Fusion and fission of membrane-bounded cells and organelles are fundamental events governing the compartmentalization of biological space. It is proposed to use video microscopy and an ultra-sensitive, electrical assay of the cell surface to study, in living cells, the mechanism of membrane fusion during exocytosis of single secretory vesicles. In particular we will explore a novel hypothesis: that the first step in membrane fusion is the formation of an ion channel (the fusion pore) that connects the inside of the vesicle to the cell exterior much like a gap junction would connect two adjacent cells, and that subsequent event in exocytosis are a consequence of electrolyte fluxes through the fusion pore. Patch clamp studies of single secretory vesicles will reveal the type of ion channels present in the vesicle membrane. We will also explore """"""""fusion pores"""""""" possibly formed by virus fusion proteins stably expressed in a fibroblast-derived cell line, in order to test whether there are similarities between fusion events induced by viruses and fusion events during exocytosis. Lastly, we plan to explore the role of cytosolic (Ca++) and other cytoplasmic messengers in the control of pinocytosis and membrane turnover.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039520-03
Application #
3296585
Study Section
Physiology Study Section (PHY)
Project Start
1988-02-01
Project End
1993-01-31
Budget Start
1990-02-01
Budget End
1991-01-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Tse, F W; Iwata, A; Almers, W (1993) Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion. J Cell Biol 121:543-52
Almers, W; Breckenridge, L J; Iwata, A et al. (1991) Millisecond studies of single membrane fusion events. Ann N Y Acad Sci 635:318-27
Spruce, A E; Iwata, A; Almers, W (1991) The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion. Proc Natl Acad Sci U S A 88:3623-7
Spruce, A E; Breckenridge, L J; Lee, A K et al. (1990) Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle. Neuron 4:643-54
Almers, W (1990) Exocytosis. Annu Rev Physiol 52:607-24
Almers, W; Tse, F W (1990) Transmitter release from synapses: does a preassembled fusion pore initiate exocytosis? Neuron 4:813-8
Almers, W; Breckenridge, L J; Spruce, A E (1989) The mechanism of exocytosis during secretion in mast cells. Soc Gen Physiol Ser 44:269-82