JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: August 24th, 2016

DOI:

10.3791/54349

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Reagents
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
Buffers
HEPES-KOH buffer (pH 7.4) 25 mM HEPES-KOH, 140 mM KCl, 100 μM EGTA, 1 mM DTT
Solvents
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) http://www.hamiltoncompany.com
Glass tubes Pyrex Vista 11 ml, 16x100 mm screw cap culture tube Pyrex  70825-16 clean thoroughly, rinse with chloroform http://catalog2.corning.com/LifeSciences/
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 http://www.avantilipids.com/
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 https://www.thermofisher.com/ 
Rotavapor R-210 Buchi R-210 heat bath above Tm of lipids used http://www.buchi.com/
OG n-Octyl-β-D-Glucopyranoside Affymetrix 0311 store at -20°C, let come to RT before opening https://www.anatrace.com/
Shaker - Eppendorf Thermomixer R Eppendorf https://www.eppendorf.com/
Slide-A-Lyze Dialysis Cassettes, 20K MWCO, 3 mL life technologies 66003 https://www.lifetechnologies.com/
Bio-Beads SM-2 Adsorbents Bio-Rad 1523920 http://www.bio-rad.com/
OptiPrep Density Gradient Medium Sigma-Aldrich D1556 http://www.sigmaaldrich.com/
Ultracentrifugation tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm  Beckman Coulter 41121703 https://www.beckmancoulter.com/
Beckman SW41 Ti rotor
SuflorhodamineB Molecular Probes S-1307 https://www.thermofisher.com/ 
Econo-Column Chromatography Columns, 2.5 × 10 cm Bio-Rad 7372512 http://www.bio-rad.com/
Sepharose CL-4B GE Healthcare 17-0150-01 http://www.gelifesciences.com/
SYPRO Orange Protein Gel Stain Molecular Probes S-6650 5,000X Concentrate in DMSO https://www.lifetechnologies.com/
PDMS block
Sylgard 184 Silicone elastomer kit, PDMS Dow Corning 3097358-1004 http://www.dowcorning.com/
Pyrex glass petri dish, 150 x 20 mm, complete with cover Corning 3160-152 http://catalog2.corning.com/LifeSciences/
Hole puncher - Reusable Biopsy Punch, 0.75mm World Precision Instruments 504529 http://www.wpi-europe.com/
Manual Hole Punching Machine  SYNEO MHPM-UNV http://www.syneoco.com/
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 http://www.coleparmer.com/
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 http://www.us.schott.com/
plasma cleaner Harrick PDC-32G http://harrickplasma.com/
pTIRF setup and accessories
IX81 microscope body Olympus IX81 http://www.olympus-lifescience.com/en/
EM CCD camera Andor ixon-ultra-897 http://www.andor.com/
Thermo Plate, heated microscope stage Tokai Hit MATS-U52RA26 http://www.tokaihit.com/
1 ml hamilton glass syringes (4x) Hamilton 81365 http://www.hamiltoncompany.com
syringe pump kd Scientific KDS-230 http://www.kdscientific.com/

  1. Sudhof, T. C., Rothman, J. E. Membrane fusion: grappling with SNARE and SM proteins. Science. 323, 474-477 (2009).
  2. Wickner, W., Schekman, R. Membrane fusion. Nat Struct Mol Biol. 15, 658-664 (2008).
  3. Harrison, S. C. Viral membrane fusion. Nat Struct Mol Biol. 15, 690-698 (2008).
  4. Jahn, R., Scheller, R. H. SNAREs--engines for membrane fusion. Nat Rev Mol Cell Biol. 7, 631-643 (2006).
  5. Lindau, M., Alvarez de Toledo, G. The fusion pore. Biochim Biophys Acta. 1641, 167-173 (2003).
  6. Staal, R. G., Mosharov, E. V., Sulzer, D. Dopamine neurons release transmitter via a flickering fusion pore. Nat Neurosci. 7, 341-346 (2004).
