Our goal is to identify the cellular elements responsible for mediating Ca++- triggered exocytosis. As an experimental system, we have chosen to use a cortex fraction derived from the sea urchin egg. When bathed in micromolar concentrations of free Ca++, egg cortex preparations have been shown to undergo an in vitro reaction that morphologically and biochemically approximates exocytosis. There is no other in vitro model for exocytosis that shares this essential feature. We have devised sensitive biochemical assays for this reaction and have identified a number of potentially useful inhibitors. Recently, we have developed a reconstituted exocytotic system based on the recombination of purified cortical secretory vesicle (CV) and egg plasma membrane (PM) fractions. Although we have not yet completed characterization of this preparation the evidence obtained, to date, strongly suggests that we have succeeded in reconstituting cortical exocytosis. We plan to continue our characterization: 1) by utilizing both electron microscopy and an immunofluorescent protection assay to demonstrate vectorial transport of CV contents across the egg plasma membrane, and 2) by utilizing a binding assay, based on the quantitation of a CV enzyme, to analyze the specificity of CV binding to the plasma membrane. We propose to use our reconstitution technology to investigate the proteins required for exocytosis and those that comprise the CV-plasma membrane junction. Two general approaches will be followed: 1) a biochemical approach in which we will a) test the ability of cytoskeletal components to enhance CV binding, b) attempt to inhibit CV binding with cytoskeletal toxins, and c) attempt to subfractionate, then reconstitute, the binding activity of the CV and PM fractions themselves; and 2) an immunological approach in which polyclonal and monoclonal antibodies (raised to cytoskeletal proteins, and to the CV and PM fractions) will be used to inhibit CV binding and/or exocytosis. Although we will utilize sea urchin eggs for this study, the fundamental similarity of Ca++-triggered exocytosis in all species suggests that it will be possible to extrapolate the results obtained to higher species. Elucidation of the molecular mechanism of exocytosis will be invaluable in the diagnosis and treatment of secretory dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026763-09
Application #
3274210
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1979-07-01
Project End
1991-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
9
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Koopmann Jr, W R; Jackson, R C (1990) Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C. Biochem J 265:365-73
Jackson, R C; Modern, P A (1990) N-ethylmaleimide-sensitive protein(s) involved in cortical exocytosis in the sea urchin egg: localization to both cortical vesicles and plasma membrane. J Cell Sci 96 ( Pt 2):313-21
Vater, C A; Jackson, R C (1990) Immunolocalization of hyalin in sea urchin eggs and embryos using an antihyalin-specific monoclonal antibody. Mol Reprod Dev 25:215-26
Jackson, R C; Modern, P A (1990) Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: assessment of binding specificity. J Membr Biol 115:83-93
Vater, C A; Jackson, R C (1989) Purification and characterization of a cortical secretory vesicle membrane fraction. Dev Biol 135:111-23
Vater, C A; Jackson, R C (1989) Identification of a major polypeptide component of the sea urchin fertilization envelope. Dev Biol 132:113-29
Crabb, J H; Modern, P A; Jackson, R C (1987) In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles. Biosci Rep 7:399-409
Crabb, J H; Jackson, R C (1986) Polycation inhibition of exocytosis from sea urchin egg cortex. J Membr Biol 91:85-96
Crabb, J H; Jackson, R C (1985) In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles. J Cell Biol 101:2263-73
Stern, J B; Jackson, R C (1985) Peptide products of the cleavage of bovine preprolactin by signal peptidase. Arch Biochem Biophys 237:244-52

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