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.

Playing with the Molecules of Life

“…65, 68, 233–236 Live cell labeling and super resolution imaging have been further facilitated by the development of bright small molecule fluorophores that enter mammalian cells and have minimal background fluorescence. 208, 237 While the refinement and optimization of labeling and imaging approaches is still on going, early examples of applications that are not possible by other methods have already emerged. 238–240 …”

“…B) Genetic incorporation of the strained alkyne into vimentin for super-resolution, live cell imaging of proteins. 208 …”

Chemically tunable mucin chimeras assembled on living cells

“…5A). In mammalian cells, Chin and coworkers expressed a membrane-resident epidermal growth factor receptor (EGFR)-green fluorescent protein (GFP) fusion construct featuring an extracellular norbornene-AA (50,51). The cell surface norbornene moiety was labeled with a tetrazine probe based on colocalization of GFP with the probe.…”

The use of unnatural amino acids to study and engineer protein function

“…This is especially important for super-resolution microscopy, which is presently limited by labelling quality. Optimization of incorporation efficiency, coupling chemistry and labelling protocols recently paved the way to the application of UAA technology in super-resolution microscopy [75][76][77].…”