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Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT
Published on: May 27, 2012
Novel approach to measure the size of plasma-membrane nanodomains in single molecule localization microscopy
Iwona Ziomkiewicz1, Jon Sporring2, Thomas Günther Pomorski1
1Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.
Insights
Researchers used single molecule localization microscopy (SMLM) to analyze glycosylphosphatidylinositol (GPI)-anchor protein nanodomains in plant cells. The domain area (DA) method best estimates nanodomain size, accommodating irregular shapes.
Area of Science:
- Plant cell biology
- Membrane biophysics
- Microscopy
Background:
- Plasma membranes feature protein-rich nanodomains.
- These domains are crucial for cellular functions.
- Lipid composition is thought to define nanodomains.
Purpose of the Study:
- To immunolocalize a GPI-anchor protein in plant cell nanodomains using SMLM.
- To compare three methods for estimating nanodomain size.
- To develop automated analysis for domain size and shape.
Main Methods:
- Single Molecule Localization Microscopy (SMLM) for high-resolution imaging.
- Immunolocalization of a glycosylphosphatidylinositol (GPI)-anchor protein.
- Development of a MatLab program for automated analysis of domain size and shape (FWHM, FWMin, DA).
Main Results:
- GPI-anchor protein nanodomains exhibit approximate elliptical shapes.
- Direct vs. indirect immunolabeling showed significant differences in apparent domain size.
- The domain area (DA) method provided the most accurate size estimation for both regular and irregular nanodomain shapes.
Conclusions:
- The domain area (DA) method is superior for quantifying nanodomain sizes, especially irregular ones.
- Immunolabeling techniques can influence apparent nanodomain size measurements.
- Automated analysis using SMLM is effective for characterizing nanodomain properties.
Abstract:
Many membrane proteins are not evenly distributed over the plasma membrane, but gathered in domains assumed to have a particular lipid composition. Using single molecule localization microscopy (SMLM) we have immunolocalized a glycosylphosphatidylinositol (GPI)-anchor protein that labels nanodomains in a specialized plant cell type, and compared the suitability of three methods to estimate their size. As conventional methods full width at half maximum (FWHM) and the full diameter (FWMin) of domains were used. A boundary detection method of the domain area (DA) was performed in order to take irregular shapes into account. In order to compare the influence of the chosen measurement methods, we have developed a MatLab program that allows for automated analysis of domain sizes from multiple SMLM images and provides the statistics of three key features of domains: FWHM and FWMin along their long and short axes as well as the DA, derived from the molecular density. Domains formed by the GPI-anchor protein are approximating elliptical shapes. Direct and indirect immunolabeling resulted in a statistically significant difference in apparent domain size, reflecting the fact that the secondary antibody molecules extend the uncertainty along the nanodomain border. FWMin values along the long and short axis give good estimates of regular, geometrically centred domain shapes, while the DA value matches regular as well as irregular shapes best, as derived from computer-generated, irregular point clusters.
