The principle long-term objective of this project is to provide a detailed biophysical and molecular understanding of exocytotic vesicle fusion and transmitter release in endocrine cells and nerve terminals. Upon electrical stimulation nerve terminals and endocrine cells release a variety of neurotransmitters and neuropeptides by an exocytotic mechanism. This process of neurosecretion is of outstanding importance in a broad range of physiological processes. A detailed understanding of the exocytotic event is thus of substantial interest, in particular for those diseases where neurosecretion is impaired. In exocytosis the vesicles dock at the plasma membrane and fuse with the plasma membrane allowing for release of the vesicle contents through the fusion pore. It is proposed to study individual fusion events and individual docking events using biophysical techniques. Simultaneous measurements of fusion and release will be performed by """"""""patch amperometry"""""""", a method which allows simultaneous recording of small capacitance changes and amperometric detection of released transmitter molecules. Experiments in """"""""cell-attached"""""""" patched provide information on the requirements of cytosolic conditions and components. To investigate the docking process which is thought to precede fusion in vitro, the applicant will develop a novel approach using an optical trap. They will characterize the physicochemical mechanisms determining docking and transmitter release. These methods will be complemented with biochemical techniques which will be used to characterize the role of specific fusion pore formation (the kiss-and-run mechanism) or alternatively by full fusion followed by conventional endocytosis via coated pits. The long-term aim is to obtain an understanding of the molecular components directly involved in fusion pore formation. Demonstration that the fusion pore in a reconstituted system resembles the fusion poreforming during exocytosis will eventually be of a significance analogous to that demonstrating that specific membrane proteins reconstituted in lipid membranes resemble the properties of specific ion channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS038200-02
Application #
6394048
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Talley, Edmund M
Project Start
2000-04-20
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
2
Fiscal Year
2001
Total Cost
$267,152
Indirect Cost
Name
Cornell University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Zhao, Ying; Fang, Qinghua; Straub, Susanne G et al. (2016) Prostaglandin E1 inhibits endocytosis in the ?-cell endocytosis. J Endocrinol 229:287-94
Kisler, Kassandra; Kim, Brian N; Liu, Xin et al. (2012) Transparent Electrode Materials for Simultaneous Amperometric Detection of Exocytosis and Fluorescence Microscopy. J Biomater Nanobiotechnol 3:243-253
Zhao, Ying; Fang, Qinghua; Straub, Susanne G et al. (2010) Hormonal inhibition of endocytosis: novel roles for noradrenaline and G protein G(z). J Physiol 588:3499-509
Zhao, Ying; Fang, Qinghua; Straub, Susanne G et al. (2010) Noradrenaline inhibits exocytosis via the G protein ýýýý subunit and refilling of the readily releasable granule pool via the ýý(i1/2) subunit. J Physiol 588:3485-98
Ngatchou, Annita N; Kisler, Kassandra; Fang, Qinghua et al. (2010) Role of the synaptobrevin C terminus in fusion pore formation. Proc Natl Acad Sci U S A 107:18463-8
Berberian, Khajak; Torres, Alexis J; Fang, Qinghua et al. (2009) F-actin and myosin II accelerate catecholamine release from chromaffin granules. J Neurosci 29:863-70
Fang, Qinghua; Berberian, Khajak; Gong, Liang-Wei et al. (2008) The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics. Proc Natl Acad Sci U S A 105:15388-92
Valero, Vicente; Nevian, Thomas; Ho, Dominik et al. (2008) Tethering forces of secretory granules measured with optical tweezers. Biophys J 95:4972-8
Gong, Liang-Wei; de Toledo, Guillermo Alvarez; Lindau, Manfred (2007) Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore. Nat Cell Biol 9:915-22
Ayers, Sunitha; Gillis, Kevin D; Lindau, Manfred et al. (2007) Design of a CMOS Potentiostat Circuit for Electrochemical Detector Arrays. IEEE Trans Circuits Syst I Regul Pap 54:736-744

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