EM 3D contour maps provide protein assembly at the nanoscale within the neuronal porosome complex.

The neuronal porosome complex, the secretory machinery at the plasma membrane of nerve terminals, is a 12-17-nm cup-shaped lipoprotein structure possessing a central plug. Since the porosome is a membrane associated, multi-protein complex measuring >650 kD, it has precluded generation of 3D crystals for x-ray diffraction studies, nor structural analysis at the atomic level using solution magnetic resonance spectroscopy. These limitations were partially overcome in the current studies, furthering our understanding of the porosome structure. Using atomic force microscopy, electron microscopy and electron density and 3D contour mapping, finally provides at the nanoscale, the structure and assembly of proteins within the neuronal porosome complex. Results from this study demonstrate a set of eight protein units lining the porosome cup, each connected via spoke-like elements to a central plug region within the structure. The isolation of intact porosomes for near-atomic resolution using cryo-electron diffraction measurements, is finally possible.

Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells.

Secretion and membrane fusion are fundamental cellular processes involved in the physiology of health and disease. Studies within the past decade reveal the molecular mechanism of secretion and membrane fusion in cells. Studies reveal that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized plasma membrane structures. Swelling of secretory vesicles result in a build-up of intravesicular pressure, which allows expulsion of vesicular contents. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution, are discussed. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, have been resolved. With these findings a new understanding of cell secretion has emerged and confirmed by a number of laboratories.

Fusion pore or porosome: structure and dynamics.

Electrophysiological measurements on live secretory cells almost a decade ago suggested the presence of fusion pores at the cell plasma membrane. Membrane-bound secretory vesicles were hypothesized to dock and fuse at these sites, to release their contents. Our studies using atomic force microscopy on live exocrine and neuroendocrine cells demonstrate the presence of such plasma membrane pores, revealing their morphology and dynamics at near nm resolution and in real time.

New structure involved in transient membrane fusion and exocytosis.

Our studies using atomic force microscopy (AFM) reveal a new group of plasma membrane structures involved in exocytosis in live pancreatic acinar cells. These studies demonstrate that "pits" and "depressions" are sites at the apical plasma membrane in live cells, where membrane-bound secretory vesicles dock and transiently fuse to release vesicular contents.

Impaired hepatocyte glucose transport protein (GLUT2) internalization in chronic pancreatitis.

Chronic pancreatitis (CP) is associated with impaired glucose tolerance and with reduced hepatic sensitivity to insulin. We have previously shown that in normal and sham-operated rats, insulin suppresses hepatic glucose production, and this suppression is associated with a decrease in the hepatocyte plasma membrane-bound quantity of the facilitative glucose transport protein GLUT2. The insulin-mediated reduction in membrane-bound GLUT2 is impaired in CP, and may play a role in the glucose intolerance associated with CP. To determine whether GLUT2 is actively internalized and whether this mechanism is disordered in CP, livers from fed and fasting rats in whom CP had been induced 2-3 months earlier by pancreatic duct oleic acid infusion, and in sham-operated (sham) rats, were fractionated to yield endosome (E)- and plasma membrane (PM)-enriched fractions. Forty-five minutes after duodenal intubation alone (fasting) or intubation plus duodenal feeding, livers were removed, homogenized and ultracentrifuged, and microsomal pellets were separated by sucrose density gradient ultracentrifugation. GLUT2 content of fractions was determined by Western blotting and scanning densitometry. The E:PM ratio of GLUT2 increased from 0.68 +/- 0.11 (mean +/- SEM) in fasting sham livers (n = 8) to 1.04 +/- 0.09 in fed sham livers (n = 8; p < 0.05). However, there was no change in the E:PM ratio of GLUT2 in CP livers after duodenal feeding (0.90 +/- 0.12 vs. 0.86 +/- 0.10; n = 8,8; p = NS). To test our findings using confocal laser scanning microscopy, liver specimens from fed and fasting CP and sham rats were minced, fixed in 4% paraformaldehyde, sectioned, and stained with rabbit antirat GLUT2 antibody followed by rhodamine-labeled secondary antibody. GLUT2 was quantified by mean pixel intensity in an 8 x 16-pixel area of PM and a 16 x 16-pixel area of cytosol (CYT) in each of 30 random cells/field (400x) in each of three rats per group. As in the fractionation study, duodenal feeding increased the CYT:PM ratio of GLUT2 from 0.75 +/- 0.01 in fasting sham liver to 0.86 +/- 0.01 in fed sham liver (p < 0.0001), while the CYT:PM ratio in CP remained unchanged. We conclude that feeding induces a shift in GLUT2 from the plasma membrane to the endosomal pool. The feeding-induced internalization of GLUT2 is absent in livers from rats with CP and may play a role in the glucose intolerance associated with CP.

