Research Highlights

Using Click-EM for Metabolic Labeling to Track and Image Tagged Non-protein Biomolecules by Light and Electron Microscopy

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26-04-2016 La Jolla

Electron microscopy (EM) has long been the preferred technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Because EM and light microscopy (LM) offer different but synergistic capabilities, it is common for researchers to incorporate both in their work into what is called correlated light and electron microscopy, or CLEM.

In this context, combined efforts from the Roger Y. Tsien and Mark Ellisman laboratories at UC San Diego, in collaboration with scientists at UC Berkeley and Stanford, led to the development of a new labeling technique, called Click-EM, to correlate LM and EM imaging of non-protein biomolecules. With this approach, metabolic labeling substrates are provided to cells and the normal cellular machinery incorporates them into various cellular constituents. Their unique chemical functionality makes it possible to selectively attach reactive oxygen-generating fluorescent dyes to them via a technique called click chemistry. (Click chemistry describes a class of biocompatible reactions intended to join small modular chemical units together) When these dye-labeled structures are illuminated, they generate reactive oxygen species that locally catalyze the polymerization of diaminobenzidine (DAB) into a reaction product that is visible by both light and EM.

Genetic tags such as MiniSOG polymerize DAB by photogenerating reactive oxygen species that polymerizes DAB into an insoluble reaction product that can be stained with osmium tetroxide and, therefore, distinguished by EM. DAB can also be polymerized enzymatically using a genetically encoded peroxidase-based tag such as APEX2. Together, these tags represent a versatile toolkit for imaging genetically tagged proteins by EM.

What remained missing from this toolkit, however, were analogous tools to image non-protein biomolecules, such as glycans, nucleic acids, and lipids, and to do this metabolically. Non-protein biomolecules comprise a significant fraction of living matter, so a simple, generalizable method for visualizing them by EM would substantially enhance researchers’ ability to dissect cellular biochemistry at the nanometer scale. This is the niche Click-EM fills. In work leading to the paper described here, the team applied Click-EM to image metabolically tagged DNA, RNA, and lipids in cultured mammalian cells and neurons, and to track peptidoglycan synthesis in the bacterium Listeria monocytogenes.

The first step in this project was to find dyes capable of efficiently photooxidizing DABthat could also be selectively conjugated to metabolically tagged biomolecules in fixed cells. In an initial screen, the team developed and/or tested 13 dyes with varying excitation wavelengths ranging from blue to far-red, including previously characterized fluorescent dyes known to photogenerate substantial levels of reactive oxygen. After five minutes of illumination, successful DAB photooxidation was indicated by the appearance of a dense reaction product visible under transmitted light. Of the 13, 10 tested positive. Of those, dibromofluorescein (DBF)-azide was found to be among the most efficient photooxidizing dyes. It was shown to be capable of efficiently photooxidizing DAB and able to be conjugated to alkyne-labeled biomolecules with excellent specificity.

Following live-cell labeling of DNA using incorporation of 5-ethynyl-2´-deoxyuridine (EdU) into DNA, cells were chemically fixed and the dye DBF-azide was attached via Cu(I)-catalyzed azide–alkyne cycloaddition (a click chemistry reaction) and the dye used to photooxidize DAB in labeled HeLa cells. The cells were then post-fixed with osmium tetroxide, embed in epoxy resin, and cut into 80nm thin sections before imaging by transmission electron microscopy (TEM). In electron micrographs, the osmium-stained DAB reaction product appeared with discernable contrast in patterns that correlated with DBF-azide fluorescence and were consistent with localization of DNA. The team captured images of the labeled cells occupying various stages of the cell cycle by confocal microscopy and then performed DAB photooxidation to visualize labeled chromosomes by EM. Additionally, the team reconstructed entire labeled HEK293 cells using serial blockface scanning EM, a technique in which sequential surface images are collected from a bulk sample containing labeled cells of interest embedded in resin. The NIGMS-supported research team could then use the stain contrast to reconstruct the chromosomes in 3D in their entirety.

The team also was able to visualize nascent RNA using metabolically incorporated 5-ethynyl-uridine (EU) in HeLa cells and choline-rich lipids using azidocholine in HEK293 cells and in neurons to demonstrate the potential of Click-EM to visualize non-protein molecules with excellent temporal and spatial resolution.

They also applied this technique to image bacterial peptidoglycan (PG), an essential cell component of most bacterial species that is also the target of many antibiotics. The superb resolution of Click-EM enabled them to distinguish extracellular PG from its intracellular intermediates, which are separated by a mere 7 nm.

In short, the Click-EM approach provides a simple and direct means to determine the detailed cellular distribution of metabolically tagged nonprotein biomolecules. It is preferred to more conventional techniques because the biomolecules of interest are incorporated prior to fixation, and all auxiliary reagents required for generating contrast are small molecules that readily diffuse into fixed cells. Moreover, Click-EM enables high-quality preservation of EM-visible landmarks whose integrity is essential to assigning precise location to the labeled biomolecules.

Click-EM should find widespread application in ultrastructural localization of diverse classes of biomolecules. Global incorporation of azide- or alkyne-containing amino acids in combination with Click-EM may be useful to image localized neuronal protein synthesis in dendrites, a neuronal subregion that is difficult to resolve by light microscopy. Additional promising applications for Click-EM include imaging of labeled biomolecules in other cellular subregions and structures that are difficult to resolve using LM, such as axon terminals, mitochondria, synaptic vesicles, and autophagosomes.

 

Citation: Ngo, John T., Adams, Stephen R., Deerinck, Thomas J., Boassa, Daniela, Rodriguez-Rivera, Frances, Palida, Sakina F., Bertozzi, Carolyn R., Ellisman, Mark H., and Tsien, Roger Y. 2016. Click-EM for imaging metabolically tagged non-protein biomolecules. Nat Chem Bio, June 2016.