title: Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes.
abstract: Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein "diffusion law" can be obtained directly from imaging, in the form of a mean-square displacement vs. time-delay plot, with no need for interpretative models. This approach is tested with several simulated 2D diffusion conditions and in live Chinese hamster ovary cells with a GFP-tagged transmembrane transferrin receptor, a well-known benchmark of membrane-skeleton-dependent transiently confined diffusion. This approach does not require extraction of the individual trajectories and can be used also with dim and dense molecules. We argue that it represents a powerful tool for the determination of kinetic and thermodynamic parameters over very wide spatial and temporal scales.
authors.affiliation: Center for Nanotechnology Innovation at National Enterprise for nanoScience and nanoTechnology, Istituto Italiano di Tecnologia, 56127 Pisa, Italy.
authors.forename: Carmine
authors.initials: C
authors.lastname: Di Rienzo
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010316:Particle Size
chemical_list: D008565:Membrane Proteins
keywords: fluorescence; membrane heterogeneity; protein dynamics; single molecule; transient confinement