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.

1. Preparation of a PDMS Block to Form the Microfluidic Channel
<img alt="Figure 1" src="/files/ftp_upload/54349/54349fig1.jpg" /> 
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...

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...

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 fusion reaction and occurs within a millisecond after the arrival of an action potential1,4,5. The fusion pore, the initial connection between the two fusing compartments, can flicker open and closed multiple times before resealing or expanding irreversibly5-7. The former results in transient, &#34;kiss &#38; run&#34; fusion, while the latter leads to full fusion. Factors governing the balance between these two modes of fusion and mechanisms regulating pore flickering are not well understood5,8.
SNARE proteins are required for exocytosis; synaptic vesicle fusion is abolished upon cleavage of SNAREs by neurotoxins9. Bulk fusion experiments using small unilamellar vesicles (SUVs) showed that SNAREs are not only required, but also sufficient to drive membrane fusion10. In this bulk assay, SUVs reconstituted with v-SNAREs (v-SUV) were doped with fluorescent phospholipids (N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-phosphoethanolamine (NBD-PE) and (N-(lissamine rhodamine B sulfonyl)-phosphoethanolamine (LR-PE) and mixed with unlabeled vesicles containing t-SNAREs (t-SUV). Initially the fluorescence of NBD-PE in v-SUVs is quenched by F&#246;rster resonance energy transfer (FRET) to LR-PE. As labeled v-SUVs fuse with unlabeled t-SUVs, the fluorophore surface density in the now combined membrane is reduced and the resulting increase in NBD-PE fluorescence reports the extent of lipid mixing10. As the bulk assay is easy to set up and analyze, it has been widely used to study mechanisms of SNARE-mediated fusion10-14. However, it has several limitations, such as low sensitivity and poor time resolution. Most importantly, as an ensemble measurement, it averages results over all events making discrimination between docking and fusion, as well as detection of hemifusion intermediates difficult.
Over the past decade several groups, including ours, have developed new assays to monitor fusion events at the single vesicle level15-27. Ha and colleagues used v-SUVs tethered onto a surface and monitored their fusion with free t-SUVs18,19. Lipid mixing was monitored using FRET between a pair of lipid-bound fluorophores embedded in the v- and t-SUVs, respectively, using total internal reflection fluorescence (TIRF) microscopy18. Later, Brunger&#39;s laboratory used a single lipid-label species together with a contents marker for simultaneous detection of lipid and contents mixing20,28. Both the lipid and the contents markers were included at high, self-quenching concentrations; fusion with unlabeled SUVs resulted in fluorescence dequenching20,28.
Others have fused v-SUVs to planar bilayers reconstituted with t-SNAREs15-17,21-27,29. The planar geometry of the target (t-SNARE containing) bilayer better mimics the physiological fusion process of small, highly curved vesicles with a flat plasma membrane. The Steinem group employed pore-spanning membranes reconstituted with t-SNAREs, suspended over a porous silicon nitride substrate and detected fusion with individual v-SUVs using confocal laser scanning microscopy23. Others fused v-SUVs to planar bilayers reconstituted with t-SNAREs, supported on a glass substrate15-17,21,22,24-27,29. The great advantage of using supported bilayers (SBLs) is that TIRF microscopy can be used to detect docking and fusion events with excellent signal-to-noise ratio and without interference from free v-SUVs, although using microfluidics also provides single-event resolution using standard far-field epifluorescence microscopy24.
A major concern is whether and how substrate-bilayer interactions affect supported bilayer quality and the fusion process. Early work made use of planar SBLs that were directly supported on a glass or quartz substrate15-17. These SBLs were made by adsorption, bursting, spreading and fusion of t-SUV membranes on the substrate. It was soon realized, however, that omitting a key t-SNARE component, SNAP25, from SBLs prepared in this manner resulted in v-SUV docking and fusion kinetics indistinguishable from those obtained using the complete t-SNAREs17. Because SNAP25 is absolutely required for fusion in vivo30,31, the physiological relevance of these early attempts was put into question. Tamm&#39;s group overcame this challenge by using better controlled supported bilayer formation21. It used Langmuir-Blodgett deposition for the protein-free first leaflet of the SBL, followed by fusion of that monolayer with t-SUVs21. This resulted in SNAP25-dependent fusion.
