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.

The authors declare that they have no competing financial interests.

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><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Milli-Q (MQ) water</td><td>Millipore</td><td></td><td></td></tr><tr><td>KOH</td><td>J.T. Baker</td><td>3040-05</td><td></td></tr><tr><td>Ethanol 190 Proof</td><td>Decon</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>A416P4</td><td></td></tr><tr><td>HEPES</td><td>AmericanBio</td><td>AB00892</td><td></td></tr><tr><td>Sodium Cholride (KCl)</td><td>AmericanBio</td><td>AB01915</td><td></td></tr><tr><td>Dithiothreitol</td><td>AmericanBio</td><td>AB00490</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-[2-hydroxyethyl] piperazine-&lt;em&gt;N&#039;&lt;/em&gt;-[2-ethanesulfonic acid] (HEPES)</td><td>AmericanBio</td><td>AB00892</td><td></td></tr><tr><td>EGTA</td><td>Acros Organics</td><td>409911000</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Buffers&lt;/strong&gt;</td></tr><tr><td>HEPES-KOH buffer (pH 7.4)</td><td></td><td></td><td>25 mM HEPES-KOH, 140&amp;nbsp;mM KCl, 100&amp;nbsp;&amp;mu;M EGTA, 1&amp;nbsp;mM DTT</td></tr><tr><td colspan="4">&lt;strong&gt;Solvents&lt;/strong&gt;</td></tr><tr><td>Chloroform&amp;nbsp;</td><td>J.T. Baker</td><td>9180-01</td><td>in glass bottle, CAUTION, wear PPE</td></tr><tr><td>Methanol</td><td>J.T. Baker</td><td>9070-03</td><td>in glass bottle, CAUTION, wear PPE</td></tr><tr><td colspan="4">&lt;strong&gt;Liposome preparation&lt;/strong&gt;</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></tr><tr><td>Glass tubes Pyrex Vista 11 ml, 16x100 mm screw cap culture tube</td><td>Pyrex&amp;nbsp;</td><td>70825-16</td><td>clean thoroughly, rinse with chloroform</td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine, 16:0-18:1 PC (POPC)</td><td>Avanti Polar Lipids</td><td>850457</td><td>Lipids come dissolved in CHCl&lt;sub&gt;3&lt;/sub&gt; or as lyphilized powder in sealed vials. Aliquot upon opening. Store extra as dried lipid films under inert atmosphere at -20 &amp;deg;C. Keep stocks in CHCl&lt;sub&gt;3&lt;/sub&gt;/MeOH (2:1, v/v) at -20 &amp;deg;C. let come to RT before opening</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-L-serine (sodium salt), 18:1 PS (DOPS)</td><td>Avanti Polar Lipids</td><td>840035</td><td>Same as above.</td></tr><tr><td>1-stearoyl-2-arachidonoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine, 18:0-20:4 PE (SAPE)</td><td>Avanti Polar Lipids</td><td>850804</td><td>Same as above.</td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt), Brain PI(4,5)P&lt;sub&gt;2&lt;/sub&gt;</td><td>Avanti Polar Lipids</td><td>840046</td><td>Same as above.</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), 18:1 NBD PE</td><td>Avanti Polar Lipids</td><td>810145</td><td>Same as above.</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-[methoxy(polyethylene glycol)-2000] (ammonium salt), 18:1 PEG2000 PE</td><td>Avanti Polar Lipids</td><td>880130</td><td>Same as above.</td></tr><tr><td>cholesterol (ovine wool, &gt;98%)</td><td>Avanti Polar Lipids</td><td>700000</td><td>Same as above.