title: Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).
abstract: Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.
authors.affiliation: Department of Biomedical Engineering, Oregon Health and Science University; Knight Cancer Institute, Oregon Health and Science University; OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University.
authors.forename: Andrew
authors.initials: A
authors.lastname: Nickerson
authors.affiliation: Department of Biomedical Engineering, Oregon Health and Science University; Knight Cancer Institute, Oregon Health and Science University; OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University.
authors.forename: Tao
authors.initials: T
authors.lastname: Huang
authors.affiliation: Department of Biomedical Engineering, Oregon Health and Science University; Knight Cancer Institute, Oregon Health and Science University; OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University.
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Lin
authors.affiliation: Department of Biomedical Engineering, Oregon Health and Science University; Knight Cancer Institute, Oregon Health and Science University; OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University; nan@ohsu.edu.
authors.forename: Xioalin
authors.initials: X
authors.lastname: Nan
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D060066:Protein Interaction Maps; D048490:raf Kinases / Q000737:chemistry* / Q000378:metabolism*; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D048490:raf Kinases; D018631:ras Proteins