To avoid potential artefacts associated with a bilayer directly supported on a glass substrate without need to use Langmuir-Blodgett methods, Karatekin et al. introduced a soft, hydrated poly(ethylene glycol) (PEG) cushion between the bilayer and the substrate24. This modification also resulted in SNAP25-dependent fusion24. Bilayers cushioned on a soft polymer layer had been known to better preserve transmembrane protein mobility and function32, and had been used in fusion studies with viruses33. In addition, PEGylated bilayers seem to retain some ability to self-heal and are very robust34,35. First, a fraction of commercially available, lipid-linked PEG chains are included in the t-SUV membrane. When these t-SUVs burst and form a planar bilayer on a glass substrate, a PEG brush covers both leaflets of the planar bilayer. Because planar bilayer formation is driven by adhesion of the PEG chains surrounding t-SUVs onto the hydrophilic glass surface, liposome bursting and planar bilayer formation are relatively insensitive to the lipid composition used. However, when large amounts of cholesterol are included, increasing the cohesive properties of the SUVs, the SUVs may not burst spontaneously. If this is the case, osmotic shock or divalent ions can be employed to help planar bilayer formation25.
As mentioned above, in this approach a PEG brush covers both sides of the planar, supported bilayer. The brush facing the microfluidic flow channel helps to prevent nonspecific adhesion of incoming v-SUVs which are also usually covered with a PEG layer. Formation of v- and t-SNARE complexes starts from the membrane-distal N-termini and proceeds in stages toward the membrane-proximal domains36. For the v-SUVs to interact with the t-SBL, the v- and t-SNARE N-termini need to protrude above the PEG brushes, which seems to be the case under the conditions of the assay. Brush height can be adapted to study proteins other than SNAREs by varying the density of PEGylated lipids and the PEG chain length37,38. Another benefit of the PEG brushes covering the proximal surfaces of the fusing bilayers is that they mimic the crowded environment of biological membranes which are packed with 30,000-40,000 integral membrane proteins per square micron39. Just like the PEG chains in this assay, the repulsive protein layer covering biological membranes needs to be pushed aside to allow for contact between the two phospholipid bilayers for fusion to occur.
Microfluidic flow channels are used in this assay, as they offer unique advantages. First, microfluidic flow enables more uniform deposition of t-SUVs to spread and fuse to form the t-SBL. Second, the small channel volume (&#60; 1 &#181;l) minimizes sample consumption. Third, the small volumes required allow the entire experiment to be conducted under constant flow. Flow removes weakly, presumably non-specifically, adhered v-SUVs from the SBL16. It also maintains a constant density of v-SUVs above the t-SBL, simplifying kinetic analysis17. Finally, docked vesicles are easily distinguished from free ones carried by the flow25. Fourth, several microfluidic channels can be used on the same coverslip, each probing a different condition. This allows comparison of conditions during the same experimental run. A similar approach has been used by the van Oijen group to study fusion between influenza virus and cushioned SBLs33.
In TIRF microscopy, the exponential decay of the evanescent field (with a decay constant ~100 nm) confines fluorescence excitation to those molecules that are in very close proximity of the glass-buffer interface. This minimizes contribution of fluorescent molecules that are further away, increases the signal-to-noise ratio, and allows single molecule sensitivity with frame exposure times of 10-40 msec. The evanescent field also leads to a signal increase upon fusion: as the labeled lipids transfer from the SUV into the SBL, they find themselves, on average, in a stronger excitation field. This increase in fluorescence is stronger for larger liposomes.
If polarized light is used to generate the evanescent field, additional effects contribute to changes in fluorescence upon transfer of labels from the SUV into the SBL. Some lipid dyes have a transition dipole oriented with a preferred mean angle with respect to the bilayer in which they are embedded. This creates a difference in the amount of fluorescence emitted by the fluorophores when they are in the SUV versus the SBL, since the polarized beam will excite dyes in the two membranes differently. For the former, the excitation beam will interact with transition dipoles oriented around the spherical SUV, whereas for the latter, dipole orientations will be confined by the flat SBL geometry. For example, when s-polarized incident light (polarized normal to the plane of incidence) is used, excitation is more efficient when the dye is in the SBL than in the SUV for a lipid dye transition dipole oriented parallel to the membrane29,40 (such as that of DiI or DiD41-43). A SUV doped with such a fluorophore appears dim when it docks onto the SBL (Figure 7, Representative Results). As a fusion pore opens and connects the SUV and SBL membranes, fluorescent probes diffuse into the SBL and become more likely to be excited by the s-polarized evanescent field25,27,29. Consequently, the fluorescence signal integrated around the fusion site increases sharply during dye transfer from the SUV into the SBL27 (Figure 3 and Figure 7). An additional factor that contributes to signal changes that accompany fusion is dequenching of fluorescent labels as they are diluted when transferred into the SBL. The contribution of dequenching is usually minor compared to evanescent field decay and polarization effects in the assay described here, because only a small fraction (<img alt="Equation 1" src="/files/ftp_upload/54349/54349eq1.jpg" />) of the lipids are labeled.
The signal increase upon fusion can be exploited to deduce fusion pore properties by comparing the time, <img alt="Equation 1" src="/files/ftp_upload/54349/54349eq2.jpg" /> , required for a lipid to escape through a pore that is freely permeable to lipids to the actual release time, <img alt="Equation 1" src="/files/ftp_upload/54349/54349eq3.jpg" />. If the two time scales are comparable, it would be concluded that the pore presents little resistance to lipid flow. However, if the actual release time is significantly longer than the time for diffusion-limited release, this would indicate a process, such as pore flickering, retarding lipid release. The diffusion-limited release time, <img alt="Equation 1" src="/files/ftp_upload/54349/54349eq2.jpg" />, depends on the size of the fusing liposome and lipid-diffusivity; its estimation requires these two parameters to be quantified. The single molecule sensitivity of the assay allows lipid diffusivity to be measured by tracking several single lipid fluorophores after their release into the SBL for every fusion event26. The size of every fusing vesicle can be estimated27 by combining (i) the intensity of a single lipid dye, (ii) the change in the total fluorescence around a docking site after all fluorophores are transferred into the SBL upon fusion, (iii) the known labeling density of SUV lipids, and (iv) the area per lipid. For many fusion events, the actual lipid release times were found to be much slower than expected by diffusion-controlled release27, as was noted previously assuming uniform SUV size44. Assuming retardation of lipid release is due to pore flickering, a quantitative model allows estimation of &#34;pore openness&#34;, the fraction of time the pore remains open during fusion27.
Whenever practical, it is important to test fusion mechanisms using both lipid and soluble contents labels. For example, lipid release could be retarded by processes other than pore flickering, such as restriction of lipid diffusion by the SNARE proteins that surround the pore. If this were the case, then release of contents would precede release of lipid labels, provided the pore is large enough to allow passage of soluble probes. A more fundamental flaw in the approach could be in the assumption that the transfer of labeled lipids to the SBL occurs through a narrow fusion pore connecting the SBL to a vesicle that has largely retained its pre-fusion shape. Lipid transfer into the SBL could also result from rapid dilation of the fusion pore with a concomitant, extremely rapid collapse of the SUV into the SBL membrane, as previously suggested based on lipid release data alone29. Monitoring both lipid and contents release simultaneously, it was found that many pores resealed after releasing all their lipid labels, but retained some of their soluble cargo27. This indicates that at least some liposomes do not collapse into the SBL after fusion, and that the lipid dye transfer into the SBL occurs through a fusion pore. In addition, lipid and contents release occurred simultaneously27, making it unlikely that retardation of lipid release was due to hindrance of lipid diffusion by the SNARE proteins surrounding the pore45.
A SUV-SBL fusion protocol that did not monitor soluble contents release was previously published by Karatekin and Rothman25. Here, more recent developments are included, namely simultaneous monitoring of lipid and contents release and estimation of SUV, lipid, and fusion pore properties27. The protocol starts with instructions for preparing the microfluidic cells, made by bonding a poly(dimethyl siloxane) (PDMS) elastomer block containing grooves with a glass coverslip25. Next, preparation of v-SUVs with both lipid and contents markers is explained. Sections 4 and 5 provide instructions for assembling the microfluidic cells, forming the SBLs in situ and checking for defects and fluidity, introduction of v-SUVs into the flow cells and detection of fusion events. Section 6 provides instructions for data analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q (MQ) water</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>J.T. Baker</td>
			<td>3040-05</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 190 Proof</td>
			<td>Decon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemical</td>
			<td>A416P4</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td rowspan="3">AmericanBio</td>
			<td>AB00892</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cholride (KCl)</td>
			<td>AB01915</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>AB00490</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-[2-hydroxyethyl] piperazine-N&#39;-[2-ethanesulfonic acid] (HEPES)</td>
			<td>AmericanBio</td>
			<td>AB00892</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros Organics</td>
			<td>409911000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES-KOH buffer (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>25 mM HEPES-KOH, 140 mM KCl, 100 &#956;M EGTA, 1 mM DTT</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solvents</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform&#160;</td>
			<td>J.T. Baker</td>
			<td>9180-01</td>
			<td>in glass bottle, CAUTION, wear PPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T. Baker</td>
			<td>9070-03</td>
			<td>in glass bottle, CAUTION, wear PPE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liposome preparation&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gastight Hamilton syringe</td>
			<td>Hamilton</td>
			<td>var. sizes</td>
			<td>only use glass sringe with solents (Chlorophorm/ Methanon, 2:1, v/v)</td>
			<td><a href="http://www.hamiltoncompany.com/">http://www.hamiltoncompany.com</a></td>
		</tr>
		<tr>
			<td>Glass tubes Pyrex Vista 11 ml, 16x100 mm screw cap culture tube</td>
			<td>Pyrex&#160;</td>
			<td>70825-16</td>
			<td>clean thoroughly, rinse with chloroform</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/">http://catalog2.corning.com/LifeSciences/</a></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 16:0-18:1 PC (POPC)</td>
			<td rowspan="7">Avanti Polar Lipids</td>
			<td>850457</td>
			<td rowspan="7">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 &#176;C. Keep stocks in CHCl3/MeOH (2:1, v/v) at -20 &#176;C. let come to RT before opening</td>
			<td rowspan="7"><a href="http://www.avantilipids.com/">http://www.avantilipids.com/</a></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 18:1 PS (DOPS)</td>
			<td>840035</td>
		</tr>
		<tr>
			<td>1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine, 18:0-20:4 PE (SAPE)</td>
			<td>850804</td>
		</tr>
		<tr>
			<td>L-&#945;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt), Brain PI(4,5)P2</td>
			<td>840046</td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), 18:1 NBD PE</td>
			<td>810145</td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 18:1 PEG2000 PE</td>
			<td>880130</td>
		</tr>
		<tr>
			<td>cholesterol (ovine wool, &#62;98%)</td>
			<td>700000</td>
		</tr>
		<tr>
			<td>DiD&#39; oil; DiIC18(5) oil (1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-Tetramethylindodicarbocyanine Perchlorate)</td>
			<td>Molecular Probes</td>
			<td>D-307</td>
			<td></td>
			<td><a href="https://www.thermofisher.com/">https://www.thermofisher.com/&#160;</a></td>
		</tr>
		<tr>
			<td>Rotavapor R-210</td>
			<td>Buchi</td>
			<td>R-210</td>
			<td>heat bath above Tm of lipids used</td>
			<td><a href="http://www.buchi.com/">http://www.buchi.com/</a></td>
		</tr>
		<tr>
			<td>OG n-Octyl-&#946;-D-Glucopyranoside</td>
			<td>Affymetrix</td>
			<td>0311</td>
			<td>store at -20&#176;C, let come to RT before opening</td>
			<td><a href="https://www.anatrace.com/">https://www.anatrace.com/</a></td>
		</tr>
		<tr>
			<td>Shaker - Eppendorf Thermomixer R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
			<td><a href="https://www.eppendorf.com/">https://www.eppendorf.com/</a></td>
