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-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.
Highlights
Visualizing the dynamic process of protein-protein interactions (PPIs) at high spatiotemporal resolution is of great importance for understanding molecular mechanisms [1, 2] in live cells
We developed bimolecular fluorescence complementation (BiFC) assay to achieve high resolution, real time detection of PPIs in live cells based on split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein with fast and efficient maturation previously applied in RESOLFT nanoscopy [15]
To determine the optimal cleavage site for generating non-fluorescent fragments of rsEGFP2 for BiFC imaging, we referred to EGFP which was successfully used in BiFC with a cleavage site at 158Q [22, 23] as the amino acids sequence of rsEGFP2 shares 98% identity with that of EGFP (Fig. 1(a))
Summary
Visualizing the dynamic process of protein-protein interactions (PPIs) at high spatiotemporal resolution is of great importance for understanding molecular mechanisms [1, 2] in live cells. We developed BiFC assay to achieve high resolution, real time detection of PPIs in live cells based on split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein with fast and efficient maturation previously applied in RESOLFT nanoscopy [15]. As a proof of concept, through combing with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of rsEGFP2-based BiFC assay by visualizing the heterodimerization interactions between members of the Bcl-2 family proteins Bcl-xL and Bak [19], as well as the dynamics of the complex on mitochondria membrane in live HeLa cells. Our newly developed reversibly photoswitchable fluorescent protein rsEGFP2-based BiFC assay expands the fluorescent protein toolbox available for BiFC and facilitates the detection and visualization of PPIs at super-resolution level in live cells
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