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Sample records for docks secretory vesicles

  1. Morphological docking of secretory vesicles

    PubMed Central

    2010-01-01

    Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses. PMID:20577884

  2. Docking of Secretory Vesicles Is Syntaxin Dependent

    PubMed Central

    de Wit, Heidi; Cornelisse, L. Niels; Toonen, Ruud F.G.; Verhage, Matthijs

    2006-01-01

    Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones. PMID:17205130

  3. Dissecting docking and tethering of secretory vesicles at the target membrane

    PubMed Central

    Toonen, Ruud F; Kochubey, Olexiy; de Wit, Heidi; Gulyas-Kovacs, Attila; Konijnenburg, Bas; Sørensen, Jakob B; Klingauf, Jurgen; Verhage, Matthijs

    2006-01-01

    Secretory vesicles dock at their target in preparation for fusion. Using single-vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18-1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking-deficient munc18-1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t-SNARE and Munc18-1 binding partner syntaxin, but not the v-SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18-1/syntaxin-dependent, strongly tethered, fusion-competent state. PMID:16902411

  4. All three components of the neuronal SNARE complex contribute to secretory vesicle docking

    PubMed Central

    Wu, Yao; Gu, Yiwen; Morphew, Mary K.; Yao, Jun; Yeh, Felix L.; Dong, Min

    2012-01-01

    Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs. PMID:22869597

  5. Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes.

    PubMed

    de Wit, Heidi; Walter, Alexander M; Milosevic, Ira; Gulyás-Kovács, Attila; Riedel, Dietmar; Sørensen, Jakob B; Verhage, Matthijs

    2009-09-04

    Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. Here, using adrenal chromaffin cells, we identify the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, we show that the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, lead us to propose that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes.

  6. The Role of Rab3a in Secretory Vesicle Docking Requires Association/Dissociation of Guanidine Phosphates and Munc18-1

    PubMed Central

    van Weering, Jan R.T.; Toonen, Ruud F.; Verhage, Matthijs

    2007-01-01

    Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1. PMID:17637832

  7. Open Syntaxin Docks Synaptic Vesicles

    PubMed Central

    Olsen, Shawn; Jorgensen, Erik M

    2007-01-01

    Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking. PMID:17645391

  8. Role of aquaporins and regulation of secretory vesicle volume in cell secretion

    PubMed Central

    Sugiya, H; Matsuki-Fukushima, M; Hashimoto, S

    2008-01-01

    Abstract In exocrine glands, secretory proteins synthesized in the rough endoplasmic reticulum (RER) exhibit vectorial transport from ER through a succession of membrane-bounded components such as Golgi complex, condensing vacuoles and secretory granules. The secretory granules migrate to particular locations within the cell close to the apical membrane prior to the release of their contents into the acinar lumen. Currently, to release intragranular contents, secretory granules have been demonstrated to transiently dock and fuse at ‘porosome’, a permanent cup-shaped structures at the cell membranes. Then swelling of secretory granules occurs to allow explusion of intragranular contents. In this process, water and ion fluxes in the granule membrane appear to contribute to maintain secretory granule integrity and morphology via osmoregulation in secretory granules. Aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins, which function as channels to permeate water and small solutes. The AQPs reside constitutively at the plasma membrane in most cell types. However, recent studies have demonstrated that the AQPs are present in secretory granules in exocrine glands, synaptic vesicles and intracellular vesicles in liver and kidney, implying that AQPs in secretory granules and vesicles are involved in their volume regulation. This paper reviews the possible role of AQPs on secretory granules, especially in exocrine glands, in secretory function. PMID:18194450

  9. ATP: The crucial component of secretory vesicles.

    PubMed

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  10. ATP: The crucial component of secretory vesicles

    PubMed Central

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R.; González-Santana, Ayoze; Westhead, Edward W.; Borges, Ricardo; Machado, José David

    2016-01-01

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission. PMID:27342860

  11. Variable priming of a docked synaptic vesicle

    PubMed Central

    Jung, Jae Hoon; Szule, Joseph A.; Marshall, Robert M.; McMahan, Uel J.

    2016-01-01

    The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM–PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM–PM contact area is dynamic and in equilibrium. The extent of VM–PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca2+ channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium. PMID:26858418

  12. Observations of Calcium Dynamics in Cortical Secretory Vesicles

    PubMed Central

    Raveh, Adi; Valitsky, Michael; Shani, Liora; Coorssen, Jens R.; Blank, Paul S.; Zimmerberg, Joshua; Rahamimoff, Rami

    2012-01-01

    SUMMARY Calcium (Ca2+) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca2+ concentration in the secretory vesicles was estimated to be in the range of ~10 – 100 μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca2+ dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca2+ signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Cav2.1) Ca2+ channels. Blocking the p-type Ca2+ channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca2+ dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca2+ indicator fluorescence. The results suggest that the free Ca2+ content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process. PMID:22831912

  13. Alignment of synaptic vesicle macromolecules with the macromolecules in active zone material that direct vesicle docking.

    PubMed

    Harlow, Mark L; Szule, Joseph A; Xu, Jing; Jung, Jae Hoon; Marshall, Robert M; McMahan, Uel J

    2013-01-01

    Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron's axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle's luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly's chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly's shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking

  14. Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

    PubMed Central

    Xu, Jing; Jung, Jae Hoon; Marshall, Robert M.; McMahan, Uel J.

    2013-01-01

    Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for

  15. Fusion competent synaptic vesicles persist upon active zone disruption and loss of vesicle docking

    PubMed Central

    Wang, Shan Shan H.; Held, Richard G.; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S.

    2016-01-01

    In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. PMID:27537483

  16. Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

    PubMed

    Kanagaraj, Palsamy; Gautier-Stein, Amandine; Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-06-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

  17. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    PubMed Central

    Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

  18. Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

    PubMed Central

    Kabachinski, Greg; Kielar-Grevstad, D. Michelle; Zhang, Xingmin; James, Declan J.; Martin, Thomas F. J.

    2016-01-01

    The Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. PMID:26700319

  19. In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

    PubMed Central

    Arrastua, Lorena; San Sebastian, Eider; Quincoces, Ana F; Antony, Claude; Ugalde, Unai

    2003-01-01

    The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study. PMID:12435271

  20. Toward the Inner Nanostructure of a Secretory Vesicle.

    PubMed

    Nieto-González, José Luis; Fernández-Chacón, Rafael

    2017-04-25

    The release of chemical mediators is an essential element of cell-to-cell communication. Signaling molecules such as neurotransmitters and hormones are stored in membrane-bound organelles called secretory vesicles. Some of these organelles can store molecules at high concentrations, overcoming the osmotic shock that could burst the organelle. These organelles contain a proteinaceous matrix that traps the molecules and avoids high intravesicular osmotic pressure. The functional nanostructure and internal organization of the matrix is not well understood. A report by Lovrić et al. in this issue of ACS Nano provides insight into the storage of a small molecule-dopamine-within the intraluminal compartments of a secretory vesicle. Lovrić et al. used a powerful combination of high spatial resolution mass spectrometry and transmission electron microscopy in conjunction with amperometric measurements of exocytotic release to delineate the temporal and spatial fate of intravesicular dopamine and its interaction with the matrix.

