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PubMed2006

Analysis of transient behavior in complex trajectories: application to secretory vesicle dynamics.

Analysis of trajectories of dynamical biological objects, such as breeding ants or cell organelles, is essential to reveal the interactions they develop with their environments. Many previous works used a global characterization based on parameters calculated for entire trajectories. In cases where transient behavior was detected, this usually concerned only a particular type, such as confinement or directed motion. However, these approaches are not appropriate in situations in which the tracked objects may display many different types of transient motion. We have developed a method to exhaustively analyze different kinds of transient behavior that the tracked objects may exhibit. The method discriminates stalled periods, constrained and directed motions from random dynamics by evaluating the diffusion coefficient, the mean-square displacement curvature, and the trajectory asymmetry along individual trajectories. To detect transient motions of various durations, these parameters are calculated along trajectories using a rolling analysis window whose width is variable. The method was applied to the study of secretory vesicle dynamics in the subplasmalemmal region of human carcinoid BON cells. Analysis of transitions between transient motion periods, combined with plausible assumptions about the origin of each motion type, leads to a model of dynamical subplasmalemmal organization.

Cell Line↗
PubMed2006

Cytosolic PLA2 in zymogen granule fusion and amylase release: inhibition of GTP-induced fusion by arachidonyl trifluoromethyl ketone points to cPLA2 in G-protein-mediated secretory vesicle fusion.

Previously we reported that the G-protein Galphai3 localized in pancreatic zymogen granule (ZG) membrane participates in vesicular fusion at the cell plasma membrane (PM). In the present study, the presence of cytosolic phosholipase A2 (cPLA2) in rat ZGs was demonstrated and its potential role in G-protein-mediated ZG-PM fusion was investigated. In vitro fusion assays utilizing both enzymatic and fluorimetric techniques demonstrate that ZGs fuse with PM with a greater potency in the presence of GTP. Arachidonyl trifluoromethyl ketone (AACOCF3) at 40 microM reduces GTP-induced ZG-PM fusion by 25-50%. Anti-cPLA2 antibody reduces ZG-PM fusion in a dose-dependent manner and a 50% reduction of the fusion takes place in the range of 0.48-0.64 ratios of cPLA2 antibody to ZG proteins. PLAP, a cPLA2 activator synthetic peptide increases ZG-PM fusion in a limited dose-dependent manner and tends to inhibit at higher concentrations. Exogenous arachidonic acid inhibits GTP-induced ZG-PM fusion in a dose-dependent manner. Furthermore, a non-hydrolysable GTP analogue, Gpp(NH)p, reduces PLAP effect in ZG-PM fusion; and the net effect of Gpp(NH)p and PLAP differs significantly from the net effect of GTP and PLAP on ZG-PM fusion suggesting that cPLA2 is involved in G-protein-mediated secretory vesicle fusion.

Amylases↗
PubMed1990

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: assessment of binding specificity.

An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding of S. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4 +/- 3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bind S. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74 +/- 9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.

Animals↗
PubMed2006

Cytokeratin-20 and seminal vesicle secretory protein VI as possible marker proteins in urinary bladder preneoplastic lesions induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

BACKGROUND: A simple and non-invasive methods for the diagnosis of transitional cell carcinoma of the bladder are needed for the prevention of invasive tumor. A proteomic technology has recently been developed to facilitate protein profiling of biological mixtures. We tried to detect the marker proteins by proteomic approach during the initiation stages on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder carcinogenesis in rats. METHODS: Ten rats in group A were given 0.05% BBN in drinking water for 12 weeks. Other 10 rats in group B with 10 rats were designated as a control group and were not given BBN. Whole urinary bladders of all rats were excised at 12 weeks from the beginning of the experiment. Conventional proteomics was performed with high resolution 2-dimension gel electrophoresis followed by computational image analysis and protein identification using mass spectrometry. RESULTS: A comparison of urinary bladder hyperplasia tissue with control tissue showed that five proteins; actin gamma2 propeptide, cytokeratin-20 (CK-20), proapolipoprotein, alpha2 actin (alpha-cardiac actin) and heat shock 27 kDa protein-1 were over-expressed in hyperplastic tissues. Three proteins; transcription factor myocardin, seminal vesicle secretory protein VI (SVS-VI) precursor and hypothetical protein RMT-7 were under-expressed in hyperplastic tissues. CONCLUSION: In our animal mode, BBN-induced urinary bladder mucosal hyperplasia resulted in an increase in the expression of five proteins and a decrease in the expression of three proteins. Of these, CK-20 and SVS-VI seem to be of particular interest. However other method such as Western blotting seems to be needed for confirmation of these proteins and more information on human bladder tissue is needed for clinical application.

Animals↗
PubMed2003

Defective sorting to secretory vesicles in trans-Golgi network is partly responsible for protein C deficiency: molecular mechanisms of impaired secretion of abnormal protein C R169W, R352W, and G376D.

Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Animals↗
PubMed1997

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens.

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.

Animals↗
PubMed1990

Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae.

CDP-diacylglycerol synthase, phosphatidylinositol synthase, and phosphatidylinositol kinase activities were associated with post-Golgi apparatus secretory vesicles destined for the plasma membrane of Saccharomyces cerevisiae. These results suggest that the plasma membrane is capable of synthesizing both CDP-diacylglycerol and phosphatidylinositol as well as phosphorylating phosphatidylinositol.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Inhibition of neurotransmitter and hormone transport into secretory vesicles by 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-alpha-ergocryptine: both compounds act as uncouplers and dissipate the electrochemical gradient of protons.

2-(4-Phenylpiperidino)cyclohexanol (AH-5183) and 2-bromo-alpha-ergocryptine, known inhibitors of the transport of acetylcholine and L-glutamate, respectively, into synaptic vesicles, inhibited the ATP-dependent uptake of dopamine in parallel with the dissipation of the electrochemical gradient of protons in chromaffin granule membrane vesicles. These compounds induced the release of accumulated dopamine from the vesicles. They also inhibited the ATP-dependent formation of the electrochemical gradient of protons in liposomes reconstituted with chromaffin H(+)-ATPase without affecting the activities for ATP hydrolysis, and ATP-dependent uptakes of dopamine, gamma-aminobutyrate, and glutamate into synaptic vesicles. These results indicated that 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-alpha-ergocryptine acted as uncouplers in the secretory vesicles.

Animals↗
PubMed1996

Acidification of serotonin-containing secretory vesicles induced by a plasma membrane calcium receptor.

Parafollicular (PF) cells secrete 5-hydroxytryptamine in response to increased extracellular Ca2+ ([Ca2+]e). This stimulus causes Cl- channels in PF secretory vesicles to open, leading to vesicle acidification. PF cells express a plasmalemmal heptahelical receptor (CaR) that binds Ca2+, Gd3+, and Ba2+. We now report that the CaR mediates vesicle acidification. Ca2+, Gd3+, and Ba2+ induced vesicle acidification, which was independent of channel-mediated Ca2+ entry. Agonist-induced vesicle acidification was blocked by pertussis toxin, inhibitors of phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl cyclase, or protein kinase G. PF cells contained NO synthase immunoreactivity, and vesicles were acidified by NO donors and dibutyryl cGMP. [Ca2+]e, and Gd3+ mobilized thapsigargin-sensitive internal Ca2+ stores. [35S]G alpha i and [35S]G alpha q were immunoprecipitated from PF membranes incubated with agonists in the presence of [35S]adenosine 5'-O-(thiotriphosphate). Labeling of G alpha i but not G alpha q was antagonized by pertussis toxin. Vesicles acidified in response to activation of protein kinase C; however, protein kinase C inhibition blocked calcium channel- but not CaR-dependent acidification. We propose the following signal transduction pathway: CaR -> Gi -> phosphatidylinositol-phospholipase C -> inositol 1,4,5-trisphosphate -> [Ca2+]i -> Ca2+/calmodulin -> NO synthase -> NO -> guanylyl cyclase -> cGMP -> protein kinase G -> opens vesicular Cl- channel.

Animals↗
PubMed1980

Role of microtubules in milk secretion--action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells.

Effect of colchicine on microtubules was studied in mammary epithelial cells treated both in vivo and in vitro with the alkaloid. Three hours after the intramammary infusion of colchicine, secretory activity of mammary epithelia ceased, milk constituents accumulated and were randomly distributed within the cytoplasm, sometimes leaking into the perialveolar connective tissue, and autophagic vacuoles were prevalent. It appeared that an accelerated involutionary process was occurring. No microtubules were observed after this treatment. In vitro treated cells appeared to be less affected by the alkaloid. Although numerous casein-containing secretory vesicles accumulated in the cytoplasm, lipid droplet accumulation was less, and fewer autophagic vacuoles were observed, although lysosomes were commonly observed. Occasionally, obliquely sectioned microtubules were found in cells treated with low concentrations of colchicine but were absent at higher colchicine concentrations; however, paracrystalline inclusions (tubulin aggregates) were observed in some cells at all concentrations of the drug. These observations provide evidence that drugs which interfere with microtubule integrity reduce the secretory activity in mammary epithelia. This evidence is consistent with the concept of an association of the microtubular system and the secretory process.

Animals↗
PubMed1995

Ca2+ and secretory-vesicle dynamics.

