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Biomedical subjects

Bhanu P Jena

Publications and source records attributed to Bhanu P Jena.

At least 19 recordsLinked to original sources
PubMed2006

Cholesterol is critical to the integrity of neuronal porosome/fusion pore.

Secretion is one of the most fundamental cellular processes. Porosomes have been demonstrated as the universal secretory machinery in cells. Earlier studies determine the presence of a number of proteins in porosomes, among them the N- and P/Q-type calcium channels, actin, syntaxin-1, synaptotagmin-1, vimentin, the N-ethylmaleimide-sensitive factor (NSF), the chloride channel CLC-3, and the alpha subunit of the heterotrimeric GTP-binding protein G(o). Studies demonstrate that t-SNAREs localize at the base of porosomes, and directly interact with calcium channels. In the present study, we demonstrate that Syntaxin-1 co-localizes with cholesterol in solubilized synaptosomal membrane preparations. Depletion of cholesterol, results in the dissociation of both Syntaxin-1 and N-type calcium channel from neuronal porosomes. Thus, cholesterol participates as an integral component of the neuronal porosome complex, and is required for its stability.

Animals↗
PubMed2006

Cell secretion machinery: studies using the AFM.

A new field in biology, 'nano-cell biology', has emerged from the successful use of force microscopy in understanding the structure and dynamics of cells and biomolecules, at nm resolution and in real time. Atomic force microscopy, in combination with conventional tools and approaches (electron microscopy, electrophysiology, X-ray diffraction, photon correlation spectroscopy, mass spectroscopy, biochemistry, and molecular biology), has revealed for the first time, the universal molecular machinery and mechanism of secretion in cells. Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them, dock and fuse at plasma membrane-associated supramolecular structures called Porosome, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle swelling, and the fusion of opposing bilayers, i.e., the fusion of secretory vesicle membrane at the base of the porosome membrane, has also been resolved.

Animals↗
PubMed2006

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy.

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 microm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

Actins↗
PubMed2006

Energy-dependent disassembly of self-assembled SNARE complex: observation at nanometer resolution using atomic force microscopy.

Full-length v-SNARE protein reconstituted in lipid vesicles, when exposed to t-SNARE-reconstituted lipid membrane, results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores and the establishment of continuity between the opposing bilayers. In contrast, when v-SNARE protein alone (without liposomes) is exposed to t-SNARE-reconstituted lipid membrane, they also self-assemble to form t-/v-SNARE complexes, although such complexes fail to possess the characteristic ring pattern, nor do they help in the establishment of continuity between the opposing bilayers. Hence, t-SNAREs and v-SNARE need to be membrane-associated to interact in a circular array to form conducting pores in the presence of calcium. This study demonstrates that, irrespective of their arrangement, both forms of the SNARE complex can be disassembled in the presence of NSF-ATP.

Adenosine Triphosphate↗
PubMed2006

Porosome: the fusion pore revealed by multiple imaging modalities.

Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a buildup of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes and vesicle swelling have also been resolved. These findings reveal the molecular machinery and mechanism of cell secretion. In this chapter, the discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane are discussed.

Animals↗
PubMed2006

Secretory vesicle swelling by atomic force microscopy.

The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remained unknown. Earlier studies from our laboratory demonstrated the association of the alpha-subunit of heterotrimeric GTP-binding protein G(alphai3) with zymogen granule membrane and implicated its involvement in vesicle swelling. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and found to be KCl sensitive. However, Ca2+ had no effect on zymogen granule size. Taken together, these results indicated that zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G(alphai3) protein. Subsequently, our studies demonstrated that the water channel aquaporin-1 (AQP1) is also present at the zymogen granule membrane and participates in rapid GTP-induced and G(alphai3)-mediated vesicular water gating and swelling. Isolated zymogen granules exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of zymogen granules with the known water channel inhibitor 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-terminal domain of AQP1 blocked GTP-stimulable swelling of vesicles. Our results demonstrate that AQPI associated at the zymogen granule membrane is involved in basal GTP-induced and G(alphai3)-mediated rapid gating of water into zymogen granules of the exocrine pancreas.