Insights on membrane fusion.

Membrane fusion is a fundamental cellular process regulating intracellular transport, neurotransmission, enzyme secretion, hormone release, and the entry/exit of viruses, to name a few. Knowledge of how opposing bilayers fuse, besides advancing our understanding of these cellular processes, will provide us with the facts to ameliorate secretory defects and prevent cellular entry or exit of pathogenic viruses. In the last few years, great strides have been made in our understanding of the molecular machinery and mechanism of membrane fusion. In this Special Issue of Cell Biology International, entitled 'Membrane fusion: machinery and mechanism', we have tried to cover several aspects of this vital cellular process, providing insights on the machinery, mechanism and dynamics of the process. Membrane fusion studies reported in this Special Issue have been performed on whole cells, synaptic terminals, viruses, and fusion proteins.

Continuous detection of extracellular ATP on living cells by using atomic force microscopy.

Atomic force microscopy is a powerful technique used to investigate the surface of living cells under physiological conditions. The resolution of the instrument is mainly limited by the softness of living cells and the interactions with the scanning tip (cantilever). Atomic force microscopy, in combination with myosin-functionalized cantilevers, was used in the detection of ATP concentrations in solution and on living cells. Functionally active tips were used to scan the surface of cells in culture and to show that the CFTR+ cell line (S9) had a basal surface ATP concentration that could be detected with atomic force microscopy (n = 10). ATP-dependent signals were not detectable in cells scanned with noncoated or heat-inactivated enzyme-coated tips (n = 9). Enzymatically active tips may serve as a model for future development of atomic force microscopy biosensors that can simultaneously detect topographical and biologically important compounds at the surface microenvironment of living cells.

Binding contribution between synaptic vesicle membrane and plasma membrane proteins in neurons: an AFM study.

The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes.

The native membrane fusion machinery in cells.

Recombinant SNAREs have been demonstrated as the minimal membrane fusion machinery. The participation of additional proteins in the regulation of membrane fusion has been suggested. In this study we provide nanometer-resolution images of native NSF oligomers and SNARE complexes isolated from neurons and the pancreas. Our study reveals the presence of new coiled rod-like structures in association with the SNARE complex only in neuronal tissue. Neuronal SNAREs were found coiled and super-coiled with these structures. The existence of NSF as pentamers in its native state is also demonstrated. The extent of coiling and super-coiling of SNAREs may regulate the potency and efficacy of membrane fusion in cells.

Gi regulation of secretory vesicle swelling examined by atomic force microscopy.

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. 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 KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that G(alpha i3) protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Localization of SH-PTP1 to synaptic vesicles: a possible role in neurotransmission.

The tyrosine kinase pp60src is known to phosphorylate synaptophysin and in doing so may regulate neurotransmitter release. The tyrosyl phosphorylated state of synaptophysin is dependent on pp60src kinase and the unknown protein tyrosine phosphate phosphohydrolase (PTPase, EC 3.1.3.48). Here we report the protein tyrosine phosphate phosphohydrolase SH-PTP1, to associate with synaptic vesicles and interact with synaptophysin. These studies identify SH-PTP1 as a new member of the secretory machinery at the nerve terminal and suggest its involvement in neurotransmission.

Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis.

The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily due to limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. "Pits" measuring 500-2000 nm and containing 3-20 depressions measuring 100-180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in "depression" size correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be the fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time.

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 +/- 172 fl. Cell volume was found to increase by 28% 5 min 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.

Effect of tyrosine kinase inhibition on basal and epidermal growth factor-stimulated human Caco-2 enterocyte sheet migration and proliferation.

Mucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation-blocked cell migration using human Caco-2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2,5-dihydroxymethylcinnamate, which block kinase ATP-binding and substrate-binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF-stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2,5-dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation-blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein-linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5-dihydroxymethylcinnamate blocked changes in the alpha 2 integrin subunit organization which may modulate EGF-stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase-dependent modulation of cell-matrix interaction inhibits EGF-stimulated migration.

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis.

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

Acidic residues are involved in substrate recognition by two soluble protein tyrosine phosphatases, PTP-5 and rrbPTP-1.

