title: Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.
abstract: Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Chayasith
authors.initials: C
authors.lastname: Uttamapinant
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Howe
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Václav
authors.initials: V
authors.lastname: Beránek
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Lloyd
authors.initials: L
authors.lastname: Davis
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Mohan
authors.initials: M
authors.lastname: Mahesh
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Barry
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000199:Actins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D005815:Genetic Code / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine; D061848:Optical Imaging; D015202:Protein Engineering; D014746:Vimentin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000199:Actins; D014746:Vimentin; D008239:Lysine
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