Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms

Dev Cell. 2015 Nov 23;35(4):513-25. doi: 10.1016/j.devcel.2015.10.016. Epub 2015 Nov 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified / growth & development
  • Animals, Genetically Modified / metabolism*
  • Ascorbate Peroxidases / metabolism*
  • Cricetinae
  • Green Fluorescent Proteins / metabolism*
  • High-Throughput Screening Assays / methods*
  • Kidney / cytology
  • Kidney / metabolism*
  • Microscopy, Electron / methods*
  • Protein Transport
  • Soybeans / enzymology
  • Subcellular Fractions
  • Zebrafish / growth & development
  • Zebrafish / metabolism*

Substances

  • Green Fluorescent Proteins
  • Ascorbate Peroxidases