Functional Insights into Membrane Bound α-Synuclein: Surface Density, Conformation, and Localization inside Cells

Research output: ThesisPhD Thesis - Research UT, graduation UT

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Abstract

Alpha-synuclein (αS), an intrinsically disordered protein, is thought to be the major player in synucleinopathies such as Parkinson’s disease. The exact function of αS is not known. In vitro experiments show that αS is disordered in buffer and that in the presence of model lipid membranes it can adopt alpha-helical structure. In cells αS has been shown to colocalize with membranes. Taking into account the membrane bound structure found in vitro, one would expect that αS adopts alpha-helical structure on cellular lipid bilayers. In contrast, NMR and EPR studies indicate that αS remains disordered inside the cell.
We address this controversy. We showed that cellular αS is associated with membranes inside neuron like cells. We used ultrasensitive microscopy and photobleaching to quantify the number of αS-GFP on vesicles. We found a number of 70 αS-GFP/vesicle which is an extraordinary high number. To identify if this membrane bound αS adopts α-helical structures, we microinjected cells with small amounts of αS labelled with a FRET pair sensitive to the membrane bound conformation (Fig. 1 top). We use the FRET signal as a readout for protein conformation and observed two significant different conformations of αS in cells, one in the cytoplasm and the other on cellular vesicles (Fig. 1 middle and bottom panels). This clearly shows that there are at least two structurally distinct subensembles of αS inside cells: 1) a disordered form in the cytosol, 2) a membrane associated form. Our data shows that the disordered nature of monomeric αS is not fully preserved in cells.
Original languageEnglish
Awarding Institution
  • University of Twente
Supervisors/Advisors
  • Claessens, Mireille M.A.E., Supervisor
  • Blum, Christian , Co-Supervisor
Award date7 Nov 2018
Place of PublicationEnschede
Publisher
Print ISBNs978-94-632-3367-5
DOIs
Publication statusPublished - 7 Nov 2018

Keywords

  • Synuclein
  • Disordered protein
  • Membrane
  • Counting
  • FRET
  • Single molecule
  • Endocytosis

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