  1. Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells

    PubMed Central

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A.; Rubinsztein-Dunlop, Halina; Meunier, Frederic A.

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this “conveyor belt” towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation. PMID:24489879

  2. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  3. Developmental genetics of secretory vesicle acidification during Caenorhabditis elegans spermatogenesis.

    PubMed

    Gleason, Elizabeth J; Hartley, Paul D; Henderson, Melissa; Hill-Harfe, Katherine L; Price, Paul W; Weimer, Robby M; Kroft, Tim L; Zhu, Guang-Dan; Cordovado, Suzanne; L'Hernault, Steven W

    2012-06-01

    Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.

  4. Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material.

    PubMed

    Szule, Joseph A; Harlow, Mark L; Jung, Jae Hoon; De-Miguel, Francisco F; Marshall, Robert M; McMahan, Uel J

    2012-01-01

    The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.

  5. Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites*

    PubMed Central

    Smyth, Annya M.; Yang, Lei; Martin, Kirsty J.; Hamilton, Charlotte; Lu, Weiping; Cousin, Michael A.; Rickman, Colin; Duncan, Rory R.

    2013-01-01

    Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites. PMID:23223447

  6. Regulation of Synaptic Vesicle Docking by Different Classes of Macromolecules in Active Zone Material

    PubMed Central

    Szule, Joseph A.; Harlow, Mark L.; Jung, Jae Hoon; De-Miguel, Francisco F.; Marshall, Robert M.; McMahan, Uel J.

    2012-01-01

    The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10–15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles’ distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry. PMID:22438915

  7. Calcium dynamics in catecholamine-containing secretory vesicles.

    PubMed

    Moreno, Alfredo; Lobatón, Carmen D; Santodomingo, Jaime; Vay, Laura; Hernández-SanMiguel, Esther; Rizzuto, Rosario; Montero, Mayte; Alvarez, Javier

    2005-06-01

    We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.

  8. Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

    PubMed Central

    Wang, Tzu-Ming; Hilgemann, Donald W.

    2008-01-01

    We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP

  9. Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

    PubMed Central

    Chiang, Lilian; Karvar, Serhan; Hamm-Alvarez, Sarah F.

    2012-01-01

    This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules. PMID:22363735

  10. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    PubMed

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2011-04-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

  11. P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

    PubMed Central

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R.

    2011-01-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. PMID:21292765

  12. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    PubMed Central

    Klemm, Robin W.; Ejsing, Christer S.; Surma, Michal A.; Kaiser, Hermann-Josef; Gerl, Mathias J.; Sampaio, Julio L.; de Robillard, Quentin; Ferguson, Charles; Proszynski, Tomasz J.; Shevchenko, Andrej

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery. PMID:19433450

  13. Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

    PubMed Central

    Wang, Zhao; Liu, Huisheng; Gu, Yiwen

    2011-01-01

    The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems. PMID:22184197

  14. Secretory vesicle rebound hyperacidification and increased quantal size resulting from prolonged methamphetamine exposure.

    PubMed

    Markov, Dmitriy; Mosharov, Eugene V; Setlik, Wanda; Gershon, Michael D; Sulzer, David

    2008-12-01

    Acute exposure to amphetamines (AMPHs) collapses secretory vesicle pH gradients, which increases cytosolic catecholamine levels while decreasing the quantal size of catecholamine release during fusion events. AMPH and methamphetamine (METH), however, are retained in tissues over long durations. We used optical and electron microscopic probes to measure the effects of long-term METH exposure on secretory vesicle pH, and amperometry and intracellular patch electrochemistry to observe the effects on neurosecretion and cytosolic catecholamines in cultured rat chromaffin cells. In contrast to acute METH effects, exposure to the drug for 6-48 h at 10 microM and higher concentrations produced a concentration-dependent rebound hyperacidification of secretory vesicles. At 5-10 microM levels, prolonged METH increased the quantal size and reinstated exocytotic catecholamine release, although very high (> 100 microM) levels of the drug, while continuing to produce rebound hyperacidification, did not increase quantal size. Secretory vesicle rebound hyperacidification was temperature dependent with optimal response at approximately 37 degrees C, was not blocked by the transcription inhibitor, puromycin, and appears to be a general compensatory response to prolonged exposure with membranophilic weak bases, including AMPHs, methylphenidate, cocaine, and ammonia. Thus, under some conditions of prolonged exposure, AMPHs and other weak bases can enhance, rather than deplete, the vesicular release of catecholamines via a compensatory response resulting in vesicle acidification.

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

    PubMed

    Kinney, A J; Carman, G M

    1990-07-01

    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.

  16. A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking

    PubMed Central

    Chen, Xiaobing; Reese, Thomas S.

    2016-01-01

    Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone. SIGNIFICANCE STATEMENT The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release. PMID:26985032

  17. A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking.

    PubMed

    Cole, Andy A; Chen, Xiaobing; Reese, Thomas S

    2016-03-16

    Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone. The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release. Copyright © 2016 the authors 0270-6474/16/363222-09$15.00/0.

  18. PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

    PubMed Central

    Jansen, Anna M.; Jin, Chunyu; Rickhag, Mattias; Lund, Viktor K.; Jensen, Morten; Bhatia, Vikram; Sørensen, Gunnar; Madsen, Andreas N.; Xue, Zhichao; Møller, Siri K.; Woldbye, David; Qvortrup, Klaus; Huganir, Richard; Stamou, Dimitrios; Kjærulff, Ole; Gether, Ulrik

    2013-01-01

    Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine

  19. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

    PubMed

    Holst, Birgitte; Madsen, Kenneth L; Jansen, Anna M; Jin, Chunyu; Rickhag, Mattias; Lund, Viktor K; Jensen, Morten; Bhatia, Vikram; Sørensen, Gunnar; Madsen, Andreas N; Xue, Zhichao; Møller, Siri K; Woldbye, David; Qvortrup, Klaus; Huganir, Richard; Stamou, Dimitrios; Kjærulff, Ole; Gether, Ulrik

    2013-01-01

    Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine

  20. Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1.

    PubMed

    Gracheva, Elena O; Maryon, Ed B; Berthelot-Grosjean, Martine; Richmond, Janet E

    2010-01-01

    The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.

