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Mol Biol Cell. 2017 Jun 1;28(11):1507-1518. doi: 10.1091/mbc.E16-07-0536. Epub 2017 Apr 12.

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS.

Author information

1
MRC Human Immunology Unit, University of Oxford, Oxford OX39DS, United Kingdom.
2
Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX39DS, United Kingdom.
3
MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark.
4
Izmir International Biomedicine and Genome Institute, Dokuz Eylul University Medical School, Inciralti-Balcova, 35340 Izmir, Turkey.
5
Department of Medical Biology and Genetics, Dokuz Eylul University Medical School, Inciralti-Balcova, 35340 Izmir, Turkey.
6
MRC Human Immunology Unit, University of Oxford, Oxford OX39DS, United Kingdom erdinc.sezgin@rdm.ox.ac.uk christian.eggeling@rdm.ox.ac.uk.

Abstract

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signaling and are suggested to be strongly associated with the actin cytoskeleton. Here we use superresolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live-cell plasma membrane and in actin cytoskeleton-free, cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids is abolished in the GPMVs, whereas transient nanodomain incorporation of ganglioside lipid GM1 is apparent in both the live-cell membrane and GPMVs. For GPI-APs, we detect two molecular pools in living cells; one pool shows high mobility with transient incorporation into nanodomains, and the other pool forms immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules and highlight a powerful experimental approach to decipher specific influences on molecular plasma membrane dynamics.

PMID:
28404749
PMCID:
PMC5449149
DOI:
10.1091/mbc.E16-07-0536
[Indexed for MEDLINE]
Free PMC Article

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