laboratoire de physique statistique
 
 
laboratoire de physique statistique

Publications

Rechercher
 
2016
1
Experimental Measurement of the Activation Energy of Phospholipid Membrane Fusion - Francois-Martin, Claire and Rothman, James E. and Pincet, Frederic
BIOPHYSICAL JOURNAL 110520A (2016) 
LPS
Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion - Wang, Yong Jian and Li, Feng and Rodriguez, Nicolas and Lafosse, Xavier and Gourier, Christine and Perez, Eric and Pincet, Frederic
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1133533-3538 (2016) 
LPS


Abstract : Many prominent biological processes are driven by protein assembling between membranes. Understanding the mechanisms then entails determining the assembling pathway of the involved proteins. Because the intermediates are by nature transient and located in the intermembrane space, this determination is generally a very difficult, not to say intractable, problem. Here, by designing a setup with sphere/plane geometry, we have been able to freeze one transient state in which the N-terminal domains of SNARE proteins are assembled. A single camera frame is sufficient to obtain the complete probability of this state with the transmembrane distance. We show that it forms when membranes are 20 nm apart and stabilizes by further assembling of the SNAREs at 8 nm. This setup that fixes the intermembrane distance, and thereby the transient states, while optically probing the level of molecular assembly by Forster resonance energy transfer (FRET) can be used to characterize any other transient transmembrane complexes.
A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion - Xu, Weiming and Nathwani, Bhavik and Lin, Chenxiang and Wan, Jing and Karatekin, Erdem and Pincet, Frederic and Shih, William and Rothman, James E.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 1384439-4447 (2016) 
LPS


Abstract : Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.
Control of plasma membrane lipid homeostasis by the extended synaptotagmins - Saheki, Yasunori and Bian, Xin and Schauder, Curtis M. and Sawaki, Yujin and Surma, Michal A. and Klose, Christian and Pincet, Frederic and Reinisch, Karin M. and De Camilli, Pietro
NATURE CELL BIOLOGY 18504+ (2016) 
LPS


Abstract : Acute metabolic changes in plasma membrane (PM) lipids, such as those mediating signalling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the extended synaptotagmins (E-Syts), endoplasmic reticulum (ER) proteins that function as PtdIns(4,5)P-2- and Ca2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro, and this transfer requires Ca2+ and their lipid-harbouring SMP domain. Genome-edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but exhibit enhanced and sustained accumulation of PM diacylglycerol following PtdIns(4,5)P-2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syt-dependent ER-PM tethers in response to stimuli that cleave PtdIns(4,5)P-2 and elevate Ca2+ may help reverse accumulation of diacylglycerol in the PM by transferring it to the ER for metabolic recycling.
On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets - Molino, D. and Quignard, S. and Gruget, C. and Pincet, F. and Chen, Y. and Piel, M. and Fattaccioli, J.
SCIENTIFIC REPORTS 6 (2016) 
LPS


Abstract : The ability of immune cells to migrate within narrow and crowded spaces is a critical feature involved in various physiological processes from immune response to metastasis. Several in-vitro techniques have been developed so far to study the behaviour of migrating cells, the most recent being based on the fabrication of microchannels within which cells move. To address the question of the mechanical stress a cell is able to produce during the encounter of an obstacle while migrating, we developed a hybrid microchip made of parallel PDMS channels in which oil droplets are sparsely distributed and serve as deformable obstacles. We thus show that cells strongly deform droplets while passing them. Then, we show that the microdevice can be used to study the influence of drugs on migration at the population level. Finally, we describe a quantitative analysis method of the droplet deformation that allows measuring in real-time the mechanical stress exerted by a single cell. The method presented herein thus constitutes a powerful analytical tool for cell migration studies under confinement.
FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments - Pincet, Frederic and Adrien, Vladimir and Yang, Rong and Delacotte, Jerome and Rothman, James E. and Urbach, Wladimir and Tareste, David
PLOS ONE 11 (2016) 
LPS


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 Munc 18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.
Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion - Li, Feng and Tiwari, Neeraj and Rothman, James E. and Pincet, Frederic
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 11310536-10541 (2016) 
LPS


Abstract : Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.
Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex - Zhang, Xinming and Rebane, Aleksander A. and Ma, Lu and Li, Feng and Jiao, Junyi and Qu, Hong and Pincet, Frederic and Rothman, James E. and Zhang, Yongli
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113E8031-E8040 (2016) 
LPS


Abstract : Synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of similar to 17 k(B)T, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.
 