		</tr>
		<tr>
			<td>Slide-A-Lyze Dialysis Cassettes, 20K MWCO, 3 mL</td>
			<td>life technologies</td>
			<td>66003</td>
			<td></td>
			<td><a href="https://www.lifetechnologies.com/">https://www.lifetechnologies.com/</a></td>
		</tr>
		<tr>
			<td>Bio-Beads SM-2 Adsorbents</td>
			<td>Bio-Rad</td>
			<td>1523920</td>
			<td></td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
		</tr>
		<tr>
			<td>OptiPrep&#160;Density Gradient Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr>
			<td>Ultracentrifugation tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm&#160;</td>
			<td>Beckman Coulter</td>
			<td>41121703</td>
			<td></td>
			<td><a href="https://www.beckmancoulter.com/">https://www.beckmancoulter.com/</a></td>
		</tr>
		<tr>
			<td>Beckman SW41 Ti rotor</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuflorhodamineB</td>
			<td>Molecular Probes</td>
			<td>S-1307</td>
			<td></td>
			<td><a href="https://www.thermofisher.com/">https://www.thermofisher.com/&#160;</a></td>
		</tr>
		<tr>
			<td>Econo-Column&#160;Chromatography Columns, 2.5 &#215; 10 cm</td>
			<td>Bio-Rad</td>
			<td>7372512</td>
			<td></td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
		</tr>
		<tr>
			<td>Sepharose CL-4B</td>
			<td>GE Healthcare</td>
			<td>17-0150-01</td>
			<td></td>
			<td><a href="http://www.gelifesciences.com/">http://www.gelifesciences.com/</a></td>
		</tr>
		<tr>
			<td>SYPRO Orange Protein Gel Stain</td>
			<td>Molecular Probes</td>
			<td>S-6650</td>
			<td>5,000X Concentrate in DMSO</td>
			<td><a href="https://www.lifetechnologies.com/">https://www.lifetechnologies.com/</a></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS block</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone elastomer kit, PDMS</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td></td>
			<td><a href="http://www.dowcorning.com/">http://www.dowcorning.com/</a></td>
		</tr>
		<tr>
			<td>Pyrex glass petri dish, 150 x 20 mm, complete with cover</td>
			<td>Corning</td>
			<td>3160-152</td>
			<td></td>
			<td><a href="http://catalog2.corning.com/LifeSciences/">http://catalog2.corning.com/LifeSciences/</a></td>
		</tr>
		<tr>
			<td>Hole puncher - Reusable Biopsy Punch, 0.75mm</td>
			<td>World Precision Instruments</td>
			<td>504529</td>
			<td></td>
			<td><a href="http://www.wpi-europe.com/">http://www.wpi-europe.com/</a></td>
		</tr>
		<tr>
			<td>Manual Hole Punching Machine&#160;</td>
			<td rowspan="2">SYNEO</td>
			<td>MHPM-UNV</td>
			<td></td>
			<td rowspan="2">http://www.syneoco.com/</td>
		</tr>
		<tr>
			<td>Drill .035 x .026 x 1.5 304 SS TiN coated round punch</td>
			<td>CR0350265N20R4</td>
			<td>drill diameter: 0.9 mm</td>
		</tr>
		<tr>
			<td>Tygon Microbore tubing, 0.25 mm ID, 0.76 mm OD</td>
			<td rowspan="2">Cole-Parmer</td>
			<td>06419-00</td>
			<td>0.010&#34; ID, 0.030&#34; OD</td>
			<td rowspan="2"><a href="http://www.coleparmer.com/">http://www.coleparmer.com/</a></td>
		</tr>
		<tr>
			<td>Silicone Tubing (0.51 mm ID, 2.1 mm OD</td>
			<td>95802-00</td>
			<td>0.020&#34; ID, 0.083&#34; OD</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass -&#160; cleanroom cleaned</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schott Nexterion cover slip glass D</td>
			<td>Schott</td>
			<td>1472305</td>
			<td></td>
			<td><a href="http://www.us.schott.com/">http://www.us.schott.com/</a></td>
		</tr>
		<tr>
			<td>plasma cleaner</td>
			<td>Harrick</td>
			<td>PDC-32G</td>
			<td></td>
			<td><a href="http://harrickplasma.com/">http://harrickplasma.com/</a></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pTIRF setup and accessories</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX81 microscope body</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
			<td><a href="http://www.olympus-lifescience.com/en/">http://www.olympus-lifescience.com/en/</a></td>
		</tr>
		<tr>
			<td>EM CCD camera</td>
			<td>Andor</td>
			<td>ixon-ultra-897</td>
			<td></td>
			<td><a href="http://www.andor.com/">http://www.andor.com/</a></td>
		</tr>
		<tr>
			<td>Thermo Plate, heated microscope stage</td>
			<td>Tokai Hit</td>
			<td>MATS-U52RA26</td>
			<td></td>
			<td><a href="http://www.tokaihit.com/">http://www.tokaihit.com/</a></td>
		</tr>
		<tr>
			<td>1 ml hamilton glass syringes (4x)</td>
			<td>Hamilton</td>
			<td>81365</td>
			<td></td>
			<td><a href="http://www.hamiltoncompany.com/">http://www.hamiltoncompany.com</a></td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>kd Scientific</td>
			<td>KDS-230</td>
			<td></td>
			<td><a href="http://www.kdscientific.com/">http://www.kdscientific.com/</a></td>
		</tr>
	</tbody>
</table>