Exocytosis in neurones and neuroendocrine cells is triggered by an increase in the cytosolic concentration of Ca2+, and is followed by endocytotic membrane retrieval. Electrophysiological studies have characterized the nature of the Ca2+ signal that is required for exocytosis, and have defined the Ca(2+)-dependent steps in exocytotic and endocytotic vesicle cycling. In parallel, biochemical approaches have led to the discovery of a range of proteins that appears to function in synaptic- and secretory-vesicle dynamics. The nature of the Ca(2+)-binding proteins, and how they interact with the identified components of the exocytotic and endocytotic machinery, remain key unresolved issues. However, it is apparent that exocytosis involves multiple Ca(2+)-binding proteins with different affinities, and that the Ca2+ sensor involved in the final membrane-fusion step has different affinities for Ca2+ in synapses and neuroendocrine cells.

Animals↗
PubMed2001

Morphological and functional characterization of beige mouse adrenomedullary secretory vesicles.

We tested whether the giant secretory granules observed in the mast cells of the naturally occurring mutant beige mouse (BM) (C57BL/6N-bg) were also present in the adrenal chromaffin cells. The presence of large chromaffin granules (CG) would be a valuable tool for the study of exocytosis in neuronal tissues. Conversely, the observation of large vesicles within chromaffin cells that are different from CG could indicate that CG are of a different origin than granules of mast cells. Ultrastructural analysis demonstrated the presence of large lysososmal-like vesicles in the BM, and also a discrete increase in the number of CG with diameters larger than 240 nm but not of giant CG. In addition, amperometric measurements of single-event exocytosis, using carbon fiber microelectrodes, showed no differences between the quantal size of secretory events from BM and wildtype or bovine chromaffin cells. Minor but significant differences were found between the kinetics of exocytosis in BM cells andwild-type mouse cells. We conclude that CG, but not the abnormal-sized vesicles found in BM chromaffin cells contribute to the catecholamine secretion and that abnormal secretory granules are not present in adrenergic cell lineage.

Animals↗
PubMed1999

Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice.

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.

Animals↗
PubMed1993

Giardia lamblia: absence of cyst antigens and reduced secretory vesicle formation and bile salt uptake in an encystation-deficient subline.

Encystation of Giardia lamblia entails the appearance of a number of new antigens, as well as formation of a novel class of large encystation-specific secretory vesicles (ESV) that transport stage-specific proteins to the nascent cyst wall. The monoclonal antibody GCSA-1, which was raised against purified cyst walls, recognizes protein species of approximately 26-46 kDa that are regulated by exposure to bile (plus lactic acid) and alkaline pH, the factors that induce encystation. The GCSA-1 epitope is maximally expressed after approximately 14 hr of encystation and localizes to the interior, but not the membrane of the ESV as shown by frozen section immunoelectron microscopy. To further understand the process of encystation, we compared two sublines of strain WB that differ in their ability to encyst in vitro. Water-resistant cysts were not detected in subline A6 under conditions in which subline C6 formed approximately 2 x 10(5) cysts/ml. Moreover, subline A6 did not form ESV efficiently or detectably express antigens recognized by mAb GCSA-1 or by polyclonal anti-cyst sera. Finally, uptake of the bile salt taurocholate by A6 was reduced 4- to 20-fold, compared with that of C6, although transport by both strains was sodium-dependent and regulated by bile salt starvation. The decrease in bile salt uptake by A6 may be related to its defect in encystation.

Animals↗
PubMed1998

The cysteine-string domain of the secretory vesicle cysteine-string protein is required for membrane targeting.

The post-translational addition of palmitic acid residues to cysteine-string protein (Csp) was originally thought to form the basis for membrane association of this secretory-vesicle protein. However, subsequent work showed that chemical depalmitoylation of Csp does not result in its release from membranes. We have confirmed these findings and employed [3H]palmitate labelling of PC12 cells to demonstrate that Csp1 remains associated with membranes following the complete removal of palmitic acid residues. Although palmitoylation is not essential for the stable membrane association of Csp, its role in membrane targeting has not been assessed. To examine this, we constructed a Csp mutant protein with seven cysteines replaced by serines in the cysteine-string domain. In contrast to wild-type Csps, this mutant protein was not targeted to membranes when expressed in PC12 or HeLa cells. We conclude that although a palmitoylated cysteine-string domain is not required for stable membrane association of Csp, it is essential for initial membrane targeting.

Animals↗
PubMed1999

Glucose transporter Glut3 is targeted to secretory vesicles in neurons and PC12 cells.

In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.

Animals↗
PubMed1998

Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug-resistance protein 1.

Intracellular glutathione-conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300x doxorubicin-resistant, MRP1-over-expressing, GLC4-Adr subline. Transport of non-toxic concentrations of monochlorobimane and 5-chloro-methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC4-Adr. Immunotransmission electron microscopy showed that MRP1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1-chloro-2,4-dinitrobenzene could be detected. Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans-Golgi network. In GLC4-Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex. Our results indicate that MRP1 functions as a glutathione-conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles.

ATP-Binding Cassette Transporters↗
PubMed1994

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C-peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.

Ammonium Chloride↗