Exocytosis; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Molecular Biology; Physiology; Secretory Vesicles; Microfluidics
We are studying membrane fusion, a fundamental process required for trafficking of proteins in the cell, as well as secretion of physiological mediators like hormones and neurotransmitters.
A protein complex named the the SNARE complex is a key and common component of many types of fusion reactions in the cell. However, how the SNARE proteins drive membrane fusion, or how other proteins regulate this process is not well understood. We have reconstituted SNARE proteins into artificial, model membranes in order to study their function under well-controlled conditions, without interference from other components found in the complex intracellular environment.
The model membranes allow us to visualize single vesicles docking to and fusing with planar membranes resting on a support, a geometry that mimics the fusion of synaptic vesicles with the plasma membrane. By controlling different parameters and adding/removing components, we are learning about their contributions. For example, we have measured the minimum number of SNARE complexes required for fusion.
Specialized Terms: Membrane fusion; Exocytosis; Secretory vesicle dynamics; Fluorescence microscopy; Image analysis; Microfluidics; Supported bilayers; Proteoliposomes
Extensive Research Description
SNARE proteins constitute the core of the eukaryotic fusion machinery, yet
how SNAREs accomplish fusion and the role of regulatory proteins remain
unsettled. Novel in vitro fusion assays which can detect single docking and
fusion events have great potential for unraveling mechanistic details, but have
been suffering from reproducibility and reliability problems. In addition,
almost all past in vitro work used small vesicles (SUVs, diameters ~50 nm) which
may be good mimics for the smallest organelles such as synaptic vesicles, but
are not likely to be good models for fusion reactions involving large organelles
such as yeast vacuoles. For the fusion of large membranes, membrane undulations
and large numbers of SNARE proteins may be involved, making the adhesion/fusion
process qualitatively different from the case for small vesicles for which
undulations are lacking and only a few complexes can drive fusion.
I have been developing novel in vitro fusion assays involving three types of membrane structure: supported bilayers (SBLs) which are "infinitely" large membranes which lack undulations due to their interactions with an underlying substrate, SUVs, and giant unilamellar vesicles (GUVs, diameters ~10-50 microns) which provide freely suspended, large membranes. Various combinations of these three types of membranes reconstituted with SNARE proteins allow us to obtain complementary information and to mimic the vast length and time scales of fusion found in nature.
This work was initiated when I was at the Institut de Biologie Physico-Chimique, CNRS FRE 3146, Paris, France, in collaboration with the group of Michael Seagar at Université de la Mediterranée-Aix Marseille 2 and INSERM U641, Marseilles, France. I am currently on leave from the CNRS to continue the work here. On theoretical aspects, we collaborate with Prof. Ben O'Shaughnessy and Jason Warner at Columbia University.
- Z. Wu, S. M. Auclair, O. Bello, W. Vennekate, S. Krishnakumar, and E. Karatekin*, “Control of fusion pore nucleation and dynamics by SNARE protein transmembrane domains”, submitted.
- B. S. Stratton, Z. Wu, J. M. Warner, G. Wei, E. Karatekin*, and Ben O'Shaughnessy*, “SNARE-Mediated Fusion Pore Dynamics from Quantitative TIRF Microscopy” submitted.
- 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.