TY - JOUR

T1 - Novel approach to measure the size of plasma-membrane nanodomains in single molecule localization microscopy

AU - Ziomkiewicz, Iwona

AU - Sporring, Jon

AU - Pomorski, Thomas Günther

AU - Schulz, A.

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

KW - dSTORM

KW - ENOD-like proteins

KW - GPI-anchor proteins

KW - protein clusters

KW - single molecule localization microscopy

KW - super-resolution microscopy

U2 - 10.1002/cyto.a.22708

DO - 10.1002/cyto.a.22708

M3 - Journal article

C2 - 26109552

VL - 87

SP - 868

EP - 877

JO - Cytometry. Supplement : the journal of the Society for Analytical Cytology

JF - Cytometry. Supplement : the journal of the Society for Analytical Cytology

SN - 1046-7386

IS - 9

ER -