PUBLICATION
Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms
- Authors
- Ariotti, N., Hall, T.E., Rae, J., Ferguson, C., McMahon, K.A., Martel, N., Webb, R.E., Webb, R.I., Teasdale, R.D., Parton, R.G.
- ID
- ZDB-PUB-151121-10
- Date
- 2015
- Source
- Developmental Cell 35(4): 513-25 (Journal)
- Registered Authors
- Hall, Thomas, Parton, Robert G.
- Keywords
- none
- MeSH Terms
-
- Animals
- Animals, Genetically Modified/growth & development
- Animals, Genetically Modified/metabolism*
- Ascorbate Peroxidases/metabolism*
- Cricetinae
- Glycine max/enzymology
- Green Fluorescent Proteins/metabolism*
- High-Throughput Screening Assays/methods*
- Kidney/cytology
- Kidney/metabolism*
- Microscopy, Electron/methods*
- Protein Transport
- Subcellular Fractions
- Zebrafish/growth & development
- Zebrafish/metabolism*
- PubMed
- 26585296 Full text @ Dev. Cell
Citation
Ariotti, N., Hall, T.E., Rae, J., Ferguson, C., McMahon, K.A., Martel, N., Webb, R.E., Webb, R.I., Teasdale, R.D., Parton, R.G. (2015) Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms. Developmental Cell. 35(4):513-25.
Abstract
Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping