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A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo
- Source :
- Nature Communications, Vol 10, Iss 1, Pp 1-16 (2019), Nature Communications
- Publication Year :
- 2019
-
Abstract
- To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.<br />Expression of genetically encoded antibodies for cell labelling is often limited by folding issues. Here, the authors engineer an anti-HA scFv antibody that works in the cellular environment and use it to track mRNA translation dynamics in living cells and to label proteins in live zebrafish embryos.
- Subjects :
- 0301 basic medicine
Recombinant Fusion Proteins/isolation & purification/*metabolism
Embryo, Nonmammalian
Neurons/metabolism
General Physics and Astronomy
02 engineering and technology
Epitope
Epitopes
Protein biosynthesis
lcsh:Science
Zebrafish
Neurons
Multidisciplinary
Chemistry
Translation (biology)
021001 nanoscience & nanotechnology
Single Molecule Imaging
3. Good health
Cell biology
0210 nano-technology
Molecular Probes/*metabolism
Cell type
RNA, Messenger/genetics/metabolism
Science
Recombinant Fusion Proteins
Green Fluorescent Proteins
General Biochemistry, Genetics and Molecular Biology
Article
03 medical and health sciences
Single-molecule biophysics
In vivo
Cell Line, Tumor
Animals
RNA, Messenger
Green Fluorescent Proteins/metabolism
Staining and Labeling
General Chemistry
030104 developmental biology
Cell culture
Cytoplasm
Molecular Probes
Protein Biosynthesis
Epitopes/*metabolism
Single-Chain Antibodies/metabolism
lcsh:Q
Zebrafish/embryology
Single-Chain Antibodies
Embryo, Nonmammalian/metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 20411723
- Volume :
- 10
- Issue :
- No. 1
- Database :
- OpenAIRE
- Journal :
- Nat Commun
- Accession number :
- edsair.doi.dedup.....da3cb1b5966d3bc5f03941dc03e19d59