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PLoS One. 2016 Jul 7;11(7):e0158457. doi: 10.1371/journal.pone.0158457. eCollection 2016.

FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.

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Laboratoire de Physique Statistique, Ecole Normale Supérieure, CNRS UMR 8550, Université Pierre et Marie Curie, Sorbonne Universités, Paris, France.
Department of Cell Biology, School of Medicine, Yale University, New Haven, CT, United States of America.
Laboratoire de Cristallographie et RMN Biologiques, CNRS UMR 8015, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Department of Physiology and Cellular Biophysics, Columbia University, New York, United States of America.
UFR Biomédicale, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Membrane Traffic in Health & Disease, INSERM ERL U950, Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris, France.


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.

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