  1. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    PubMed Central

    Jethwaney, Deepa; Islam, Md Rafiqul; Leidal, Kevin G; de Bernabe, Daniel Beltran-Valero; Campbell, Kevin P; Nauseef, William M; Gibson, Bradford W

    2007-01-01

    Background Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli. PMID:17692124

  2. Secretory vesicle priming by CAPS is independent of its SNARE-binding MUN domain.

    PubMed

    Nguyen Truong, Cuc Quynh; Nestvogel, Dennis; Ratai, Olga; Schirra, Claudia; Stevens, David R; Brose, Nils; Rhee, JeongSeop; Rettig, Jens

    2014-11-06

    Priming of secretory vesicles is a prerequisite for their Ca(2+)-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca(2+)-dependent activator protein for secretion (CAPS) also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

  3. SNAP-25 gene family members differentially support secretory vesicle fusion.

    PubMed

    Arora, Swati; Saarloos, Ingrid; Kooistra, Robbelien; van de Bospoort, Rhea; Verhage, Matthijs; Toonen, Ruud F

    2017-06-01

    Neuronal dense-core vesicles (DCVs) transport and secrete neuropeptides necessary for development, plasticity and survival, but little is known about their fusion mechanism. We show that Snap-25-null mutant (SNAP-25 KO) neurons, previously shown to degenerate after 4 days in vitro (DIV), contain fewer DCVs and have reduced DCV fusion probability in surviving neurons at DIV14. At DIV3, before degeneration, SNAP-25 KO neurons show normal DCV fusion, but one day later fusion is significantly reduced. To test if other SNAP homologs support DCV fusion, we expressed SNAP-23, SNAP-29 or SNAP-47 in SNAP-25 KO neurons. SNAP-23 and SNAP-29 rescued viability and supported DCV fusion in SNAP-25 KO neurons, but SNAP-23 did so more efficiently. SNAP-23 also rescued synaptic vesicle (SV) fusion while SNAP-29 did not. SNAP-47 failed to rescue viability and did not support DCV or SV fusion. These data demonstrate a developmental switch, in hippocampal neurons between DIV3 and DIV4, where DCV fusion becomes SNAP-25 dependent. Furthermore, SNAP-25 homologs support DCV and SV fusion and neuronal viability to variable extents - SNAP-23 most effectively, SNAP-29 less so and SNAP-47 ineffectively. © 2017. Published by The Company of Biologists Ltd.

  4. Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.

    PubMed

    de Wit, Joris; Toonen, Ruud F; Verhage, Matthijs

    2009-01-07

    Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent fusion events. Transient events represented full release followed by cargo diffusion or incomplete release followed by vesicle retrieval, as previously observed in neuroendocrine cells. Unexpectedly, we also observed that certain cargo, such as Semaphorin 3A (Sema3A), was delivered at the cell surface as stable deposits. Stable deposits and transient events were observed for single cargo and both were SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and calcium dependent. The ratio between stable and transient events did not depend on cargo size, subcellular localization (synaptic vs extrasynaptic secretion), or the presence of the extracellular matrix. Instead, the ratio is cargo specific and depends on an interaction with the vesicle matrix through a basic domain in the cargo protein. Inhibition of this interaction through deletion of the basic domain in Sema3A abolished stable deposits and rendered all events transient. Strikingly, cargo favoring transient release was stably deposited after corelease with cargo favoring stable deposit. These data argue against cargo diffusion after exocytosis as a general principle. Instead, the vesicle matrix retains secreted signals, probably for focal signaling at the cell surface.

  5. Deciphering dead-end docking of large dense core vesicles in bovine chromaffin cells.

    PubMed

    Hugo, Sandra; Dembla, Ekta; Halimani, Mahantappa; Matti, Ulf; Rettig, Jens; Becherer, Ute

    2013-10-23

    Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 μm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.

  6. Complexin Activates Exocytosis of Distinct Secretory Vesicles Controlled by Different Synaptotagmins

    PubMed Central

    Cao, Peng; Yang, Xiaofei; Südhof, Thomas C.

    2013-01-01

    Complexins are SNARE-complex binding proteins essential for the Ca2+-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a co-factor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca2+-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin-adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved. PMID:23345244

  7. Complexin activates exocytosis of distinct secretory vesicles controlled by different synaptotagmins.

    PubMed

    Cao, Peng; Yang, Xiaofei; Südhof, Thomas C

    2013-01-23

    Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.

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

    PubMed Central

    1994-01-01

    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. PMID:8063854

  9. The role of Sec3p in secretory vesicle targeting and exocyst complex assembly.

    PubMed

    Luo, Guangzuo; Zhang, Jian; Guo, Wei

    2014-11-15

    During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis. © 2014 Luo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. ATP-dependent transport of reduced glutathione in yeast secretory vesicles.

    PubMed Central

    Rebbeor, J F; Connolly, G C; Dumont, M E; Ballatori, N

    1998-01-01

    Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19+/-5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2, 4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18+/-2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter. PMID:9729482

  11. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    SciTech Connect

    Somanath, Sangeeta; Partridge, Christopher J.; Marshall, Catriona; Rowe, Tony; Turner, Mark D.

    2016-04-29

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

  12. A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.

    PubMed

    Moreno, Alfredo; SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Alvarez, Javier

    2010-12-01

    Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH(3) cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques.

  13. Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.

    PubMed

    Wu, Xin-Sheng; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Luo, Fujun; Wu, Ling-Gang

    2014-05-22

    Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

  14. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

    PubMed

    Burke, Michael C; Li, Feng-Qian; Cyge, Benjamin; Arashiro, Takeshi; Brechbuhl, Heather M; Chen, Xingwang; Siller, Saul S; Weiss, Matthew A; O'Connell, Christopher B; Love, Damon; Westlake, Christopher J; Reynolds, Susan D; Kuriyama, Ryoko; Takemaru, Ken-Ichi

    2014-10-13

    Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

  15. A continuum model of docking of synaptic vesicle to plasma membrane

    PubMed Central

    Liu, Tianshu; Singh, Pankaj; Jenkins, James T.; Jagota, Anand; Bykhovskaia, Maria; Hui, Chung-Yuen

    2015-01-01

    Neurotransmitter release from neuronal terminals is governed by synaptic vesicle fusion. Vesicles filled with transmitters are docked at the neuronal membrane by means of the SNARE machinery. After a series of events leading up to the fusion pore formation, neurotransmitters are released into the synaptic cleft. In this paper, we study the mechanics of the docking process. A continuum model is used to determine the deformation of a spherical vesicle and a plasma membrane, under the influence of SNARE-machinery forces and electrostatic repulsion. Our analysis provides information on the variation of in-plane stress in the membranes, which is known to affect fusion. Also, a simple model is proposed to study hemifusion. PMID:25551140