snare-mediated fusion of single proteoliposomes with tethered supported bilayers in a microfluidic flow cell monitored by polarized tirf microscopy

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, &#34;kiss-and-run&#34; 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 &#34;openness&#34;, 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.

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...

Watch this Scientific Journal Video about SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy at JoVE.co

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

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. ...

The overall goal of this assay is to observe individual membrane fusion events driven by neuronal and exocytotic SNARE proteins to understand fundamental mechanisms of neurotransmitter and hormone release. This method can help answer key questions in the membrane fusion field, such as how cargo release occurs through a dynamic fusion pore, and the fate of the pore after fusion. The main advantage of this technique is that distinct stages of vesicle docking, fusion for opening, and cargo release kinetics can be observed for individual events within biochemically defined environments.Though this method can provide insight into exocytosis, it can also be applied to other membrane-fusion reactions, such as the fusion of envelope viruses with target cell membranes during infection. Generally, individuals new to this method will struggle because many different steps need to be mastered from microfabrication to protein purification and imaging. To begin, prepare a silicone master mold using standard photolithography techniques and the template shown here.Next, add 100 milliliters of PDMS base and 10 milliliters of the curing agent into a disposable plastic cup, and stir the mixture well. Then, place the cup into a vacuum desiccator. Apply a vacuum, but watch it closely to prevent the mixture from overflowing.Once degassed, pour a large drop of the degassed PDMS mixture into a glass petri dish, and press the mold onto the PDMS with the template side facing up to avoid trapping bubbles beneath the wafer. Now, pour degassed PDMS onto the template until it is covered by five to eight millimeters of the polymer. If an air bubble occurs, gently remove it with a pipette tip.Place the PDMS-covered template on the level surface of an oven, and bake it for three hours at 60 degrees Celsius. Then, use a new scalpel blade to cut out a PDMS block containing the molded channel structures. Note that the cut out block needs to fit onto a cover slip.Next, place the side of the channel grooves up, and use a hole-punch to drill through the block in one continuous motion. Be sure to remove the punched-out piece from each of the eight hole locations. When finished, stick the block grooves down onto a clean piece of aluminum foil.Now, cut tubing so that it has a slight slant at one end, and use a pair of tweezers to push the tubing about one-third of the way into the punched holes. Cut the tubes so that they are able to reach the buffer reservoir and the syringe pump. To connect the tubing to the syringes on the syringe pump, cut a short piece from larger silicone tubing, and into one side, insert the thinner tube.This ready-to-use block of PDMS can be kept for a few months. First, place the PDMS block under high vacuum for at least 20 minutes to remove dissolved gases and reduce the risk of air bubbles in the microfluidic channels during the fusion experiment. Next, make sure that the microscope stage area is set to the desired temperature.While the temperature equilibrates, prepare the NBD-PE fluorescently labeled small unilamellar vesicles containing t-SNARE proteins, or protein-free controls, by diluting 30 microliters of the vesicles with 60 microliters of buffer. Then, draw up the solution into a three-milliliter syringe. Press out most of the air above the sample while holding the syringe vertically.Then, seal the tip using paraffin film, and create a vacuum by pulling down the plunger. While pulling a vacuum, tap the syringe barrel to accelerate degassing of the solution. Repeat this process a few times until no more air bubbles occur when the vacuum is applied.Next, use a hypodermic needle with a diameter slightly larger than the microfluidic tubing to punch a hole in the cap of a microcentrifuge tube. Fill the degassed small unilamellar vesicles into the microcentrifuge tube, place it into the holder on the microscope stage, and allow the solution to equilibrate to the set temperature. Place a clean cover slip into a plasma cleaner, and run air plasma over the cover slip for about five minutes.Then, remove the cover slip and place it, treated side up, on top of a few lint-free tissues serving as a cushion. Next, place the PDMS block, patterned side facing down, on top of the cover slip. Use a pair of tweezers to gently press down on the block, but do not press so hard as to break the glass.Place the assembled flow cell onto the microscope stage. Then, connect the tubes to the small unilamellar vesicle reservoirs and to the syringe pump. Start aspirating the small unilamellar vesicles at three microliters per minute until the solution fills the