2015
Re-visiting the trans insertion model for complexin clamping - Krishnakumar, Shyam S. and Li, Feng and Coleman, Jeff and Schauder, Curtis M. and Kuemmel, Daniel and Pincet, Frederic and Rothman, James E. and Reinisch, Karin M.
ELIFE 4 (2015) 
LPS


Abstract : We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.
DOI
10
Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock - Motta, Isabelle and Gohlke, Andrea and Adrien, Vladimir and Li, Feng and Gardavot, Helene and Rothman, James E. and Pincet, Frederic
LANGMUIR 317091-7099 (2015) 
LPS


Abstract : Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However, the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes with an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even larger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation. The resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins (To1C and the neuronal t-SNARE), and one lipid-anchored peripheral protein (GABARAP-Like 1 (GL1)). In each case, we verified that the protein remains active after incorporation and in its correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.
DOI
11
The Energy of COPI for Budding Membranes - Thiam, Abdou Rachid and Pincet, Frederic
PLOS ONE 10 (2015) 
LPS


Abstract : As a major actor of cellular trafficking, COPI coat proteins assemble on membranes and locally bend them to bud 60 nm-size coated particles. Budding requires the energy of the coat assembly to overcome the one necessary to deform the membrane which primarily depends on the bending modulus and surface tension, gamma. Using a COPI-induced oil nano-droplet formation approach, we modulated the budding of nanodroplets using various amounts and types of surfactant. We found a Heaviside-like dependence between the budding efficiency and.: budding was only dependent on. and occurred beneath 1.3 mN/m. With the sole contribution of. to the membrane deformation energy, we assessed that COPI supplies similar to 1500 k(B)T for budding particles from membranes, which is consistent with common membrane deformation energies. Our results highlight how a simple remodeling of the composition of membranes could mechanically modulate budding in cells.
DOI
12
Accelerating SNARE-Mediated Membrane Fusion by DNA-Lipid Tethers - Xu, Weiming and Wang, Jing and Rothman, James E. and Pincet, Frederic
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 5414388-14392 (2015) 
LPS


Abstract : SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid-conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA-lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base-pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE-mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA-lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.
 
2014
DOI
13
Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting - Wilfling, Florian and Thiam, Abdou Rachid and Olarte, Maria-Jesus and Wang, Jing and Beck, Rainer and Gould, Travis J. and Allgeyer, Edward S. and Pincet, Frederic and Bewersdorf, Joerg and Farese, Jr., Robert V. and Walther, Tobias C.
ELIFE 3 (2014) 
LPS


Abstract : Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (similar to 60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism.
DOI
14
A Half-Zippered SNARE Complex Represents a Functional Intermediate in Membrane Fusion - Li, Feng and Kuemmel, Daniel and Coleman, Jeff and Reinisch, Karin M. and Rothman, James E. and Pincet, Frederic
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 1363456-3464 (2014) 
LPS


Abstract : SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate fusion by pulling biological membranes together via a zippering mechanism. Recent biophysical studies have shown that t- and v-SNAREs can assemble in multiple stages from the N-termini toward the C-termini. Here we show that functionally, membrane fusion requires a sequential, two-step folding pathway and assign specific and distinct functions for each step. First, the N-terminal domain (NTD) of the v-SNARE docks to the t-SNARE, which leads to a conformational rearrangement into an activated half-zippered SNARE complex. This partially assembled SNARE complex locks the C-terminal (CTD) portion of the t-SNARE into the same structure as in the postfusion 4-helix bundle, thereby creating the binding site for the CTD of the v-SNARE and enabling fusion. Then zippering of the remaining CTD, the membrane-proximal linker (LD), and transmembrane (TMD) domains is required and sufficient to trigger fusion. This intrinsic property of the SNAREs fits well with the action of physiologically vital regulators such as complexin. We also report that NTD assembly is the rate-limiting step. Our findings provide a refined framework for delineating the molecular mechanism of SNARE-mediated membrane fusion and action of regulatory proteins.
DOI
15
Interfacial pressure and phospholipid density at emulsion droplet interface using fluorescence microscopy - Delacotte, Jerome and Gourier, Christine and Pincet, Frederic
COLLOIDS AND SURFACES B-BIOINTERFACES 117545-548 (2014) 
LPS