  7. Wu, Z., et al. Nanodisc-cell fusion: Control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains. Sci. Rep. 6, 27287 (2016).
  8. Alabi, A. A., Tsien, R. W. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Ann Rev Physiol. 75, 393-422 (2013).
  9. Rossetto, O., Pirazzini, M., Montecucco, C. Botulinum neurotoxins: genetic, structural and mechanistic insights. Nature Rev Microbiol. 12, 535-549 (2014).
  10. Weber, T., et al. SNAREpins: minimal machinery for membrane fusion. Cell. 92, 759-772 (1998).
  11. Nickel, W., et al. Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs. Proc Natl Acad Sci U S A. 96, 12571-12576 (1999).
  12. McNew, J. A., et al. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature. 407, 153-159 (2000).
  13. Melia, T. J., You, D. Q., Tareste, D. C., Rothman, J. E. Lipidic antagonists to SNARE-mediated fusion. J Biol Chem. 281, 29597-29605 (2006).
  14. Hernandez, J. M., et al. Membrane fusion intermediates via directional and full assembly of the SNARE complex. Science. 336, 1581-1584 (2012).
  15. Fix, M., et al. Imaging single membrane fusion events mediated by SNARE proteins. Proc Natl Acad Sci U S A. 101, 7311-7316 (2004).
  16. Bowen, M. E., Weninger, K., Brunger, A. T., Chu, S. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). Biophys J. 87, 3569-3584 (2004).
  17. Liu, T., Tucker, W. C., Bhalla, A., Chapman, E. R., Weisshaar, J. C. SNARE-driven, 25-millisecond vesicle fusion in vitro. Biophys J. 89, 2458-2472 (2005).
  18. Yoon, T. Y., Okumus, B., Zhang, F., Shin, Y. K., Ha, T. Multiple intermediates in SNARE-induced membrane fusion. Proc Natl Acad Sci U S A. 103, 19731-19736 (2006).
  19. Diao, J., et al. A single-vesicle content mixing assay for SNARE-mediated membrane fusion. Nat Commun. 1, 1-6 (2010).
  20. Kyoung, M., et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc Natl Acad Sci U S A. 108, E304-E313 (2011).
  21. Domanska, M. K., Kiessling, V., Stein, A., Fasshauer, D., Tamm, L. K. Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J Biol Chem. 284, 32158-32166 (2009).
  22. Kreutzberger, A. J., Kiessling, V., Tamm, L. K. High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion. Biophys J. 109, 319-329 (2015).
  23. Schwenen, L. L., et al. Resolving single membrane fusion events on planar pore-spanning membranes. Sci Rep. 5, 12006 (2015).
  24. Karatekin, E., et al. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc Natl Acad Sci U S A. 107, 3517-3521 (2010).
  25. Karatekin, E., Rothman, J. E. Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells. Nat Protoc. 7, 903-920 (2012).
  26. Smith, M. B., et al. Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys J. 101, 1794-1804 (2011).
  27. Stratton, B. S., et al. Cholesterol Increases the Openness of SNARE-mediated Flickering Fusion Pores. Biophysical journal. 110, (2016).
  28. Diao, J., et al. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. Elife. 1, e00109 (2012).
  29. Kiessling, V., Domanska, M. K., Tamm, L. K. Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM. Biophys J. 99, 4047-4055 (2010).
  30. Blasi, J., et al. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature. 365, 160-163 (1993).
  31. Washbourne, P., et al. Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat Neurosci. 5, 19-26 (2002).
  32. Diaz, A. J., Albertorio, F., Daniel, S., Cremer, P. S. Double cushions preserve transmembrane protein mobility in supported bilayer systems. Langmuir. 24, 6820-6826 (2008).