</td></tr><tr><td>DiD&#039; oil; DiIC18(5) oil (1,1&#039;-Dioctadecyl-3,3,3&#039;,3&#039;-Tetramethylindodicarbocyanine Perchlorate)</td><td>Molecular Probes</td><td>D-307</td><td></td></tr><tr><td>Rotavapor R-210</td><td>Buchi</td><td>R-210</td><td>heat bath above Tm of lipids used</td></tr><tr><td>OG n-Octyl-&amp;beta;-D-Glucopyranoside</td><td>Affymetrix</td><td>0311</td><td>store at -20 &amp;deg;C, let come to RT before opening</td></tr><tr><td>Shaker - Eppendorf Thermomixer R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Slide-A-Lyze Dialysis Cassettes, 20k MWCO, 3&amp;nbsp;ml</td><td>life technologies</td><td>66003</td><td></td></tr><tr><td>Bio-Beads SM-2 Adsorbents</td><td>Bio-Rad</td><td>1523920</td><td></td></tr><tr><td>OptiPrep&amp;nbsp;Density Gradient Medium</td><td>Sigma-Aldrich</td><td>D1556</td><td></td></tr><tr><td>Ultracentrifugation tube, Thinwall, Ultra-Clear, 13.2&amp;nbsp;ml, 14 x 89 mm</td><td>Beckman Coulter</td><td>41121703</td><td></td></tr><tr><td>Beckman SW41 Ti rotor</td><td></td><td></td><td></td></tr><tr><td>SuflorhodamineB</td><td>Molecular Probes</td><td>S-1307</td><td></td></tr><tr><td>Econo-Column&amp;nbsp;Chromatography Columns, 2.5 &amp;times; 10 cm</td><td>Bio-Rad</td><td>7372512</td><td></td></tr><tr><td>Sepharose CL-4B</td><td>GE Healthcare</td><td>17-0150-01</td><td></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></tr><tr><td colspan="4">&lt;strong&gt;PDMS block&lt;/strong&gt;</td></tr><tr><td>Sylgard 184 Silicone elastomer kit, PDMS</td><td>Dow Corning</td><td>3097358-1004</td><td></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></tr><tr><td>Hole puncher - Reusable Biopsy Punch, 0.75&amp;nbsp;mm</td><td>World Precision Instruments</td><td>504529</td><td></td></tr><tr><td>Manual Hole Punching Machine&amp;nbsp;</td><td>SYNEO</td><td>MHPM-UNV</td><td></td></tr><tr><td>Drill .035 x .026 x 1.5 304 SS TiN coated round punch</td><td>SYNEO</td><td>CR0350265N20R4</td><td>drill diameter: 0.9 mm, punch bore size: 0.74 mm</td></tr><tr><td>Tygon Microbore tubing, 0.25 mm ID, 0.76 mm OD</td><td>Cole-Parmer</td><td>06419-00</td><td>0.010&quot; ID, 0.030&quot; OD</td></tr><tr><td>Silicone Tubing (0.51 mm ID, 2.1 mm OD</td><td>Cole-Parmer</td><td>95802-00</td><td>0.020&quot; ID, 0.083&quot; OD</td></tr><tr><td colspan="4">&lt;strong&gt;Cover glass - cleanroom cleaned&lt;/strong&gt;</td></tr><tr><td>Schott Nexterion cover slip glass D</td><td>Schott</td><td>1472305</td><td></td></tr><tr><td>plasma cleaner</td><td>Harrick</td><td>PDC-32G</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;pTIRF setup and accessories&lt;/strong&gt;</td></tr><tr><td>IX81 microscope body</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>EM CCD camera</td><td>Andor</td><td>ixon-ultra-897</td><td></td></tr><tr><td>Thermo Plate, heated microscope stage</td><td>Tokai Hit</td><td>MATS-U52RA26</td><td></td></tr><tr><td>1 ml hamilton glass syringes (4x)</td><td>Hamilton</td><td>81365</td><td></td></tr><tr><td>syringe pump</td><td>kd Scientific</td><td>KDS-230</td><td></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. ...

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

Research

JoVE Journal

Neuroscience

10.8K Views.  Yale University School of Medicine.  Hier presenteren we een protocol om enkele, SNARE-bemiddelde fusie gebeurtenissen tussen liposomen en ondersteund dubbellagen in microfluïdische kanalen met gepolariseerde TIRFM, met 'single molecule gevoeligheid en ~ 15 msec tijdsresolutie te detecteren. Lipide en oplosbare lading afgifte kan gelijktijdig worden gedetecteerd. Liposoom grootte, lipide diffusie, en fusie porie eigenschappen worden gemeten. 

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

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Biochemistry

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Biology

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Bioengineering

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Immunology and Infection

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://www.jove.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...

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

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