The long term objective of this proposal is to understand the cellular mechanisms of sustained secretion in neuroendocrine cells. Using a combination of new imaging, electrophysiological, and molecular biological techniques, different aspects of the secretion cycle in pancreatic beta-cells will be examined. In particular, single secretory granules and single secretory endosomes will be separately identified and tracked in living cells under a variety of conditions. Specifically, the aims are to test the following hypotheses: (1) Trafficking of secretory granules and secretory-derived endosomes is regulated by cytoplasmic Ca2+ and protein phosphorylation-signals which themselves are generated by cell stimulation from neuronal and hormonal inputs. (2) The readily releasable pool of secretory granules is replenished from predocked granules, without the need for mobilization of granules from the cytoplasm. Sustained secretion, however, requires movement of granules from deep in the cell interior to the plasma membrane. (3) Secretory granules are directly retrieved during endocytosis, and can be recycled to the releasable pool for subsequent exocytosis. Single secretory granules will be identified because they are labeled with a granule specific green fluorescent protein chimera. Single endosomes derived from secretory granules that undergo exo-endocytosis will be additionally labeled with styryl dyes FM1-43 and FM4-64. These optical assays of membrane trafficking in living cells will be combined with patch clamp recording of membrane capacitance and electron microscopy to establish a firm physiological understanding of the mechanisms that allow for sustained secretion from neuroendocrine cells. These studies will lay the ground work for an understanding of how these processes may be altered in diseases of neuroendocrine cells such as diabetes mellitus that demonstrate an apparent disregulation of secretory granule biogenesis, trafficking or recycling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK057999-05
Application #
6603222
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Haft, Carol R
Project Start
1999-09-30
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2005-07-31
Support Year
5
Fiscal Year
2003
Total Cost
$192,439
Indirect Cost
Name
University of Denver
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
007431760
City
Denver
State
CO
Country
United States
Zip Code
80208
Overlease, Ruth L; Bauer, Roslyn A; Angleson, Joseph K (2005) Retention of peptide hormones during partial secretion in pituitary somatotrophs and corticotrophs. Pflugers Arch 449:458-62
Bauer, Roslyn A; Khera, Rebecca S; Lieber, Janet L et al. (2004) Recycling of intact dense core vesicles in neurites of NGF-treated PC12 cells. FEBS Lett 571:107-11
Bauer, Roslyn A; Overlease, Ruth L; Lieber, Janet L et al. (2004) Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells. J Cell Sci 117:2193-202
Vo, Yen P; Hutton, John C; Angleson, Joseph K (2004) Recycling of the dense-core vesicle membrane protein phogrin in Min6 beta-cells. Biochem Biophys Res Commun 324:1004-10
Brumback, Audrey C; Lieber, Janet L; Angleson, Joseph K et al. (2004) Using FM1-43 to study neuropeptide granule dynamics and exocytosis. Methods 33:287-94