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

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 la...

Full description

Saved in:
Bibliographic Details
Published in:PloS one 2013-12, Vol.8 (12), p.e78096
Main Authors: Arnspang, Eva C, Schwartzentruber, Jeremy, Clausen, Mathias P, Wiseman, Paul W, Lagerholm, B Christoffer
Format: Article
Language:English
Subjects:
Animals
Biotin - chemistry
Cell Line
Cell Membrane - chemistry
Cell Membrane - metabolism
Cell membranes
Correlation analysis
Data analysis
Data processing
Datasets
Diffusion
Diffusion rate
Dynamics
Fibroblasts - chemistry
Fluorescence
Fluorescent Dyes
Image processing
Information management
Labeling
Labelling
Lipids
Lipids - chemistry
Macromolecules
Measurement
Measurement methods
Measurement techniques
Methods
Mice
Microscopy, Fluorescence
Pharmacy
Physics
Proteins
Quantum dots
Quantum Dots - chemistry
Spectroscopy
Spectrum Analysis - methods
Staining and Labeling - methods
Stem cells
Streptavidin - chemistry
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0078096