The long-term goal of our research plan is to understand the molecular mechanism of exocytosis. Using atomic force microscopy (AFM), a new group of plasma membrane structures called 'pits' 8and 'depressions', have been identified and implicated in exocytosis in live pancreatic acinar cells. Our immediate research goal is to characterize and determine the involvement of these structures in the exocytotic process. Impairment of the exocytotic process in cells is involved in a number of disease states. Understanding this vital cellular process will eventually lead to effective diagnosis and treatment of diseases resulting from secretory defects. Our studies demonstrate 'depressions' to dilate during secretion, returning to their resting size following completion of the process. Exposure of cells to cytochalasin B, a fungal toxin that inhibits actin polymerization, results in a decrease in size of 'depressions', accompanied by a loss in enzyme secretion. We therefore hypothesize 'depressions' to be the sites where vesicle may dock and fuse to release their contents. We seek to test this hypothesis and to gain further understanding of these structures and their biochemical composition. In view of this, our Specific Aims are (1) determine if secretory dock and fuse at 'depressions', (2) determine anatomy of 'pits' and 'depressions' at angstrom resolution, and (3) determine the biochemistry of 'pits' and 'depressions'. To determine if secretory vesicles dock and fuse at 'depressions', the capability of the AFM as a force spectroscope will be utilized. AFM tip disturbance at the cell surface as a consequence of expulsion of vesicular contents will be examined to determine if 'depressions' are secretory sites. To advance our understanding of the function of 'depressions', further AFM studies on the dynamics of its size will be carried out following exposure of cells to stimulators and inhibitors of secretion. Since fixatives are known to provide rigidity to biological samples, proper fixation that retain these PM structures, will enable angstrom resolution imaging by AFM and electron microscopy. To understand the biochemistry of 'pits' and 'depressions', the AFM cantilever will be used as a nanosurgical tool to isolate them for biochemical analysis using mass spectroscopy. Since actin regulates 'depression' morphology, actin antibody affinity columns will be used to isolate and characterize 'depressions' from solubilized acinar cell plasma membrane preparations. These studies will further our understanding of the exocytotic fusion machinery in pancreatic acinar cells, contributing to our understanding of this vital cellular process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056212-04
Application #
6381605
Study Section
Surgery and Bioengineering Study Section (SB)
Program Officer
Serrano, Jose
Project Start
1999-09-01
Project End
2004-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
4
Fiscal Year
2001
Total Cost
$270,042
Indirect Cost
Name
Wayne State University
Department
Surgery
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Naik, Akshata R; Lewis, Kenneth T; Jena, Bhanu P (2016) The neuronal porosome complex in health and disease. Exp Biol Med (Maywood) 241:115-30
Naik, Akshata R; Kulkarni, Sanjana P; Lewis, Kenneth T et al. (2016) Functional Reconstitution of the Insulin-Secreting Porosome Complex in Live Cells. Endocrinology 157:54-60
Jena, Bhanu P (2015) 'Porosome' discovered nearly 20 years ago provides molecular insights into the kiss-and-run mechanism of cell secretion. J Cell Mol Med 19:1427-40
Jena, Bhanu P (2014) Neuronal Porosome-The Secretory Portal at the Nerve Terminal: It's Structure-Function, Composition, and Reconstitution. J Mol Struct 1073:187-195
Taatjes, Douglas J; Quinn, Anthony S; Rand, Jacob H et al. (2013) Atomic force microscopy: High resolution dynamic imaging of cellular and molecular structure in health and disease. J Cell Physiol 228:1949-55
Jena, Bhanu P (2009) Atomic force microscopy: Unraveling the fundamental principles governing secretion and membrane fusion in cells. Ultramicroscopy 109:1094-104
Jena, Bhanu P (2009) Porosome: the secretory portal in cells. Biochemistry 48:4009-18
Jena, Bhanu P (2007) Secretion machinery at the cell plasma membrane. Curr Opin Struct Biol 17:437-43
Cho, Sang-Joon; Jena, Bhanu P (2006) Secretory vesicle swelling by atomic force microscopy. Methods Mol Biol 319:317-30
Jena, Bhanu P (2006) Porosome: the fusion pore revealed by multiple imaging modalities. Methods Mol Biol 319:295-316

Showing the most recent 10 out of 28 publications