Studying lateral mobility of surface molecules on the plasma membrane using biophysical approaches Public Deposited

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  • March 22, 2019
Creator
  • Chen, Yun
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
Abstract
  • Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are common biophysical methods to measure lateral mobility of membrane molecules. These methods typically sample lateral mobility in micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. FRAP methodologies are described for a dedicated wide field microscope, which can be applied to confocval-based FRAP too. SPT can be applied to track single fluorescent molecules in membranes but this aspect will not be treated in detail. SPT has been a useful tool to study the micro-structure of membranes on various time scales. Using SPT, we found that by deliberately cross-linking several glycosyl-phosphatidylinositol-anchored proteins under antibody conjugated 40-nm gold particles, transient anchorage of the gold-labeled clusters occurred for periods ranging from 300 ms to 10 seconds. This phenomenon was observed with two different GPI-anchored proteins: Thy-1 and CD73, a 5' exonucleotidase. Cholesterol depletion and Src family kinase (SFK) inhibition abolished transient anchorage, indicating the involvement of cholesterol and SFKs in cross-linking dependent transient anchorage. Further evidence for the involvement of SFKs was obtained from Src, Yes, Fyn defective cells, in which transient anchorage of CD73 was not detected. Caveolin-1 knockout cells exhibited reduced transient anchorage time, suggesting the partial participation of caveolin-1. Additionally, in PI3 kinase inhibited IMR 90 cells expressing CD73, short-period transient anchorage (< 1 second) increased sharply and no long-period anchorage occurred. By contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended on interaction of its PDZ binding domain with the adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol dependent, SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.
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  • In Copyright
Advisor
  • Jacobson, Ken
Degree granting institution
  • University of North Carolina at Chapel Hill
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