FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments

RESEARCH ARTICLE FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments Frédéric Pincet1,2*, Vladimir Adrien1,3, Rong Yang4¤, Jérôme Delacotte1, James E. Rothman2, Wladimir Urbach1,5, David Tareste6,7* a11111 1 Laboratoire de Physique Statistique, Ecole Normale Supérieure, CNRS UMR 8550, Université Pierre et Marie Curie, Sorbonne Universités, Paris, France, 2 Department of Cell Biology, School of Medicine, Yale University, New Haven, CT, United States of America, 3 Laboratoire de Cristallographie et RMN Biologiques, CNRS UMR 8015, Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 4 Department of Physiology and Cellular Biophysics, Columbia University, New York, United States of America, 5 UFR Biomédicale, Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 6 Membrane Traffic in Health & Disease, INSERM ERL U950, Université Paris Diderot, Sorbonne Paris Cité, Paris, France, 7 Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris, France ¤ Current address: Adimab LLC, Lebanon, NH, United States of America * (FP); (DT) OPEN ACCESS Citation: Pincet F, Adrien V, Yang R, Delacotte J, Rothman JE, Urbach W, et al. (2016) FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments. PLoS ONE 11(7): e0158457. doi:10.1371/journal.pone.0158457 Editor: Colin Johnson, Oregon State University, UNITED STATES Received: February 20, 2016 Accepted: June 16, 2016 Published: July 7, 2016 Copyright: © 2016 Pincet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the ANR @RATION SynBioExo to J.E.R.; by the ANR Blanc ANR-12-BSV5- 0002 to F.P.; by the ANR Blanc ANR12-BSV8-0010-ASSEMBLY to W.U.; by the ANR Jeunes Chercheurs ANR-09-JCJC-0062-01 and the AFM Trampoline 16799 to D.T.; V.A. is supported by funds from the PhD Program “Frontières du Vivant (FdV) – Cursus Bettencourt”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Rong Yang worked in James Rothman's lab at Columbia 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. Introduction Living cells are highly dynamic multi-compartment systems, whose main constituents (proteins and lipids) are in constant movement within and across compartments. This permanent intracellular motion is notably important for the proper localization and lateral organization of PLOS ONE | DOI:10.1371/journal.pone.0158457 July 7, 2016 1 / 19 FRAP to Characterize Molecular Interaction University as a post-doctoral research scientist from January 2005 to October 2006. Any research work performed by this author during this period was funded by James Rothman’s lab. None of the work related to this manuscript was funded by Rong Yang’s current employer, Adimab, LLC. This current employer did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare that no competing interests exist. The current affiliation of Rong Yang to Adimab, LLC does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. membrane proteins at their site of action. Various model lipidic platforms are now available to reconstitute and study in vitro the distribution and mobility of proteins within the plane of membranes, as well as their interaction with lipids and other (membrane or soluble) proteins [1,2]. These include supported lipid bilayers, giant liposomes and sponge phases (Fig 1) that all have specific advantages and limitations (Table 1). Supported lipid bilayers formed by the Langmuir-Blodgett deposition technique can mimic the asymmetric distribution of lipids between the two leaflets, as found in biological membranes. But the presence of the underlying substrate induces some friction forces, leading to a reduction of lateral diffusion and even the absence of mobility in the case of transmembrane proteins [1]. Alternative methods have been developed to address this problem, including the formation of bilayers on polymer cushions [3] or over holes [4]. This issue can also be overcome with giant liposomes that are free standing, micromanipulable, lipid bilayers. Lipid composition asymmetry is more difficult to recapitulate in this system although some recent double-emulsion and microfluidics approaches have allowed the reconstitution of fully functional transmembrane proteins into asymmetrical giant liposomes [5]. Sponge phases consist of a network of interconnecting model bilayers whose hydrophobic thickness and separating distance can be easily modulated, by adding the appropriate (hydrophobic or aqueous) solvent [6,7]. This system thus provides a powerful tool to follow the mobility of transmembrane proteins, as well as their interactions within or across membranes [8,9]. Fluorescence recovery after photobleaching (FRAP) measurements have been widely used to monitor the mobility and the interaction of fluorescently-labeled biological molecules within living cells as well (...truncated)