期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 30, 页码 12307-12312出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1222097110
关键词
fluorescence; protein dynamics; membrane heterogeneity; transient confinement; single molecule
资金
- National Institutes of Health (NIH) [P41 P41-RRO3155, NIH P50-GM076516]
- Fondazione Monte dei Paschi di Siena grant
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.
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