channels completely.When the solutions in all of the channels start moving up the tubing on the exit side, reduce the flow to 0.5 microliters per minute, and incubate for 30 to 45 minutes. The time between plasma-treating the glass cover slip and flowing the SUVs into the channel should not exceed 10 minutes, as the effect of the plasma treatment is transient. Check the channels for any leaks using fluorescence imaging with a low magnification objective, and then, rinse all channels with degassed buffer to wash away unbound vesicles.Then, switch to the high-magnification oil-immersion objective for TIRF microscopy, and secure the flow cell by taping it onto the microscope stage. Close the field's diaphragm to about 40 micrometers in diameter, and adjust the 488-nanometer excitation light intensity using the software in order to bleach the fluorescence in the exposed area significantly, but not completely. The fluorescence intensity in the middle of the exposed area should be lower than at the edges, as the intact NBD-PE molecules enter and diffuse a certain distance before bleaching.However, if the surface-adherent SUVs fail to burst, all the fluorophores in the exposed area should bleach. To verify the results of the study state measurements, stop the illumination and restart it a few minutes later to see if the fluorescence has recovered. Degas the reconstitution buffer, and use it to dilute the v-SNARE reconstituted small unilamellar vesicle stock solution, so that the resultant solution produces 10 to 100 fusion events in the field of view over the course of 60 seconds.Too many fusion events increase the background fluorescence and make detection and analysis of the events difficult. In contrast, a fusion rate that is too low can result in poor statistics. Flow the v-SNARE reconstituted small unilamellar vesicles into the channel at a flow rate of two microliters per minute, and switch to the appropriate excitation emission settings to monitor lipid mixing.Then, adjust the TIRF angle and polarization as described in the accompanying text protocol. Continuously excite and monitor the vesicles'fluorescence using a 561-nanometer laser. As the vesicles reach the flow channel and dock onto and fuse with the supported bilayer, the background fluorescence signal starts to accumulate.Adjust the excitation laser intensity through the software to continuously bleach the background fluorescence, so that new docking and fusion events can readily be observed. Acquire several movies at different positions in a given channel. Check NBD-PE fluorescence to verify that the supported bilayer does not have defects at these positions.The fluorescence recovery after photo bleaching is shown here for a t-SNARE reconstituted supported bilayer that was relatively defect-free and fluid. The area is imaged at low light exposure to follow the fluorescence recovery by diffusion of NBD-labeled lipids. It is easier to observe docking and fusion events while monitoring fluorescence from lipid labels alone.In the sequence of images shown here, a frame was recorded every 18 milliseconds. Upon fusion, the lipid labels transfer into the supported bilayer and diffuse away from the site of fusion. In some cases, it is desirable to monitor lipid mixing and soluble cargo release simultaneously.A representative event describing simultaneous lipid mixing and soluble cargo release is shown here. At the high concentrations used for encapsulation, the soluble content fluorescence depicted in red is initially self-quenched. A sharp increase in lipid fluorescence, depicted in blue, is indicative of lipid mixing due to a fusion event as in the single-color monitoring of lipid labels shown earlier.Pore opening releases soluble content dye and relieves the dye's initial self-quenching in the vesicle lumen. This leads to an increase of the soluble cargo signal, plotted in red. Interestingly, the cargo's fluorescence reached a stable plateau.This indicates that the pore resealed after releasing all lipid labels and a fraction of the soluble cargo. At the end, the soluble content signal, depicted in red, decreases very rapidly. This is likely due to either rapid full fusion or liposome bursting.After watching this video, you should have a good understanding of how to prepare a microfluidic flow cell form a supported bilayer reconstituted with t-SNARE proteins, and monitor and analyze single vesicle fusion events using TIRF microscopy. Thanks for watching, and good luck with your experiments.

snare-mediated-fusion-single-proteoliposomes-with-tethered-supported

Research

JoVE Journal

Neuroscience

10.8K visualizações.  Yale University School of Medicine. The overall goal of this assay is to observe individual membrane fusion events driven by neuronal and exocytotic SNARE proteins to understand fundamental mechanisms of neurotransmitter and hormone release. This method can help answer key questions in the membrane fusion field, such as how cargo release occurs through a dynamic fusion pore, and the fate of the pore after fusion. The main advantage of this technique is that distinct stages of vesicle docking, fusion for opening, and cargo relea...

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