Exocytosis underlies neurotransmitter and hormone release. In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. This process is tightly regulated: release is stimulated by a local increase in the free calcium concentration following the arrival of an action potential. Hormones are released in a similar fashion using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial connection between a SV or SG and the plasma membrane is a small pore (~1 nm wide) that can open and close in succession before either closing permanently (transient, or kiss-and-run fusion) or dilating fully. There is large variabiliy in behavior between cell types (pore open times span ~100 ?s to 10s of s) and within the same cell (some pores flicker, some dilate abruptly). Pore flickering is modulated by physiological inputs such as stimulation strength, with important consequences about what is released (only small cargo can escape through a small pore), on what time course, and how exocytosis is coupled to endocytosis. Despite the fundamental importance of fusion pores in regulating neurotransmitter and hormone release, very little is understood regarding mechanisms controlling pore nucleation and dynamics. This is mainly due to difficulties in studying fusion pores in reconstituted systems with well-defined protein and membrane components that would allow isolating the role of each component. Fusion mediated by exocytotic SNARE proteins and their regulators has been reconstituted and studied for the past 15 years. However, existing methods are not able to resolve single reconstituted fusion pores and follow pore dynamics with sufficient time resolution.
We aim (1) to engineer novel experimental approaches to enable probing mechanisms of nucleation and flickering of exocytotic fusion pores. Combining electrophysiological methods, single-particle fluorescence, microfabricated devices, and artificial bilayer technologies we will develop in vitro assays that allow direct, simultaneous monitoring of single pore flickering and lipid mixing;counting protein numbers and/or probing protein-protein interactions;and controlling membrane curvature and tension. Using these assays, we will then (2) determine factors that govern nucleation and dynamics of SNARE- mediated fusion pores. We will resolve how membrane mechanics and the dynamics of fusion proteins together determine the number of SNARE complexes required for fusion. Further, we will quantify the roles of membrane tension, constraints mimicking the cytoskeleton, curvature, and mutations on pore flickering and expansion. These fundamental studies will advance our understanding of how neurotransmitter and hormone release are regulated, with potential impact on human health in the long term.

Public Health Relevance

Neurotransmitters and hormones are released each time a neurotransmitter- or hormone-filled vesicle fuses with the plasma membrane of a neuron or endocrine cell. The release process is regulated by the speed with which a fusion pore opens and the subsequent evolution of the pore (some pores flicker open and closed repetitively while others open abruptly), but little is understood about molecular mechanisms governing pore nucleation and dynamics. We aim to develop new technology that will enable studying fusion pores that are reconstituted with well-defined membrane and protein components so that the contribution of each component and the overall molecular mechanisms can be understood in detail.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM108954-01
Application #
8615066
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Chin, Jean
Project Start
2014-02-15
Project End
2019-01-31
Budget Start
2014-02-15
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$316,350
Indirect Cost
$126,350
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520