Bhanu P. Jena
George E. Palade University Professor
Department of Physiology
Wayne State University School of Medicine
Detroit, Michigan, USA
Summary of Research (1997-2017)
Research Summary: Cellular Secretion and Membrane Fusion
The molecular mechanism of fusion of membrane-bound secretory vesicles at the cell plasma membrane and subsequent release of vesicular contents, has long been a fundamental physiological problem. Important cellular events such as ER-Golgi transport, plasma membrane recycling, and the release of enzymes, hormones and neurotransmitters, all require fusion of opposing bilayers. The role of secretion and membrane fusion in health and disease, is clear. Our studies in the past several years, has enabled:
Discovery of a New Cellular Structure the POROSOME (Fusion Pore is formed at the base of the porosome following docking of the secretory vesicle), at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release vesicular contents.
Elucidation of the molecular mechanism of SNARE-induced membrane fusion.
Understanding the molecular regulation of secretory vesicle swelling, an important cellular process involved in the expulsion of vesicular contents.
1. Discovery of a New Cellular Structure the POROSOME (Fusion Pore is formed at the base of the porosome following docking of the secretory vesicle )
In early 1996, while studying live pancreatic acinar cells using the atomic force microscope (AFM), we identified at the apical cellular region, a group of circular ‘pits’ measuring 0.4mm–1.2 mm in diameter, punctuated by smaller depressions averaging150 nm in diameter [Proc. Natl. Acad. Sci. USA 1997. 94:316-321]. Stimulation of secretion caused 'depressions' to dilate and return to their resting size following completion of the process. Exposure of cells to actin depolymerizing agents resulted in collapse of 'depressions' and a consequent loss in secretion. Zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas contain the starch digesting enzyme amylase. Using amylase-specific immunogold-AFM studies, localization of amylase at 'depressions' following stimulation of secretion was demonstrated by our group [Cell Biology International. Jan. 2002, 26 (1):35-42; Cell Biology International. Jan. 2002, 26 (1):29-33 and Cover illustration of the fusion pore in pancreatic acinar cells; and Methods in Cell Biology. 2002. 68:1-415, Cover illustration of the fusion pore; NIPS Dec. 2002, 17(6):219-222 and Cover illustration; J. Endocrinology ]. 2003, 176(2):169-174]. These studies demonstrate 'depressions' to be the fusion pores in pancreatic acinar cells, where membrane-bound secretory vesicles dock and fuse to release vesicular contents. In collaboration with other groups, we have confirmed the presence of similar fusion pores in growth hormone secreting cells of the pituitary [Endocrinology 2002,143:1144-1148 (Cover illustration of the fusion pore in GH-secreting cells)] and in chromaffin cells [Annals of the New York Academy of Sciences 2002. 971:254-256]. Recently, we have succeeded in imaging the fusion pore using electron microscopy. These studies reveal fusion pores to possess a cup-shaped basket-like morphology, with t-SNAREs at the base of the cup. Partial biochemical composition of the fusion pore is also revealed from this study (Biophys. J. 2003, 84:1337-1343; Biophys. J. 2003, 85(3):2035-2043). The existence of the fusion pore suggested a number of years ago from electrophysiological measurements, was confirmed, and for the first time, its structure and dynamics determined at nm resolution and in real time. These studies besides revealing the molecular composition and architecture of the exocytotic machinery at the cell plasma membrane, provides a new perspective on cellular structure-function.
2. Molecular mechanism of SNARE-induced membrane fusion
In the last decade, several proteins have been identified in the regulation of membrane fusion. Among them, the N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated as the minimal fusion machinery, capable of fusing opposing bilayers. Target SNAREs or t-SNAREs (involving two proteins) are located at the cell plasma membrane, and vesicle SNARE or v-SNARE, (a single protein) is present at the secretory vesicle membrane. Although the crystal structure of the core complex of t-/v-SNARE is known, the molecular mechanism of SNARE participation in fusion of vesicle and target membrane, remained a mystery. In recent studies, we were able to resolve the molecular mechanism of SNARE-induced membrane fusion (Biophys. J. (2002), 83:2522-2527).
In an earlier study on native SNARE complexes isolated from neuronal tissue [Cell Biol. Int. (1999), 22:657-670. (cover illustration)], we demonstrate the coiling and supercoiling of v- and t-SNAREs, and their possible interaction in a circular array at the point of contact between the vesicle membrane and plasma membrane. This coiling and supercoiling of t-/v-SNAREs, result in the generation of centrifugal force and tight apposition between the opposing bilayers.
In a recent study (Biophys. J. (2002), 83:2522-2527), the structure and rearrangement of SNAREs associated with opposing lipid bilayers, that leads to membrane fusion was determined using AFM and electrophysiological measurements. A bilayer electrophysiological setup allowed measurements of membrane conductance and capacitance. Results from these studies demonstrate that t-SNAREs in one bilayer and v-SNARE in an opposing bilayer when brought together, interact in a circular array to form conducting pores. In contrast, addition of v-SNARE in solution when added to t-SNARE reconstituted lipid membrane, increased only the size of the globular t-SNARE oligomer, without influencing the electrical properties of the membrane. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions, leading to membrane fusion. Besides solving the molecular mechanism of SNARE-induced membrane fusion, this study reveals much more. Results from the study demonstrates that membrane proteins interact much differently in solution then when associated with membrane. Hence, crystal structures of interacting membrane proteins in solution, could reveal little information about their interactions with one another, compared to when membrane-associated.
3. Molecular regulation of secretory vesicle swelling
Vesicle swelling has been implicated to play a critical role in membrane fusion and or expulsion of vesicular contents. However, the molecular mechanism of swelling of membrane-bound secretory vesicles remained to be determined. Using AFM, the size dynamics of secretory vesicles, both within live cells and in isolated preparations, was determined. The swelling dynamics of secretory vesicles (Zymogen Granules) within live pancreatic acinar cells examined before and after stimulation of secretion, demonstrated an increase in secretory vesicle size following stimulation [Cell Biol. Int. 2002. 26(1):29-33. (Cover illustration)] Similarly, exposure of isolated zymogen granules to GTP, resulted in an increase in granule size [Proc. Natl. Acad. Sci. 1997. 94:13317-13322]. In this and other studies [J. Biochemistry 2002. 131:815-820], vesicle swelling in response to GTP was found to be mediated via a Gai3 protein and PLA2, in the vesicle membrane. Our studies further demonstrate the association of the water channel aquaporin-1 (AQP1), in the zymogen granule membrane and its participation in rapid GTP-induced water gating and vesicle swelling. Isolated zymogen granules exhibit low basal water permeability, compared to GTP exposure. Treatment of zymogen granules with Hg2+, is accompanied by a reversible loss in both the basal and GTP-stimulable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxy domain of AQP1, blocked GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the zymogen granule membrane is involved in basal and GTP-induced and Gai3-/PLA2-mediated, rapid gating of water [Proc. Natl. Acad. Sci. USA. 2002. 99(7):4720-4724, Cell Biol. Int. 2004. 28:7-17]. These studies further reveal for the first time, the molecular regulation of aquaporin.
Copyright (C), Bhanu P. Jena, PhD., All right reserved. 2014
Updated: 04/17/2017 by Christine Cupps (web manager for Jena-site)