  16. Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

    PubMed Central

    Lin, Ming-Yi; Rohan, Joyce G; Cai, Haijiang; Reim, Kerstin; Ko, Chien-Ping; Chow, Robert H

    2013-01-01

    Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels. PMID:23401610

  17. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    PubMed

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

  18. RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone

    PubMed Central

    Han, Yunyun; Kaeser, Pascal S.; Südhof, Thomas C.; Schneggenburger, Ralf

    2012-01-01

    At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles. The mechanisms that enrich Ca2+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knock-out approach at a presynaptically accessible CNS synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca2+ channel density, revealing a new role of RIM proteins in Ca2+ channel targeting. Removal of RIMs also reduced the readily-releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca2+ channel - vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release: enabling a high presynaptic Ca2+ channel density, and vesicle docking at the active zone. PMID:21262468

  19. Ca2+ dynamics in the secretory vesicles of neurosecretory PC12 and INS1 cells.

    PubMed

    SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2010-11-01

    We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca(2+) pump inhibitor benzohydroquinone induced a rapid release of Ca(2+) from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca(2+) pumps is essential to maintain the steady-state [Ca(2+)](SG). Both inositol 1,4,5-trisphosphate (InsP(3)) and caffeine produced a rapid Ca(2+) release from the granules, suggesting the presence of InsP(3) and ryanodine receptors in the granules. The response to high-K(+) depolarization was different in both cell types, a decrease in [Ca(2+)](SG) in PC12 cells and an increase in [Ca(2+)](SG) in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca(2+)](SG) in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca(2+) through SERCA-type Ca(2+) pumps and can release it through InsP(3) and ryanodine receptors, supporting the hypothesis that secretory granule Ca(2+) may be released during cell stimulation and contribute to secretion.

  20. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  1. Distinct Functions of Syntaxin-1 in Neuronal Maintenance, Synaptic Vesicle Docking, and Fusion in Mouse Neurons.

    PubMed

    Vardar, Gülçin; Chang, Shuwen; Arancillo, Marife; Wu, Yuan-Ju; Trimbuch, Thorsten; Rosenmund, Christian

    2016-07-27

    Neurotransmitter release requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes by SNARE proteins syntaxin-1 (Stx1), synaptosomal-associated protein 25 (SNAP-25), and synaptobrevin-2 (Syb2). In mammalian systems, loss of SNAP-25 or Syb2 severely impairs neurotransmitter release; however, complete loss of function studies for Stx1 have been elusive due to the functional redundancy between Stx1 isoforms Stx1A and Stx1B and the embryonic lethality of Stx1A/1B double knock-out (DKO) mice. Here, we studied the roles of Stx1 in neuronal maintenance and neurotransmitter release in mice with constitutive or conditional deletion of Stx1B on an Stx1A-null background. Both constitutive and postnatal loss of Stx1 severely compromised neuronal viability in vivo and in vitro, indicating an obligatory role of Stx1 for maintenance of developing and mature neurons. Loss of Munc18-1, a high-affinity binding partner of Stx1, also showed severely impaired neuronal viability, but with a slower time course compared with Stx1A/1B DKO neurons, and exogenous Stx1A or Stx1B expression significantly delayed Munc18-1-dependent lethality. In addition, loss of Stx1 completely abolished fusion-competent vesicles and severely impaired vesicle docking, demonstrating its essential roles in neurotransmission. Putative partial SNARE complex assembly with the SNARE motif mutant Stx1A(AV) (A240V, V244A) was not sufficient to rescue neurotransmission despite full recovery of vesicle docking and neuronal survival. Together, these data suggest that Stx1 has independent functions in neuronal maintenance and neurotransmitter release and complete SNARE complex formation is required for vesicle fusion and priming, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Syntaxin-1 (Stx1) is a component of the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE

  2. Synaptic vesicle docking: sphingosine regulates syntaxin1 interaction with Munc18.

    PubMed

    Camoletto, Paola G; Vara, Hugo; Morando, Laura; Connell, Emma; Marletto, Fabio P; Giustetto, Maurizio; Sassoè-Pognetto, Marco; Van Veldhoven, Paul P; Ledesma, Maria Dolores

    2009-01-01

    Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Consistently, electron microscopy revealed reduced number of docked vesicles. Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients.

  3. Docking, Not Fusion, as the Rate-Limiting Step in a SNARE-Driven Vesicle Fusion Assay

    PubMed Central

    Smith, Elizabeth A.; Weisshaar, James C.

    2011-01-01

    In vitro vesicle fusion assays that monitor lipid mixing between t-SNARE and v-SNARE vesicles in bulk solution exhibit remarkably slow fusion on the nonphysiological timescale of tens of minutes to several hours. Here, single-vesicle, fluorescence resonance energy transfer-based assays cleanly separate docking and fusion steps for individual vesicle pairs containing full-length SNAREs. Docking is extremely inefficient and is the rate-limiting step. Of importance, the docking and fusion kinetics are comparable in the two assays (one with v-SNARE vesicles tethered to a surface and the other with v-SNARE vesicles free in solution). Addition of the VC peptide synaptobrevin-2 (syb(57–92)) increases the docking efficiency by a factor of ∼30, but docking remains rate-limiting. In the presence of VC peptide, the fusion step occurs on a timescale of ∼10 s. In previous experiments involving bulk fusion assays in which the addition of synaptotagmin/Ca2+, Munc-18, or complexin accelerated the observed lipid-mixing rate, the enhancement may have arisen from the docking step rather than the fusion step. PMID:21539781

  4. Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle

    PubMed Central

    Deng, Yongqiang; Rivera-Molina, Felix E.; Toomre, Derek K.; Burd, Christopher G.

