title: Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.
abstract: Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.
authors.affiliation: 1] Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany [2] Centre for Neural Circuits and Behaviour, University of Oxford, Mansfield Road, Oxford OX1 3SR, UK.
authors.forename: Débora M
authors.initials: DM
authors.lastname: Andrade
authors.affiliation: 1] Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany [2] MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, UK [3] MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, Odense M,DK-5230, Denmark.
authors.forename: Mathias P
authors.initials: MP
authors.lastname: Clausen
authors.affiliation: Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Keller
authors.affiliation: Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mueller
authors.affiliation: Department of Cell &Developmental Biology, and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill NC 27599, USA.
authors.forename: Congying
authors.initials: C
authors.lastname: Wu
authors.affiliation: 1] Department of Cell &Developmental Biology, and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill NC 27599, USA [2] Howard Hughes Medical Institute, University of North Carolina, Chapel Hill NC 27599, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Bear
authors.affiliation: Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.affiliation: 1] MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, UK [2] MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, Odense M,DK-5230, Denmark.
authors.forename: B Christoffer
authors.initials: BC
authors.lastname: Lagerholm
authors.affiliation: 1] Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany [2] MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003198:Computer Simulation; D004058:Diffusion; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D055785:Gene Knockdown Techniques; D051379:Mice; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D062211:Spatio-Temporal Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D010743:Phospholipids