Recently, we established a novel membrane fusion system in which """"""""flipped"""""""" v- and t-SNAREs needed in exocytosis are expressed on the surface of two cell populations, driving cell-cell fusion thereby demonstrating that SNAREs are sufficient to fuse biological membranes. Here, we propose to capitalize on this development to ask key mechanistic questions about SNARE-dependent fusion, especially questions concerning precisely how regulatory proteins - known to function physiologically - act alone and in concert to control excocytosis at the molecular level. Rigorous studies of these questions require a simplified system of this kind in which protein composition and topology can be controlled in a biologically-relevant environment so that the kinetic effect of each regulator can be assessed when it is added (alone or in combination) to the core fusion machinery of SNAREs. Regulatory proteins will be flipped by adding signal sequences and co-expressed with v- or t-SNAREs on the surface of cells, or added as pure recombinant proteins to the medium. Fusion kinetics and transition states will be measured using established techniques originally developed for viral fusion proteins. We will initially study a well-established group of proteins known to regulate exocytosis in whole cells and organisms: synaptotagmins, Sec/Munc proteins, complexins, and tomosyns, as well as NSF and SNAP. While their general physiologic importance is clear, the molecular mechanism of action - and functional interactions among themselves - are not clear due to the dearth of mechanistic studies in minimal functional fusion systems. The long-term vision is to work our way up - protein by protein - until we can reconstitute the basic properties and fine-tuning of regulated exocytosis. Imbalances in exocytosis and related processes underly major forms of diabetes and obesity, and are likely important in learning, mood, and inflammatory disorders. Knowledge of how the regulators work will likely identify novel targets for intervention.
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| Zhang, Xinming; Rebane, Aleksander A; Ma, Lu et al. (2016) Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex. Proc Natl Acad Sci U S A 113:E8031-E8040 |
| Krishnakumar, Shyam S; Li, Feng; Coleman, Jeff et al. (2015) Re-visiting the trans insertion model for complexin clamping. Elife 4: |
| Cho, Richard W; Kümmel, Daniel; Li, Feng et al. (2014) Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo. Proc Natl Acad Sci U S A 111:10317-22 |
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| Krishnakumar, Shyam S; Kümmel, Daniel; Jones, Sunny J et al. (2013) Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin. Biophys J 105:2507-16 |
| Krishnakumar, Shyam S; Radoff, Daniel T; Kümmel, Daniel et al. (2011) A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion. Nat Struct Mol Biol 18:934-40 |
| Li, Feng; Pincet, Frédéric; Perez, Eric et al. (2011) Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state. Nat Struct Mol Biol 18:941-6 |
| Giraudo, Claudio G; Garcia-Diaz, Alejandro; Eng, William S et al. (2009) Alternative zippering as an on-off switch for SNARE-mediated fusion. Science 323:512-6 |
| Südhof, Thomas C; Rothman, James E (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474-7 |
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