    2016-01-01

    One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers. PMID:27247384

  5. Vacuolar H+-ATPase subunits Voa1 and Voa2 cooperatively regulate secretory vesicle acidification, transmitter uptake, and storage

    PubMed Central

    Saw, Ner Mu Nar; Kang, Soo-Young Ann; Parsaud, Leon; Han, Gayoung Anna; Jiang, Tiandan; Grzegorczyk, Krzysztof; Surkont, Michael; Sun-Wada, Ge-Hong; Wada, Yoh; Li, Lijun; Sugita, Shuzo

    2011-01-01

    The Vo sector of the vacuolar H+-ATPase is a multisubunit complex that forms a proteolipid pore. Among the four isoforms (a1–a4) of subunit Voa, the isoform(s) critical for secretory vesicle acidification have yet to be identified. An independent function of Voa1 in exocytosis has been suggested. Here we investigate the function of Voa isoforms in secretory vesicle acidification and exocytosis by using neurosecretory PC12 cells. Fluorescence-tagged and endogenous Voa1 are primarily localized on secretory vesicles, whereas fluorescence-tagged Voa2 and Voa3 are enriched on the Golgi and early endosomes, respectively. To elucidate the functional roles of Voa1 and Voa2, we engineered PC12 cells in which Voa1, Voa2, or both are stably down-regulated. Our results reveal significant reductions in the acidification and transmitter uptake/storage of dense-core vesicles by knockdown of Voa1 and more dramatically of Voa1/Voa2 but not of Voa2. Overexpressing knockdown-resistant Voa1 suppresses the acidification defect caused by the Voa1/Voa2 knockdown. Unexpectedly, Ca2+-dependent peptide secretion is largely unaffected in Voa1 or Voa1/Voa2 knockdown cells. Our data demonstrate that Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, whereas they might not be as critical for exocytosis as recently proposed. PMID:21795392

  6. Imaging of Dynamic Secretory Vesicles in Living Pollen Tubes of Picea meyeri Using Evanescent Wave Microscopy1[W

    PubMed Central

    Wang, Xiaohua; Teng, Yan; Wang, Qinli; Li, Xiaojuan; Sheng, Xianyong; Zheng, Maozhong; Šamaj, Jozef; Baluška, František; Lin, Jinxing

    2006-01-01

    Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility. PMID:16798949

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

    PubMed

    Abu-Hamdah, Rania; Cho, Won Jin; Hörber, J K H; Jena, Bhanu P

    2006-01-01

    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.

  8. Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

    PubMed Central

    Higgins, S J; Burchell, J M; Mainwaring, W I

    1976-01-01

    1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug. Images PLATE 1(a) PLATES 1(b), 1(c) AND 1(d) PLATE 2 PMID:985427

  9. The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure

    PubMed Central

    Fargali, Samira; Garcia, Angelo L.; Sadahiro, Masato; Jiang, Cheng; Janssen, William G.; Lin, Wei-Jye; Cogliani, Valeria; Elste, Alice; Mortillo, Steven; Cero, Cheryl; Veitenheimer, Britta; Graiani, Gallia; Pasinetti, Giulio M.; Mahata, Sushil K.; Osborn, John W.; Huntley, George W.; Phillips, Greg R.; Benson, Deanna L.; Bartolomucci, Alessandro; Salton, Stephen R.

    2014-01-01

    Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1–615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1–524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1–615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.—Fargali, S., Garcia, A. L., Sadahiro, M., Jiang, C., Janssen, W. G., Lin, W.-J., Cogliani, V., Elste, A., Mortillo, S., Cero, C., Veitenheimer, B., Graiani, G., Pasinetti, G. M., Mahata, S. K., Osborn, J. W., Huntley, G. W., Phillips, G. R., Benson, D. L., Bartolomucci, A., Salton, S. R. The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure. PMID:24497580

  10. Mass spectrometry-based neuropeptidomics of secretory vesicles from human adrenal medullary pheochromocytoma reveals novel peptide products of prohormone processing.

    PubMed

    Gupta, Nitin; Bark, Steven J; Lu, Weiya D; Taupenot, Laurent; O'Connor, Daniel T; Pevzner, Pavel; Hook, Vivian

    2010-10-01

    Neuropeptides are required for cell-cell communication in the regulation of physiological and pathological processes. While selected neuropeptides of known biological activities have been studied, global analyses of the endogenous profile of human peptide products derived from prohormones by proteolytic processing in vivo are largely unknown. Therefore, this study utilized the global, unbiased approach of mass spectrometry-based neuropeptidomics to define peptide profiles in secretory vesicles, isolated from human adrenal medullary pheochromocytoma of the sympathetic nervous system. The low molecular weight pool of secretory vesicle peptides was subjected to nano-LC-MS/MS with ion trap and QTOF mass spectrometry analyzed by different database search tools (InsPecT and Spectrum Mill). Peptides were generated by processing of prohormones at dibasic cleavage sites as well as at nonbasic residues. Significantly, peptide profiling provided novel insight into newly identified peptide products derived from proenkephalin, pro-NPY, proSAAS, CgA, CgB, and SCG2 prohormones. Previously unidentified intervening peptide domains of prohormones were observed, thus providing new knowledge of human neuropeptidomes generated from precursors. The global peptidomic approach of this study demonstrates the complexity of diverse neuropeptides present in human secretory vesicles for cell-cell communication.

  11. Porosome: The Universal Secretory Portal in Cells

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu

    2012-10-01

    In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the ``all or none'' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such `all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in 1993 in a News and Views article in Nature, E. Neher wrote ``It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.'' This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the ``Porosome,'' the universal secretory machinery in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nm resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm

  12. Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity.

    PubMed

    Christensen, Sune M; Mortensen, Michael W; Stamou, Dimitrios G

    2011-02-16

    Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca(2+) dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca(2+) accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca(2+) accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t(mix) < 1 s) while an ensemble assay revealed two slow mixing processes with t(mix) ∼ 1 min and t(mix) ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Cysteine Cathepsins in the Secretory Vesicle Produce Active Peptides: Cathepsin L Generates Peptide Neurotransmitters and Cathepsin B Produces Beta-Amyloid of Alzheimer’s Disease

    PubMed Central

    Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

    2011-01-01

    Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles has been demonstrated as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β-amyloid (Aβ) peptides that accumulate in Alzheimer’s disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrasts with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin function. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. PMID:21925292

  14. Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules.

    PubMed

    Hwang, Shin-Rong; O'Neill, Audrey; Bark, Steven; Foulon, Thierry; Hook, Vivian

    2007-03-01

    Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.

  15. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

    PubMed Central

    1985-01-01

    Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins. PMID:3988801

  16. Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation in Saccharomyces cerevisiae

    PubMed Central

    Mathieson, Erin M.; Suda, Yasuyuki; Nickas, Mark; Snydsman, Brian; Davis, Trisha N.; Muller, Eric G. D.

    2010-01-01

    During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion. PMID:20826607

  17. Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

    PubMed

    Gurunathan, Sangiliyandi; David, Doris; Gerst, Jeffrey E

    2002-02-15

    Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.

  18. The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

    PubMed Central

    Kasula, Ravikiran; Bademosi, Adekunle T.; Harper, Callista B.; Morrow, Isabel C.; Hosy, Eric; Collins, Brett M.; Choquet, Daniel

    2016-01-01

    Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming. PMID:27646276

  19. Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland.

    PubMed

    Radha, Arumugam; Sree, Sreesha; Faisal, Kunnathodi; Kumar, G Pradeep; Oommen, Oommen V; Akbarsha, Mohammad A

    2014-10-01

    Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.

  20. Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering.