  33. Floyd, D. L., Ragains, J. R., Skehel, J. J., Harrison, S. C., van Oijen, A. M. Single-particle kinetics of influenza virus membrane fusion. Proc Natl Acad Sci U S A. 105, 15382-15387 (2008).
  34. Albertorio, F., et al. Fluid and air-stable lipopolymer membranes for biosensor applications. Langmuir. 21, 7476-7482 (2005).
  35. Daniel, S., Albertorio, F., Cremer, P. S. Making lipid membranes rough, tough, and ready to hit the road. Mrs Bulletin. 31, 536-540 (2006).
  36. Gao, Y., et al. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science. 337, 1340-1343 (2012).
  37. Kenworthy, A. K., Hristova, K., Needham, D., Mcintosh, T. J. Range and Magnitude of the Steric Pressure between Bilayers Containing Phospholipids with Covalently Attached Poly(Ethylene Glycol). Biophys J. 68, 1921-1936 (1995).
  38. Knoll, W., et al. Solid supported lipid membranes: New concepts for the biomimetic functionalization of solid surfaces. Biointerphases. 3, Fa125-Fa135 (2008).
  39. Quinn, P., Griffiths, G., Warren, G. Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies. J Cell Biol. 98, 2142-2147 (1984).
  40. Sund, S. E., Swanson, J. A., Axelrod, D. Cell membrane orientation visualized by polarized total internal reflection fluorescence. Biophys J. 77, 2266-2283 (1999).
  41. Johnson, D. S., Toledo-Crow, R., Mattheyses, A. L., Simon, S. M. Polarization-controlled TIRFM with focal drift and spatial field intensity correction. Biophys J. 106, 1008-1019 (2014).
  42. Anantharam, A., Onoa, B., Edwards, R. H., Holz, R. W., Axelrod, D. Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM. J Cell Biol. 188, 415-428 (2010).
  43. Axelrod, D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J. 26, 557-573 (1979).
  44. Wang, T., Smith, E. A., Chapman, E. R., Weisshaar, J. C. Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1-5 msec resolution. Biophys J. 96, 4122-4131 (2009).
  45. Chernomordik, L. V., Frolov, V. A., Leikina, E., Bronk, P., Zimmerberg, J. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J Cell Biol. 140, 1369-1382 (1998).
  46. Takamori, S., et al. Molecular anatomy of a trafficking organelle. Cell. 127, 831-846 (2006).
  47. Wilhelm, B. G., et al. Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science. 344, 1023-1028 (2014).
  48. Scott, B. L., et al. Liposome fusion assay to monitor intracellular membrane fusion machines. Methods Enzymol. 372, 274-300 (2003).
  49. Linkert, M., et al. Metadata matters: access to image data in the real world. J Cell Biol. 189, 777-782 (2010).
  50. Soumpasis, D. M. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J. 41, 95-97 (1983).
  51. Ohki, S. A mechanism of divalent ion-induced phosphatidylserine membrane fusion. Biochim Biophys Acta. 689, 1-11 (1982).
  52. Berquand, A., et al. Two-step formation of streptavidin-supported lipid bilayers by PEG-triggered vesicle fusion. Fluorescence and atomic force microscopy characterization. Langmuir. 19, 1700-1707 (2003).
  53. Tamm, L. K., McConnell, H. M. Supported phospholipid bilayers. Biophys J. 47, 105-113 (1985).
  54. Rawle, R. J., van Lengerich, B., Chung, M., Bendix, P. M., Boxer, S. G. Vesicle fusion observed by content transfer across a tethered lipid bilayer. Biophys J. 101, L37-L39 (2011).
  55. Wagner, M. L., Tamm, L. K. Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers. Biophys J. 81, 266-275 (2001).
  56. Kalb, E., Frey, S., Tamm, L. K. Formation of Supported Planar Bilayers by Fusion of Vesicles to Supported Phospholipid Monolayers. Biochimica Et Biophysica Acta. 1103, 307-316 (1992).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved