Antibody-based Probe Developed for Live-Cell Imaging in Vivo

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To expand the toolbox of imaging in living cells, researchers from Colorado State University and the Tokyo Institute of Technology have developed a genetically encoded, antibody-based probe that works with specificity in vivo. Probes built from antibodies were genetically fused with mature fluorophores. When loaded into cells, the probes bind and light up specific targets (epitopes) within the proteins of interest as soon as the epitopes are accessible. The probe, called the human influenza hemagglutinin (HA) frankenbody, can light up, in multiple colors, HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types.

Like stitching new limbs on a body — hence the name “frankenbody” — the scientists took the binding regions of a normal antibody and grafted them to a different scaffold that remains stable in live cells but retains the specificity of the antibody. With the goal of making their tool immediately useful, the scientists designed their probe to work with the classic HA tag. “For the longest time, people have been looking at HA-tagged proteins in fixed, dead cells. Now we can image the dynamics of those proteins in live cells,” professor Tim Stasevich said.

Antibody-based probe for live cell imaging, Colorado State University.
Frankenbodies (green) label nuclear proteins, mitochondria, stress fibers, and neuron membranes in living cells. Courtesy of Ning Zhao/Colorado State University.

The scientists demonstrated several applications, including single-protein tracking, single-RNA translation imaging, and amplified fluorescence imaging in zebrafish embryos. All of these experiments are more challenging when using traditional fluorescent protein tags, the team said.

The new probe could be a useful complement to the green fluorescent protein (GFP), a biochemistry tool and subject of a Nobel Prize that involves genetically fusing a light-up green tag to a protein of interest. The GFP is limited by its relatively large size and the time it takes to fluoresce. With the new frankenbody probe, the tag is smaller and becomes fluorescent faster, so the activity of a protein of interest can be captured in real time.

“We’re interested in intracellular antibodies because you can use them as imaging reagents in a live cell,” Stasevich said. “You don’t need a tag, like a green fluorescent protein, because instead you have this fluorescent antibody that will bind to your protein that you want to visualize.”

Antibody-based probe for live cell imaging, Colorado State University.
This is a frankenbody labeling mitochondria. Courtesy of Ning Zhao/Colorado State University.

The versatility of the HA frankenbody could make it a powerful tool for imaging protein dynamics in vivo, the researchers believe. Stasevich’s team is particularly interested in studying RNA translation, and the team plans to use the new system to more easily design new RNA imaging experiments.

The research was published in Nature Communications (  

Published: July 2019
Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
Research & TechnologyeducationColorado State UniversityAmericasImagingmedical imagingfluorescencefluorescent probeLight SourcesMicroscopymedicalBiophotonicsfrankenbodygreen fluorescent proteinlive-cell imagingin vivo imagingBioScan

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