    PubMed

    Mohrmann, Ralf; de Wit, Heidi; Connell, Emma; Pinheiro, Paulo S; Leese, Charlotte; Bruns, Dieter; Davletov, Bazbek; Verhage, Matthijs; Sørensen, Jakob B

    2013-09-04

    SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

  1. Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles

    PubMed Central

    Molnár, Gábor; Rózsa, Márton; Baka, Judith; Holderith, Noémi; Barzó, Pál; Nusser, Zoltan; Tamás, Gábor

    2016-01-01

    Classic theories link cognitive abilities to synaptic properties and human-specific biophysical features of synapses might contribute to the unparalleled performance of the human cerebral cortex. Paired recordings and multiple probability fluctuation analysis revealed similar quantal sizes, but 4-times more functional release sites in human pyramidal cell to fast-spiking interneuron connections compared to rats. These connections were mediated on average by three synaptic contacts in both species. Each presynaptic active zone (AZ) contains 6.2 release sites in human, but only 1.6 in rats. Electron microscopy (EM) and EM tomography showed that an AZ harbors 4 docked vesicles in human, but only a single one in rats. Consequently, a Katz’s functional release site occupies ~0.012 μm2 in the human presynaptic AZ and ~0.025 μm2 in the rat. Our results reveal a robust difference in the biophysical properties of a well-defined synaptic connection of the cortical microcircuit of human and rodents. DOI: http://dx.doi.org/10.7554/eLife.18167.001 PMID:27536876

  2. Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles.

    PubMed

    Molnár, Gábor; Rózsa, Márton; Baka, Judith; Holderith, Noémi; Barzó, Pál; Nusser, Zoltan; Tamás, Gábor

    2016-08-18

    Classic theories link cognitive abilities to synaptic properties and human-specific biophysical features of synapses might contribute to the unparalleled performance of the human cerebral cortex. Paired recordings and multiple probability fluctuation analysis revealed similar quantal sizes, but 4-times more functional release sites in human pyramidal cell to fast-spiking interneuron connections compared to rats. These connections were mediated on average by three synaptic contacts in both species. Each presynaptic active zone (AZ) contains 6.2 release sites in human, but only 1.6 in rats. Electron microscopy (EM) and EM tomography showed that an AZ harbors 4 docked vesicles in human, but only a single one in rats. Consequently, a Katz's functional release site occupies ~0.012 μm(2) in the human presynaptic AZ and ~0.025 μm(2) in the rat. Our results reveal a robust difference in the biophysical properties of a well-defined synaptic connection of the cortical microcircuit of human and rodents.

  3. Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

    PubMed

    Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

    2014-01-01

    The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

  4. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  5. Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles

    PubMed Central

    Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

    2014-01-01

    The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations. PMID:24681578

  6. Fine ultrastructure of chromaffin granules in rat adrenal medulla indicative of a vesicle-mediated secretory process.

    PubMed

    Crivellato, E; Guidolin, D; Nico, B; Nussdorfer, G G; Ribatti, D

    2006-01-01

    Observation by transmission electron microscopy, coupled with morphometric analysis and estimation procedure, revealed unique ultrastructural features in 25.94% of noradrenaline (NA)-containing granules and 16.85% of adrenaline (A)-containing granules in the rat adrenal medulla. These consisted of evaginations of the granule limiting membrane to form budding structures having different morphology and extension. In 14.8% of NA granules and 12.0% of A granules, outpouches were relatively short, looked like small blebs emerging from the granule surface and generally contained electron-dense material. A proportion of 11.2% of NA granules and 4.9% of A granules revealed the most striking ultrastructural features. These secretory organelles presented thin, elongated, tail-like or stem-like appendages, which were variably filled by chromaffin substance and terminated with spherical expansions of different electron density. A cohort of vesicles of variable size (30-150 nm in diameter) and content was found either close to them or in the intergranular cytosol. Examination of adrenal medullary cells fixed by zinc iodide-osmium tetroxide (ZIO) revealed fine electron dense precipitates in chromaffin granules, budding structures as well as cytoplasmic vesicles. These data indicate that a common constituent is revealed by the ZIO histochemical reaction in chromaffin cells. As catecholic compounds are the main tissue targets of ZIO complexes, catecholamines are good candidates to be responsible for the observed ZIO reactivity. This study adds further to the hypothesis that release of secretory material from chromaffin granules may be accomplished by a vesiclular transport mechanism typical of piecemeal degranulation.

  7. An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

    PubMed Central

    Keller, Daniel; Babai, Norbert; Kochubey, Olexiy; Han, Yunyun; Markram, Henry; Schürmann, Felix; Schneggenburger, Ralf

    2015-01-01

    The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses. PMID:25951120

  8. Titration of Syntaxin1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.

    PubMed

    Arancillo, Marife; Min, Sang-Won; Gerber, Stefan; Münster-Wandowski, Agnieszka; Wu, Yuan-Ju; Herman, Melissa; Trimbuch, Thorsten; Rah, Jong-Cheol; Ahnert-Hilger, Gudrun; Riedel, Dietmar; Südhof, Thomas C; Rosenmund, Christian

    2013-10-16

    Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.

  9. Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

    PubMed Central

    Gabel, Marion; Delavoie, Franck; Demais, Valérie; Royer, Cathy; Bailly, Yannick; Vitale, Nicolas; Bader, Marie-France

    2015-01-01

    Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites. PMID:26323692

  10. BDNF Enhances Quantal Neurotransmitter Release and Increases the Number of Docked Vesicles at the Active Zones of Hippocampal Excitatory Synapses

    PubMed Central

    Tyler, William J.; Pozzo-Miller, Lucas D.

    2009-01-01

    Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF–TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory. PMID:11404410

  11. SUMOylation Regulates Insulin Exocytosis Downstream of Secretory Granule Docking in Rodents and Humans

    PubMed Central

    Dai, Xiao-Qing; Plummer, Greg; Casimir, Marina; Kang, Youhou; Hajmrle, Catherine; Gaisano, Herbert Y.; Manning Fox, Jocelyn E.; MacDonald, Patrick E.

