The list of neurological diseases is extensive: Alzheimer's, Parkinson's, ALS, and MS, to name just a few. The goal of this work is to understand how neurons work at the molecular level. Neurons communicate with each other by releasing neurotransmitters through the plasma membrane (PM) into the synapse in a process called exocytosis. The neurotransmitter is stored in small secretory vesicles (~50 nm diameter) docked at the PM. When a neuron depolarizes, an influx of Ca2+ to the cytosolic side of the PM causes Ca2+- triggered exocytosis in less than 1 ms. Release involves fusion of the lipid bilayer of the vesicle with that of the PM. The identity of many key components of this vesicle fusion machinery is established. However, little is known at the molecular level about how the components work together to carry out exocytosis. A central component is the trans-SNARE complex, comprising the v-SNARE protein synaptobrevin (Syb) anchored in the vesicle and the t-SNARE proteins syntaxin (Syx) and SNAP-25 anchored in the PM. Synaptotagmin (Syt), also anchored in the vesicle, is probably the Ca2+ sensor that evidently triggers fusion by altering binding relationships among proteins and lipids. ? ? The fusion machinery is so complex that mechanistic inferences drawn from in vivo studies are necessarily indirect. We and others have been working to develop a reconstituted model system that faithfully captures the Ca2+-triggered fusion observed in neurons. Such a system would enable very direct study of many key mechanistic questions by adding or subtracting components one by one. We now have a model that allows direct observation of single v-SNARE vesicle docking and fusion on a planar t-SNARE/lipid bilayer in real time by widefield fluorescence microscopy. Ours is the only in vitro system thus far that exhibits SNARE-dependent fusion on a 25-ms time scale, approaching that in nature. The goals of this work are: to determine the intrinsic SNARE-driven rate of fusion by optimizing protein and lipid components; to learn about the nature of the fusion pore itself by measuring the contents release and lipid mixing time scales with 1-3 ms resolution; and to recapture Ca2+ triggering by introducing Syt into the assay. Along the way, these experiments are poised to answer a wide variety of mechanistic questions in an unusually incisive manner. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS051518-02
Application #
7164448
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Talley, Edmund M
Project Start
2006-01-01
Project End
2009-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
2
Fiscal Year
2007
Total Cost
$229,113
Indirect Cost
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Smith, Elizabeth A; Weisshaar, James C (2011) Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay. Biophys J 100:2141-50
Wang, Tingting; Ingram, Colin; Weisshaar, James C (2010) Model lipid bilayer with facile diffusion of lipids and integral membrane proteins. Langmuir 26:11157-64
Wang, Tingting; Smith, Elizabeth A; Chapman, Edwin R et al. (2009) Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1-5 ms resolution. Biophys J 96:4122-31
Yethiraj, Arun; Weisshaar, James C (2007) Why are lipid rafts not observed in vivo? Biophys J 93:3113-9