Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells

Sheng Wang; Miao Ding; Xuanze Chen; Lei Chang; Yujie Sun

doi:10.1364/BOE.8.003119

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells

Biomed Opt Express. 2017 May 31;8(6):3119-3131. doi: 10.1364/BOE.8.003119. eCollection 2017 Jun 1.

Authors

Sheng Wang^{1

2}, Miao Ding^{1

2}, Xuanze Chen^{1

3

2}, Lei Chang¹, Yujie Sun¹

Affiliations

¹ State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
² Contributed equally to this work.
³ Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China.

Abstract

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-x_L and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.

Keywords: (170.1420) Biology; (170.1530) Cell analysis; (170.2520) Fluorescence microscopy; (170.3880) Medical and biological imaging.