    2011-01-01

    OBJECTIVE The reversible attachment of small ubiquitin-like modifier (SUMO) proteins controls target localization and function. We examined an acute role for the SUMOylation pathway in downstream events mediating insulin secretion. RESEARCH DESIGN AND METHODS We studied islets and β-cells from mice and human donors, as well as INS-1 832/13 cells. Insulin secretion, intracellular Ca2+, and β-cell exocytosis were monitored after manipulation of the SUMOylation machinery. Granule localization was imaged by total internal reflection fluorescence and electron microscopy; immunoprecipitation and Western blotting were used to examine the soluble NSF attachment receptor (SNARE) complex formation and SUMO1 interaction with synaptotagmin VII. RESULTS SUMO1 impairs glucose-stimulated insulin secretion by blunting the β-cell exocytotic response to Ca2+. The effect of SUMO1 to impair insulin secretion and β-cell exocytosis is rapid and does not require altered gene expression or insulin content, is downstream of granule docking at the plasma membrane, and is dependent on SUMO-conjugation because the deSUMOylating enzyme, sentrin/SUMO-specific protease (SENP)-1, rescues exocytosis. SUMO1 coimmunoprecipitates with the Ca2+ sensor synaptotagmin VII, and this is transiently lost upon glucose stimulation. SENP1 overexpression also disrupts the association of SUMO1 with synaptotagmin VII and mimics the effect of glucose to enhance exocytosis. Conversely, SENP1 knockdown impairs exocytosis at stimulatory glucose levels and blunts glucose-dependent insulin secretion from mouse and human islets. CONCLUSIONS SUMOylation acutely regulates insulin secretion by the direct and reversible inhibition of β-cell exocytosis in response to intracellular Ca2+ elevation. The SUMO protease, SENP1, is required for glucose-dependent insulin secretion. PMID:21266332

  12. Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi.

    PubMed

    Kienle, Nickias; Kloepper, Tobias H; Fasshauer, Dirk

    2009-01-23

    In eukaryotic cells, directional transport between different compartments of the endomembrane system is mediated by vesicles that bud from a donor organelle and then fuse with an acceptor organelle. A family of integral membrane proteins, termed soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, constitute the key machineries of these different membrane fusion events. Over the past 30 years, the yeast Saccharomyces cerevisiae has served as a powerful model organism for studying the organization of the secretory and endocytic pathways, and a few years ago, its entire set of SNAREs was compiled. Here, we make use of the increasing amount of genomic data to investigate the history of the SNARE family during fungi evolution. Moreover, since different SNARE family members are thought to demarcate different organelles and vesicles, this approach allowed us to compare the organization of the endomembrane systems of yeast and animal cells. Our data corroborate the notion that fungi generally encompass a relatively simple set of SNARE proteins, mostly comprising the SNAREs of the proto-eukaryotic cell. However, all fungi contain a novel soluble SNARE protein, Vam7, which carries an N-terminal PX-domain that acts as a phosphoinositide binding module. In addition, the points in fungal evolution, at which lineage-specific duplications and diversifications occurred, could be determined. For instance, the endosomal syntaxins Pep12 and Vam3 arose from a gene duplication that occurred within the Saccharomycotina clade. Although the SNARE repertoire of baker's yeast is highly conserved, our analysis reveals that it is more deviated than the ones of basal fungi. This highlights that the trafficking pathways of baker's yeast are not only different to those in animal cells but also are somewhat different to those of many other fungi.

  13. Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi

    PubMed Central

    Kienle, Nickias; Kloepper, Tobias H; Fasshauer, Dirk

    2009-01-01

    Background In eukaryotic cells, directional transport between different compartments of the endomembrane system is mediated by vesicles that bud from a donor organelle and then fuse with an acceptor organelle. A family of integral membrane proteins, termed soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, constitute the key machineries of these different membrane fusion events. Over the past 30 years, the yeast Saccharomyces cerevisiae has served as a powerful model organism for studying the organization of the secretory and endocytic pathways, and a few years ago, its entire set of SNAREs was compiled. Results Here, we make use of the increasing amount of genomic data to investigate the history of the SNARE family during fungi evolution. Moreover, since different SNARE family members are thought to demarcate different organelles and vesicles, this approach allowed us to compare the organization of the endomembrane systems of yeast and animal cells. Our data corroborate the notion that fungi generally encompass a relatively simple set of SNARE proteins, mostly comprising the SNAREs of the proto-eukaryotic cell. However, all fungi contain a novel soluble SNARE protein, Vam7, which carries an N-terminal PX-domain that acts as a phosphoinositide binding module. In addition, the points in fungal evolution, at which lineage-specific duplications and diversifications occurred, could be determined. For instance, the endosomal syntaxins Pep12 and Vam3 arose from a gene duplication that occurred within the Saccharomycotina clade. Conclusion Although the SNARE repertoire of baker's yeast is highly conserved, our analysis reveals that it is more deviated than the ones of basal fungi. This highlights that the trafficking pathways of baker's yeast are not only different to those in animal cells but also are somewhat different to those of many other fungi. PMID:19166604

  14. Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

    PubMed

    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R; Rettig, Jens

    2010-09-20

    Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

  15. More Docked Vesicles and Larger Active Zones at Basket Cell-to-Granule Cell Synapses in a Rat Model of Temporal Lobe Epilepsy

    PubMed Central

    Yamawaki, Ruth; Thind, Khushdev

    2016-01-01

    Temporal lobe epilepsy is a common and challenging clinical problem, and its pathophysiological mechanisms remain unclear. One possibility is insufficient inhibition in the hippocampal formation where seizures tend to initiate. Normally, hippocampal basket cells provide strong and reliable synaptic inhibition at principal cell somata. In a rat model of temporal lobe epilepsy, basket cell-to-granule cell (BC→GC) synaptic transmission is more likely to fail, but the underlying cause is unknown. At some synapses, probability of release correlates with bouton size, active zone area, and number of docked vesicles. The present study tested the hypothesis that impaired GABAergic transmission at BC→GC synapses is attributable to ultrastructural changes. Boutons making axosomatic symmetric synapses in the granule cell layer were reconstructed from serial electron micrographs. BC→GC boutons were predicted to be smaller in volume, have fewer and smaller active zones, and contain fewer vesicles, including fewer docked vesicles. Results revealed the opposite. Compared with controls, epileptic pilocarpine-treated rats displayed boutons with over twice the average volume, active zone area, total vesicles, and docked vesicles and with more vesicles closer to active zones. Larger active zones in epileptic rats are consistent with previous reports of larger amplitude miniature IPSCs and larger BC→GC quantal size. Results of this study indicate that transmission failures at BC→GC synapses in epileptic pilocarpine-treated rats are not attributable to smaller boutons or fewer docked vesicles. Instead, processes following vesicle docking, including priming, Ca2+ entry, or Ca2+ coupling with exocytosis, might be responsible. SIGNIFICANCE STATEMENT One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported

  16. More Docked Vesicles and Larger Active Zones at Basket Cell-to-Granule Cell Synapses in a Rat Model of Temporal Lobe Epilepsy.