The mechanisms for substrate recognition by two cytoplasmic protein tyrosine phosphatases, PTP-5 and rrbPTP-1, were investigated. Phosphorylation sites on tyrosine-phosphorylated casein, a model PTP substrate, were characterized. Two peptides based on casein phosphorylation sites and one peptide based on the tyrosine phosphorylation site of reduced, carboxamidomethylated and maleylated (RCM) lysozyme were tested as PTP substrates. The three peptides were dephosphorylated by PTP-5 and rrbPTP-1 at rates comparable to those of the corresponding sites on the intact proteins. This indicates that peptides based on the two model PTP substrates, casein and RCM-lysozyme, contained all or most of the structural information necessary for PTP-5 and rrbPTP-1 substrate recognition. Structural elements required for substrate recognition by PTP-5 and rrbPTP-1 were also investigated. Km values for dephosphorylation of three simple aromatic phosphate esters (phosphotyrosine, p-nitrophenyl phosphate, and phenyl phosphate) by rrbPTP-1 were about 5000-fold higher than those obtained for the peptide and protein substrates. This indicates that recognition of protein and peptide substrates involves structural elements in addition to the phosphate group and the aromatic tyrosine ring of phosphotyrosine. Analysis of the effects of truncations and Ala for polar substitutions on the reactivity with PTP-5 and rrbPTP-1 of peptides based on casein, RCM-lysozyme, and angiotensin II indicated that Asp or Glu within the first five residues on the N-terminal side of phosphotyrosine increased peptide reactivity with both PTP's. Asn residues were unable or only weakly able to substitute for Asp residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct and specific GAP activities in rat pancreas act on the yeast GTP-binding proteins Ypt1 and Sec4.

Previous studies have demonstrated that Sec4, a 23.5 kDa guanine nucleotide-binding protein of the ras superfamily is required for exocytosis in the budding yeast Saccharomyces cerevisiae. Ypt1, another ras-like 23 kDa guanine nucleotide-binding protein in yeast has been found to be involved in ER-Golgi transport. A mammalian homologue of Ypt1 called rab1 has also been identified. Recent studies using purified Sec4 protein have identified a component of yeast lystate that specifically stimulates the hydrolysis of GTP bound to Sec4. In the present study, purified recombinant Sec4 and Ypt1 proteins expressed in E. coli have been used as substrates to determine if GTPase activating proteins (GAPs) directed toward these proteins are present in rat pancreas. Our studies showed that 65% of Sec4-GAP activity was associated with the 150,000 x g pancreatic particulate fraction with approximately 35% being found in the cytosol. On the other hand, more than 95% of Ypt1-GAP activity was found to associate with the particulate fraction. Sec4 and Ypt1 competition assays further demonstrated the specificity of the Sec4 and Ypt1 GAPs. The results from the present study suggest the presence of a distinct GAP in the pancreas that interacts with Sec4, and another that interacts with Ypt1.

Independent modulation of enterocyte migration and proliferation by growth factors, matrix proteins, and pharmacologic agents in an in vitro model of mucosal healing.

BACKGROUND: Gastrointestinal mucosa heals by restitution and proliferation. These are difficult to distinguish in vivo. METHODS: Human Caco-2 enterocytes were cultured on matrix proteins (collagen I, laminin, fibronectin) with growth factors (epidermal growth factor [EGF] and transforming growth factor-beta 1 [TGF-beta 1]) and the tyrosine kinase and prostaglandin inhibitors genistein and indomethacin. Healing was modeled by means of monolayer expansion, proliferation by means of 3H-thymidine uptake, and restitution by means of mitomycin-blocked migration. RESULTS: Changing matrix composition failed to alter proliferation, but collagen I stimulated migration more than laminin or fibronectin (laminin/collagen, 68% +/- 2%; p less than 0.05). EGF (30 ng/ml) increased proliferation on both collagen (225% +/- 11% of basal) and laminin (206% +/- 26%) but increased migration only over laminin (210% +/- 17%) (all, p less than 0.05). TGF-beta 1 (200 pg/ml) stimulated migration over laminin (187% +/- 18%, p less than 0.005) but inhibited migration over collagen (89% +/- 3%, p less than 0.01) and did not affect 3H-thymidine uptake. When cultured on laminin, EGF but not TGF-beta 1 altered organization of the alpha 2 integrin subunit. Genistein (100 mumol/L) inhibited basal and EGF-stimulated 3H-thymidine uptake. In addition, it prevented EGF stimulation of replication-blocked migration (81% +/- 10% vs 190% +/- 20% of basal, p less than 0.0001) without altering basal replication-blocked migration. Indomethacin (10(-5) mol/L) did not alter migration but inhibited basal and EGF-stimulated proliferation by 7% +/- 1% (each, p less than 0.005). CONCLUSIONS: Restitution and proliferation appear independently regulated by matrix and growth factors. It may be possible to individually target specific phases of mucosal healing by means of pharmacologic agents.

Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini.

Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+)-dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+)-dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers.