title: Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein-protein interactions in cells.
abstract: Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.
authors: Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University, Portland, Oregon, United States of America.
authors: Andrew
authors: A
authors: Nickerson
authors: Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University, Portland, Oregon, United States of America.
authors: Tao
authors: T
authors: Huang
authors: Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University, Portland, Oregon, United States of America.
authors: Li-Jung
authors: LJ
authors: Lin
authors: Department of Biomedical Engineering, Knight Cancer Institute, and OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University, Portland, Oregon, United States of America.
authors: Xiaolin
authors: X
authors: Nan
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D036103:Nanotechnology / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D008164:Luminescent Proteins