Pincet, F;
Adrien, V;
Yang, R;
Delacotte, J;
Rothman, JE;
Urbach, W;
Tareste, D;
(2016)
FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.
PLoS ONE
, 11
(7)
, Article e0158457. 10.1371/journal.pone.0158457.
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Abstract
Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.
| Type: | Article |
|---|---|
| Title: | FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0158457 |
| Publisher version: | https://doi.org/10.1371/journal.pone.0158457 |
| Language: | English |
| Additional information: | Copyright © 2016 Pincet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Experimental Epilepsy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1554518 |
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