Abstract : Phospholipids are widely used to stabilize oil in water micron size emulsion droplets; the interfacial phospholipid density and tension of such droplets are difficult to estimate. In the present paper, we describe a simple approach by which the measurement of a micron size oil droplet interface fluorescence intensity provides directly both the interfacial phospholipid density and the interfacial tension. This method relies on two prior calibration steps: (i) the quantitative variation of the interfacial tension with fluorescence intensity at droplets interface through micro-manipulation techniques; (ii) the variation of interfacial tension with phospholipid density through monolayer isotherm. Here, we show the validity of this approach with the example of micron size oil droplets stabilized with a phosphatidylcholine phospholipid, in aqueous buffer. (C) 2013 Elsevier B.V. All rights reserved.
DOI
16
Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins - Zorman, Sylvain and Rebane, Aleksander A. and Ma, Lu and Yang, Guangcan and Molski, Matthew A. and Coleman, Jeff and Pincet, Frederic and Rothman, James E. and Zhang, Yongli
ELIFE 3 (2014) 
LPS


Abstract : Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force dependent half-zippered intermediate and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 k(B)T) and neuronal SNARE complexes (27 k(B)T), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.
DOI
17
Calcium sensitive ring-like oligomers formed by synaptotagmin - Wang, Jing and Bello, Oscar and Auclair, Sarah M. and Wang, Jing and Coleman, Jeff and Pincet, Frederic and Krishnakumar, Shyam S. and Sindelar, Charles V. and Rothman, James E.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 11113966-13971 (2014) 
LPS


Abstract : The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded.
DOI
18
Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization - Chalbi, Myriam and Barraud-Lange, Virginie and Ravaux, Benjamin and Howan, Kevin and Rodriguez, Nicolas and Soule, Pierre and Ndzoudi, Arnaud and Boucheix, Claude and Rubinstein, Eric and Wolf, Jean Philippe and Ziyyat, Ahmed and Perez, Eric and Pincet, Frederic and Gourier, Christine
DEVELOPMENT 1413732-3739 (2014) 
LPS


Abstract : Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.
DOI
19
CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain - Ostuni, Mariano A. and Guellec, Julie and Hermand, Patricia and Durand, Pauline and Combadiere, Christophe and Pincet, Frederic and Deterre, Philippe
BIOLOGY OPEN 31173-1182 (2014) 
LPS


Abstract : The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD) to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain, and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.
 
2013
DOI
20
Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores - Shi, Lei and Howan, Kevin and Shen, Qing-Tao and Wang, Yong Jian and Rothman, James E. and Pincet, Frederic
NATURE PROTOCOLS 8935-948 (2013) 
LPS


Abstract : This protocol describes an assay that uses suspended nanomembranes called nanodiscs to analyze fusion events. A nanodisc is a lipid bilayer wrapped by membrane scaffold proteins. Fluorescent lipids and a protein that is part of a fusion machinery, VAMP2 in the example detailed herein, are included in the nanodiscs. Upon fusion of a nanodisc with a nonfluorescent liposome containing cognate proteins (for instance, the VAMP2 cognate syntaxin1/SNAP-25 complex), the fluorescent lipids are dispersed in the liposome and the increase in fluorescence, initially quenched in the nanodisc, is monitored on a plate reader. Because the scaffold proteins restrain pore expansion, the fusion pore eventually reseals. A reducing agent, such as dithionite, which can quench the fluorescence of accessible lipids, can then be used to determine the number of fusion events. A fluorescence-based approach can also be used to monitor the release of encapsulated cargo. From data on the total cargo release and the number of the much faster lipid- mixing events, the researcher may determine the amount of cargo released per fusion event. This assay requires 3 d for preparation and 4 h for data acquisition and analysis.