50nm-Scale Localization of Single Unmodified, Isotopically Enriched, Proteins in Cells

Anthony Delaune,Marc Vasse,Guillaume Legent,Camille Ripoll,Arndt Benecke,Caroline Smet-Nocca,Armelle Cabin-Flaman,Fabrice Lefebvre,David Gibouin,Paul Wrede

doi:10.1371/journal.pone.0056559

Abstract

Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when 14N isotopes are replaced with 15N. 15N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS). We describe here a novel imaging analysis algorithm to detect a few 15N-enriched proteins - and even a single protein - within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in 15N isotopic fraction due to an enriched protein and a stochastic increase due to the background. To determine the number of enriched proteins responsible for the increase in the isotopic fraction, we use sequential D-SIMS images in which we compare the measured isotopic fractions to those expected if 1, 2 or more enriched proteins are present. The number of enriched proteins is the one that gives the best fit between the measured and the expected values. We used our method to localise 15N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXRα can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXRα as a monomer or dimer and TDG only as a monomer. After 7 h, RXRα was found in the nucleus as a monomer, dimer or tetramer, whilst TDG was no longer in the nucleus and instead formed clusters in the cytoplasm. After 24 h, RXRα formed clusters in the cytoplasm, and TDG was no longer detectable. In conclusion, single unmodified proteins in cells can be counted and localised with 50 nm resolution by combining D-SIMS with our method of analysis.

Highlights

Molecules present in relatively low numbers in the cell often play a disproportionately important role for the phenotype, a phenomenon known as minority control [1]
Volume Sputtered Corresponding to a Pixel In D-Secondary Ion Mass Spectrometry (SIMS) of biological samples, sputtered nitrogen and carbon atoms recombine in the form of CN– secondary ions [11] [16,17,18,19]
The enrichment of macromolecules with one or more rare stable isotopes of its constituent elements, such as 13C and 15N, provides a powerful way to facilitate their detection in Dynamic Secondary Ion Mass Spectrometry (D-SIMS)

Summary

Introduction

Molecules present in relatively low numbers in the cell often play a disproportionately important role for the phenotype, a phenomenon known as minority control [1]. To obtain information on the role of rare macromolecules in biological variability due, for example to stochastic effects, requires single cell analysis [2]. Labelling of target macromolecules may perturb their biological activity; for example, a large fluorescent tag, like GFP, may have steric or other effects that alter the properties of the labelled macromolecule [4,5]. Antibody recognition of a macromolecule may be prevented if the epitope is masked by folding or by interactions between the macromolecule and other molecule, or if the antibody itself is attached to a fluorescent tag [6]. Detection by fluorescence of rare macromolecules may be prevented if the specific signal is masked by, for example, the autofluorescence background

Methods

Results

Conclusion