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SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Published: August 24th, 2016



1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS)

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Membrane fusion is a universal biological process required for intracellular trafficking of lipids and proteins, secretion, fertilization, development, and enveloped virus entry into host organisms1-3. For most intracellular fusion reactions including release of hormones and neurotransmitters via exocytosis, the energy to fuse two lipid bilayers is provided by formation of a four-helix bundle between cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, anchored in the vesicle (v-SNARE) and the target membrane (t-SNARE)4, respectively. Synaptic vesicle exocytosis is the most tightly regulated fus....

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1. Preparation of a PDMS Block to Form the Microfluidic Channel

Figure 1
Figure 1. Microfabrication of flow cell template and PDMS block preparation. (A) Design of a four-channel flow cell that fits onto a 24 x 60 mm glass coverslip (bottom). Six identical designs are arranged to fit onto a 10 cm silicon wafer (top). (B) Cut out block of approximately 5-8 mm thick P.......

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SBL Quality

It is crucial to verify the quality and fluidity of the SBL prior to the fusion experiment. The fluorescence at the bottom, glass side of a microfluidic channel should be uniform, without any obvious defects. If an air bubble passes though the channel, it usually leaves visible scars on the SBL. If there are such large scale scars/defects, do not use that channel. Sometimes SUVs may adhere onto the s.......

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Successful implementation of the SUV-SBL fusion assay described here depends critically on several key steps, such as functional reconstitution of proteins into liposomes, obtaining good quality SBLs, and choosing the right imaging parameters to detect single molecules. Although it may take some time and effort to succeed, once the assay is implemented successfully, it provides a wealth of information about the fusion process not available from any other in vitro fusion assay discussed in Introduction. The rates.......

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We thank Vladimir Polejaev (Yale West Campus Imaging Core) for the design and construction of the polarized TIRF microscope, David Baddeley (Yale University) for help with two-color detection instrumentation, and James E. Rothman (Yale University) and Ben O'Shaughnessy (Columbia University) and members of their groups for stimulating discussions. EK is supported by a Kavli Neuroscience Scholar Award from the Kavli Foundation and NIH grant 1R01GM108954.


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Name Company Catalog Number Comments
Milli-Q (MQ) water Millipore
KOH J.T. Baker 3040-05
Ethanol 190 Proof Decon
Isopropanol Fisher Chemical A416P4
HEPES AmericanBio AB00892
Sodium Cholride (KCl) AB01915
Dithiothreitol AB00490
N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES) AmericanBio AB00892
EGTA Acros Organics 409911000
HEPES-KOH buffer (pH 7.4) 25 mM HEPES-KOH, 140 mM KCl, 100 μM EGTA, 1 mM DTT
Chloroform  J.T. Baker 9180-01 in glass bottle, CAUTION, wear PPE
Methanol J.T. Baker 9070-03 in glass bottle, CAUTION, wear PPE
Liposome preparation 
Gastight Hamilton syringe Hamilton var. sizes only use glass sringe with solents (Chlorophorm/ Methanon, 2:1, v/v)
Glass tubes Pyrex Vista 11 ml, 16x100 mm screw cap culture tube Pyrex  70825-16 clean thoroughly, rinse with chloroform
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 16:0-18:1 PC (POPC) Avanti Polar Lipids 850457 Lipids come dissolved in CHCl3 or as lyphilized powder in sealed vials. Aliquot upon opening. Store extra as dried lipid films under inert atmosphere at -20 °C. Keep stocks in CHCl3/MeOH (2:1, v/v) at -20 °C. let come to RT before opening
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 18:1 PS (DOPS) 840035
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine, 18:0-20:4 PE (SAPE) 850804
L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt), Brain PI(4,5)P2 840046
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), 18:1 NBD PE 810145
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 18:1 PEG2000 PE 880130
cholesterol (ovine wool, >98%) 700000
DiD' oil; DiIC18(5) oil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate) Molecular Probes D-307 
Rotavapor R-210 Buchi R-210 heat bath above Tm of lipids used
OG n-Octyl-β-D-Glucopyranoside Affymetrix 0311 store at -20°C, let come to RT before opening
Shaker - Eppendorf Thermomixer R Eppendorf
Slide-A-Lyze Dialysis Cassettes, 20K MWCO, 3 mL life technologies 66003
Bio-Beads SM-2 Adsorbents Bio-Rad 1523920
OptiPrep Density Gradient Medium Sigma-Aldrich D1556
Ultracentrifugation tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm  Beckman Coulter 41121703
Beckman SW41 Ti rotor
SuflorhodamineB Molecular Probes S-1307 
Econo-Column Chromatography Columns, 2.5 × 10 cm Bio-Rad 7372512
Sepharose CL-4B GE Healthcare 17-0150-01
SYPRO Orange Protein Gel Stain Molecular Probes S-6650 5,000X Concentrate in DMSO
PDMS block
Sylgard 184 Silicone elastomer kit, PDMS Dow Corning 3097358-1004
Pyrex glass petri dish, 150 x 20 mm, complete with cover Corning 3160-152
Hole puncher - Reusable Biopsy Punch, 0.75mm World Precision Instruments 504529
Manual Hole Punching Machine  SYNEO MHPM-UNV
Drill .035 x .026 x 1.5 304 SS TiN coated round punch CR0350265N20R4 drill diameter: 0.9 mm
Tygon Microbore tubing, 0.25 mm ID, 0.76 mm OD Cole-Parmer 06419-00 0.010" ID, 0.030" OD
Silicone Tubing (0.51 mm ID, 2.1 mm OD 95802-00 0.020" ID, 0.083" OD
Cover glass -  cleanroom cleaned
Schott Nexterion cover slip glass D Schott 1472305
plasma cleaner Harrick PDC-32G
pTIRF setup and accessories
IX81 microscope body Olympus IX81
EM CCD camera Andor ixon-ultra-897
Thermo Plate, heated microscope stage Tokai Hit MATS-U52RA26
1 ml hamilton glass syringes (4x) Hamilton 81365
syringe pump kd Scientific KDS-230

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