Bhanu P. Jena

Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles

The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a Gαi3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid GTP-induced vesicular water gating and swelling. Isolated ZGs exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of ZGs with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulatable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxyl-terminal domain of AQP1 blocks GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the ZG membrane is involved in basal as well as GTP-induced rapid gating of water in ZGs of the exocrine pancreas.

Structure and Composition of the Fusion Pore

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. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, alpha-fodrin and the calcium channels alpha1c and beta3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.

SNARES in opposing bilayers interact in a circular array to form conducting pores

The process of fusion at the nerve terminal is mediated via a specialized set of proteins in the synaptic vesicles and the presynaptic membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. The structure and arrangement of these SNAREs associated with lipid bilayers were examined using atomic force microscopy. A bilayer electrophysiological setup allowed for measurements of membrane conductance and capacitance. Here we demonstrate that the interaction of these proteins to form a fusion pore is dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane. However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern and a stepwise increase in capacitance, and increase in conductance were observed. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion.

STRUCTURE AND DYNAMICS OF THE FUSION PORE IN LIVE CELLS

Atomic force microscopy reveal pit‐like structures typically containing three or four, ∼150nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane‐bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane‐bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase‐specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High‐resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.

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.

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 5–8 Å 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.

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 ∼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.

RAPID ALDOSTERONE‐INDUCED CELL VOLUME INCREASE OF ENDOTHELIAL CELLS MEASURED BY THE ATOMIC FORCE MICROSCOPE

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non‐genomic effects on intracellular pH, intracellular electrolytes and inositol‐1,4,5‐triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827±172fl. Cell volume was found to increase by 28% 5min after hormone exposure. Twenty‐five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na+/H+exchanger prevented the initial aldosterone‐induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na+/H+exchanger.

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.

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 Gαl3-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.