Exocytosis; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Molecular Biology; Physiology; Secretory Vesicles; Microfluidics
Neurotransmitters and hormones are released when a cargo-filled vesicle fuses with the plasma membrane. This requires trafficking and docking of the vesicle to the release site, priming of the vesicle to acquire fusion-competence, and a trigger signal in the form of an increase in calcium concentration (elicited by calcium influx through voltage-gated calcium channels that open in response to a depolarization). The fusion step is driven by formation of a complex between vesicular v-SNARE proteins and target membrane t-SNAREs. Other proteins are also essential. Synaptotagmin couples calcium entry to fusion with astonishing speed, complexin regulates both spontaneous and evoked release, Mun13 primes vesicles, Munc18 chaperones SNARE assembly, and other proteins dissociate post-fusion SNARE complexes for recycling.
The initial, nanometer-sized pore that opens is also subject to regulation. High resolution electrophysiological and electrochemical measurements found that the pore may fluctuate in size, and can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Mechanisms governing pore dynamics are not understood.
We have developed novel in vitro assays with biochemically defined components to study the exocytotic fusion process with single pore, or even single molecule sensitivity, and sub-ms to ~10 ms time resolution. We found pores are more stable than previously appreciated, and pore lifetimes are very sensitive to mutations in the SNARE transmembrane domains. We also found only a few SNARE complexes are required for opening a pore, but the subsequent pore dilation requires many more SNAREs. We are currently exploring how synaptotagmin couples calcium binding to fusion.
Specialized Terms: Membrane fusion; Exocytosis; Secretory vesicle dynamics; Fluorescence microscopy; Image analysis; Microfluidics; Supported bilayers; Proteoliposomes
- J. Nikolaus*, E. Karatekin*, “SNARE-mediated fusion of single proteoliposomes with tethered supported bilayers in a microfluidic flow cell monitored by polarized TIRF microscopy”, J. Vis. Exp., e54349, doi:10.3791/54349 (2016).
- W. Xu, B. Nathwani, C. Lin, J. Wang, E. Karatekin, F. Pincet, W. Shih*, J. E. Rothman*, “A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion”, J. Am.Chem. Soc., 138,4439-4447 (2016).
- Z. Wu, S. M. Auclair, O. Bello, W. Vennekate, N. Dudzinski, S. Krishnakumar, and E. Karatekin*, “Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains”, Sci. Rep., 6, 27287; doi: 10.1038/srep27287 (2016).
- B. S. Stratton, Z. Wu, J. M. Warner, G. Wei, E. Karatekin*, and Ben O'Shaughnessy*, “Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores” Biophys. J. 110, 1538–1550 (2016).
- M. R. Stachowiak, C. Laplante, H. F. Chin, B. Guirao, E. Karatekin, T. D. Pollard, and Ben O'Shaughnessy*, “Mechanism of Cytokinetic Contractile Ring Constriction in Fission Yeast”, Dev. Cell, 29, 547–561, 2014.
- T. Doan, J. Coleman, K. A. Marquis, B.M. Burton, E. Karatekin*, and D. Z. Rudner*, “FisB mediates the final membrane fission event during sporulation in Bacillus Subtilis”, Genes Dev., 27, 322-334, 2013.
- Karatekin E and Rothman JE. Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells. Nature Protocols, 7:903-920, 2012.
- Smith MB, Karatekin E, Gohlke A, Mizuno H, Watanabe N, and Vavylonis D. Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys. J. 101:1794-1804, 2011.
- Meunier A, Jouannot O, Fulcrand R, Fanget I, Bretou M, Karatekin E, Arbault S, Guille M, Darchen F, Lemaître F, and Amatore C. Coupling amperometry and total internal reflection fluorescence microscopy at ITO surfaces for monitoring exocytosis of single vesicles. Angew. Chem. Int. Ed., 50:5081-5084, 2011.
- Karatekin E, Di Giovanni J, Iborra C, Coleman J, O'Shaughnessy B, Seagar M, and Rothman JE. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc. Natl. Acad. Sci. USA. 107:3517–3521, 2010.
- Warner JM, Karatekin E, and O'Shaughnessy B. Model of SNARE-mediated membrane adhesion kinetics. PLoS One 4(8):e6375, 2009.
- Karatekin E, Tran S, Huet S, Fanget I, Cribier S, and Henry JP. A 20 nm step toward the cell membrane preceding exocytosis may correspond to docking of tethered granules. Biophys. J. 94:2891-2905, 2008.
- Tran VS, Huet S, Fanget I, Cribier S, Henry JP, and Karatekin E. Characterization of sequential exocytosis in a human neuroendocrine cell line using evanescent wave microscopy and `virtual trajectory' analysis. Eur. Biophys. J. 37:55-69, 2007.