Animals↗
PubMed2005

Size of supramolecular SNARE complex: membrane-directed self-assembly.

Full length v-SNARE protein in lipid vesicles when exposed to t-SNARE-reconstituted lipid membrane results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores, and establishing continuity between the opposing bilayers. It is known that smaller vesicles fuse more efficiently than larger ones, and hence the curvature of secretory vesicles may dictate the potency and efficacy of their fusion at the cell plasma membrane. The diameter of t- and v-SNARE vesicles may, therefore, reflect the size of the t-/v-SNARE complex formed. In the present study, this hypothesis was tested, and results from the study demonstrate that the size of the t-/v-SNARE complex is directly proportional to the vesicle diameter (R2 = 0.9725).

Lipid Bilayers↗
PubMed2005

Patch clamped single pancreatic zymogen granules: direct measurements of ion channel activities at the granule membrane.

BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.

ATP-Binding Cassette Transporters↗
PubMed2005

Cell secretion and membrane fusion.

Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a build-up of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, has also been resolved. These findings reveal the molecular mechanism of cell secretion.

Animals↗
PubMed2005

Insulin receptor (IR) and glucose transporter 2 (GLUT2) proteins form a complex on the rat hepatocyte membrane.

The hepatic glucose transporter, GLUT2, facilitates bidirectional glucose transport across the hepatocyte plasma membrane under insulin regulation. We studied the interactions of IR and GLUT2 proteins to determine whether they are physically coupled in a receptor-transporter complex. By comparing endosome and plasma membrane IR and GLUT2 ratios before and after feeding, it was determined that IR and GLUT2 are internalized in a fixed ratio. When solubilized hepatocytes were immunoprecipitated with antibodies against either IR or GLUT2, both proteins co-precipitated. The association of IR and GLUT2 was further assessed by confocal microscopy. Sections of fed liver were incubated with fluorescein-tagged anti-GLUT2 or Texas Red-tagged anti-IR. Colocalization was observed both at the plasma membrane and in the cytosol. Fluorescence-resonance energy transfer studies further confirmed this association. We conclude that IR and GLUT2 form a receptor-transporter complex in hepatocytes, which forms a mechanism of insulin-mediated hepatic glucose regulation.

Animals↗
PubMed2005

Molecular machinery and mechanism of cell secretion.

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called porosomes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamental cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.

Cell Biology↗
PubMed2005

Involvement of water channels in synaptic vesicle swelling.

Vesicle swelling is critical for secretion; however, the underlying mechanism of synaptic vesicle (SV) swelling is unknown. A G alphai3-phospholipase A2 (PLA2)-mediated involvement of the water channel aquaporin-1 (AQP1) in the regulation of secretory vesicle swelling in the exocrine pancreas has been previously reported. In the present study, the association and involvement of water channels in SV swelling was explored. Results from the study demonstrate that water channels AQP1 and AQP6, and the heterotrimeric Go protein are associated with SVs and participate in their swelling.

Animals↗
PubMed2004

Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex.

Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.

Aminobenzoates↗
PubMed2004

Calcium drives fusion of SNARE-apposed bilayers.

N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.

Calcium↗
PubMed2004

Structure, isolation, composition and reconstitution of the neuronal fusion pore.

Neuronal communication is dependent on the fusion of 40-50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5-8A resolution, the presence of 8-10 nm in diameter cup-shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.

Animals↗
PubMed2004

Vesicle swelling regulates content expulsion during secretion.

The involvement of secretory vesicle swelling has been proposed in secretion; however, little is known about its role. Using both the pancreatic acinar cell and neuronal model, we show secretory vesicle swelling in live cells. Our study reveals that vesicle swelling potentiates its fusion at the cell plasma membrane, and is required for expulsion of intravesicular contents. Since the extent of swelling is directly proportional to the amount of vesicular contents expelled, this provides cells with the ability to regulate release of secretory products. These direct observations of the requirement of secretory vesicle swelling in secretion, provides an understanding of the appearance of partially empty vesicles following the process.

Animals↗
PubMed2003

Reconstituted fusion pore.

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.

Animals↗