title: Novel approach to measure the size of plasma-membrane nanodomains in single molecule localization microscopy.
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.
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.
authors.forename: Iwona
authors.initials: I
authors.lastname: Ziomkiewicz
authors.affiliation: Department of Computer Science, University of Copenhagen, Denmark.
authors.forename: Jon
authors.initials: J
authors.lastname: Sporring
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.
authors.forename: Thomas Günther
authors.initials: TG
authors.lastname: Pomorski
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schulz
mesh_terms: D001937:Brassica / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding / Q000502:physiology
keywords: ENOD-like proteins; GPI-anchor proteins; dSTORM; protein clusters; single molecule localization microscopy; super-resolution microscopy