    PubMed

    Buckmaster, Paul S; Yamawaki, Ruth; Thind, Khushdev

    2016-03-16

    Temporal lobe epilepsy is a common and challenging clinical problem, and its pathophysiological mechanisms remain unclear. One possibility is insufficient inhibition in the hippocampal formation where seizures tend to initiate. Normally, hippocampal basket cells provide strong and reliable synaptic inhibition at principal cell somata. In a rat model of temporal lobe epilepsy, basket cell-to-granule cell (BC→GC) synaptic transmission is more likely to fail, but the underlying cause is unknown. At some synapses, probability of release correlates with bouton size, active zone area, and number of docked vesicles. The present study tested the hypothesis that impaired GABAergic transmission at BC→GC synapses is attributable to ultrastructural changes. Boutons making axosomatic symmetric synapses in the granule cell layer were reconstructed from serial electron micrographs. BC→GC boutons were predicted to be smaller in volume, have fewer and smaller active zones, and contain fewer vesicles, including fewer docked vesicles. Results revealed the opposite. Compared with controls, epileptic pilocarpine-treated rats displayed boutons with over twice the average volume, active zone area, total vesicles, and docked vesicles and with more vesicles closer to active zones. Larger active zones in epileptic rats are consistent with previous reports of larger amplitude miniature IPSCs and larger BC→GC quantal size. Results of this study indicate that transmission failures at BC→GC synapses in epileptic pilocarpine-treated rats are not attributable to smaller boutons or fewer docked vesicles. Instead, processes following vesicle docking, including priming, Ca(2+) entry, or Ca(2+) coupling with exocytosis, might be responsible. One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported impaired inhibition

  17. Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations.

    PubMed

    Correa-Basurto, José; Cuevas-Hernández, Roberto I; Phillips-Farfán, Bryan V; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L; Pérez-González, Óscar A; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

  18. Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

    PubMed Central

    Correa-Basurto, José; Cuevas-Hernández, Roberto I.; Phillips-Farfán, Bryan V.; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L.; Pérez-González, Óscar A.; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G.

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression. PMID:25914622

  19. The 11S rat seminal vesicle mRNA directs the in vitro synthesis of two precursors of the major secretory protein IV.

    PubMed Central

    Metafora, S; Guardiola, J; Paonessa, G; Abrescia, P

    1984-01-01

    The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule. Images PMID:6701092

  20. Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    PubMed Central

    Perry, Mark; Granjon, Thierry; Gonzalvez, François; Gottlieb, Eyal; Ayala-Sanmartin, Jesus; Klösgen, Beate; Schwille, Petra; Petit, Patrice X.

    2013-01-01

    Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria. PMID:23418437

  1. The secretory activity of the seminal vesicles and its relationship to sperm motility: effects of infection in the male reproductive tract.

    PubMed

    Gonzales, G F; Garcia-Hjarles, M A; Gutierrez, R; Guerra-Garcia, R

    1989-08-01

    In 146 males aged between 20 years and 40 years attending an infertility service, the secretory activity of the seminal vesicles was assessed by measurement of corrected seminal fructose concentration. This value was related to the presence of a positive semen culture, other evidence of inflammatory processes in the reproductive tract and sperm motility. Only 48% of subjects with a positive semen culture showed evidence of inflammation in the reproductive tract, as assessed by the presence of more than 20 white blood cells per high power field, and greater than 10% spermagglutination in the ejaculate. There was a relationship between the inflammatory process, hypofunction of the seminal vesicles and poor sperm motility. When the semen culture was positive but there was no evidence of inflammation neither seminal vesicle function nor sperm motility was affected. When the semen culture was negative, i.e. no evidence of inflammation and the subjects were asthenozoospermic, the corrected fructose levels were normal. It is proposed that in these conditions the cause of asthenozoospermia may be factors other than accessory sex organ dysfunction. In conclusion, there was no close relationship between the bacteriological results and evidence of inflammation of the accessory glands. A positive semen culture was related to lower levels of corrected fructose (hypofunction of the seminal vesicles) when the positive sperm culture was associated with inflammation of the reproductive tract and asthenozoospermia.

  2. A Putative Small Solute Transporter Is Responsible for the Secretion of G377 and TRAP-Containing Secretory Vesicles during Plasmodium Gamete Egress and Sporozoite Motility

    PubMed Central

    Kehrer, Jessica; Singer, Mirko; Lemgruber, Leandro; Silva, Patricia A. G. C.; Frischknecht, Friedrich; Mair, Gunnar R.

    2016-01-01

    Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission. PMID:27427910

  3. Vesicles

    MedlinePlus

    ... the top of a pin or up to 5 millimeters wide. A larger blister is called a bulla. In many cases, vesicles break easily and release their fluid onto the skin. When this fluid dries, yellow crusts may remain on the skin surface.

  4. Accumulation of secretory vesicles in the lacrimal gland epithelia is related to non-Sjögren's type dry eye in visual display terminal users.

    PubMed

    Kamoi, Mizuka; Ogawa, Yoko; Nakamura, Shigeru; Dogru, Murat; Nagai, Toshihiro; Obata, Hiroto; Ito, Masataka; Kaido, Minako; Kawakita, Tetsuya; Okada, Yasunori; Kawakami, Yutaka; Shimmura, Shigeto; Tsubota, Kazuo

    2012-01-01

    Previous observations in a rat model of a non-Sjögren's syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.

  5. Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking

    PubMed Central

    Choi, Bong-Kyu; Choi, Mal-Gi; Kim, Jae-Yeol; Yang, Yoosoo; Lai, Ying; Kweon, Dae-Hyuk; Lee, Nam Ki; Shin, Yeon-Kyun

    2013-01-01

    Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity. PMID:23431141

  6. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

    PubMed

    Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

    1999-01-29

    Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

  7. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane*

    PubMed Central

    Johnson, Jennifer L.; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B.; Zhang, Jinzhong; Catz, Sergio D.

    2016-01-01

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. PMID:26637356

  8. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

    PubMed

    Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

    2016-02-12

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

  9. The Arabidopsis P4-ATPase ALA3 Localizes to the Golgi and Requires a β-Subunit to Function in Lipid Translocation and Secretory Vesicle Formation[W

    PubMed Central

    Poulsen, Lisbeth Rosager; López-Marqués, Rosa Laura; McDowell, Stephen C.; Okkeri, Juha; Licht, Dirk; Schulz, Alexander; Pomorski, Thomas; Harper, Jeffrey F.; Palmgren, Michael Gjedde

    2008-01-01

    Vesicle budding in eukaryotes depends on the activity of lipid translocases (P4-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P4-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a β-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development. PMID:18344284

  10. Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

    PubMed Central

    Bowman, Grant R.; Elde, Nels C.; Morgan, Garry; Winey, Mark; Turkewitz, Aaron P.

    2016-01-01

    The formation of dense core secretory granules is a multi-stage process beginning in the trans-Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion. PMID:15752136

  11. Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles

    PubMed Central

    Miklavc, Pika; Ehinger, Konstantin; Sultan, Ayesha; Felder, Tatiana; Paul, