Bridging the gap between single molecule and ensemble methods for measuring lateral dynamics in the plasma membrane

PLoS One. 2013 Dec 4;8(12):e78096. doi: 10.1371/journal.pone.0078096. eCollection 2013.

Abstract

The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotin / chemistry
  • Cell Line
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Diffusion
  • Fibroblasts / chemistry
  • Fluorescent Dyes
  • Lipids / chemistry
  • Mice
  • Microscopy, Fluorescence
  • Quantum Dots / chemistry*
  • Spectrum Analysis / methods
  • Staining and Labeling / methods*
  • Streptavidin / chemistry

Substances

  • Fluorescent Dyes
  • Lipids
  • Biotin
  • Streptavidin

Grants and funding

This work was funded by the VillumKann Rasmussen Foundation (to BioNET), Leo Pharma Forskningsfond, the Lundbeck Foundation, the Novo Nordisk Foundation, the Danish Natural Research Foundation (to MEMPHYS-Center for Biomembrane Physics), and the Danish Ministry of Science (to DaMBIC – Danish Molecular Biomedical Imaging Center). PWW acknowledges grant support from the Natural Sciences and Engineering Research Council of Canada (NSERC). JS acknowledges fellowship support from NSERC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.