How can virtually the same SNARE machine operate at dramatically different speeds depending on context, often far faster than a single SNAREpin? This is one of the central questions driving the field today, and the problem it embodies stands in boldest relief at the neuronal synapse, so it is here that we focus on the structures, biophysics, and physiological properties of the key protein machinery. Our overall hypothesis is that multiple SNAREpins released synchronously, each already close to the point of triggering fusion, co-operate to achieve fusion dramatically faster than any one alone. During the current period of support we discovered that the calcium sensor Synaptotagmin (normally anchored in synaptic vesicle) can self-assemble in vitro into Ca2+- sensitive, ring-like oligomers ~30 nm in diameter and have suggested that such rings forming between the synaptic vesicle (or insulin secretory vesicle) and the plasma membrane would prevent release until they are disrupted by Ca2+. Our specific hypothesis is that such ring oligomers of Synaptotagmin (Syt) are a central organizing principle for exocytosis, enabling the clamping and rapid synchronous release of multiple SNAREpins. This hypothesis is strongly supported by recent experiments in which a targeted mutation (F349A) that de-stabilizes Syt1 rings dramatically increases spontaneous and evoked release and in hippocampal neurons, and dramatically reduces the synchronicity of release with the action potential. We propose to 1) Test the hypothesis that ring-like oligomers of Synaptotagmins regulate exocytosis; 2) Test the hypothesis that Syt1 and Syt7 play distinct structural and functional roles in synchronous and asynchronous release from the same docked vesicles; 3) Elucidate the dynamics and topology of Munc13 and its proposed oligomers and the posited dual roles as vesicle tether and outer ring chaperone templating SNAREpins; and 4) Obtain by single particle cryo-EM and cryo EM tomography high resolution structures of functional release sites in vitro and in situ trapped in defined functional states. Similar machinery mediates neuroendocrine secretory physiology, including pancreatic insulin secretion, so we expect the answers will be highly relevant to the mission of NIDDK. Further, there is little doubt in the post-leptin era of the key role of the nervous system in metabolic balance and diseases.
How can virtually the same SNARE machine operate at dramatically different speeds depending on physiological context? This basic question is key to understanding the regulation of exocytosis and stands in boldest relief at the neuronal synapse where we focus. There is no doubt in the post-leptin era of the key role the nervous system plays in metabolic diseases. For this reason and also because nearly identical machinery regulates neuroendocrine secretions, including insulin, we expect our results to be immediately relevant to the mission of NIDDK.
| Rebane, Aleksander A; Wang, Bigeng; Ma, Lu et al. (2018) Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly. J Mol Biol 430:479-490 |
| Bello, Oscar D; Jouannot, Ouardane; Chaudhuri, Arunima et al. (2018) Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis. Proc Natl Acad Sci U S A 115:E7624-E7631 |
| Rothman, James E; Krishnakumar, Shyam S; Grushin, Kirill et al. (2017) Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission. FEBS Lett 591:3459-3480 |
| Wu, Zhenyong; Bello, Oscar D; Thiyagarajan, Sathish et al. (2017) Dilation of fusion pores by crowding of SNARE proteins. Elife 6: |
| Wang, Jing; Li, Feng; Bello, Oscar D et al. (2017) Circular oligomerization is an intrinsic property of synaptotagmin. Elife 6: |
| Xu, Weiming; Nathwani, Bhavik; Lin, Chenxiang et al. (2016) A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion. J Am Chem Soc 138:4439-47 |
| Bello, Oscar D; Auclair, Sarah M; Rothman, James E et al. (2016) Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores. Langmuir 32:3015-23 |
| Wu, Zhenyong; Auclair, Sarah M; Bello, Oscar et al. (2016) Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains. Sci Rep 6:27287 |
| 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 |
| Yang, Yang; Wang, Jing; Shigematsu, Hideki et al. (2016) Self-assembly of size-controlled liposomes on DNA nanotemplates. Nat Chem 8:476-83 |
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