualisation and identification of the interaction between stim1s in resting cells可视化和识别stim1s在静息细胞之间的互动.pdfVIP

Visualisation and Identification of the Interaction between STIM1s in Resting Cells 1 2 1 1 Jun He *, Tao Yu , Jingying Pan , He Li * 1 Division of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2 Clinic Laboratory of Wuhan Children’s Hospital, Wuhan, China Abstract Store-operated Ca2+ channels are a major Ca2+ 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 Ca2+ release- activated Ca2+ (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca2+ 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 Ca2+ stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca2+ stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1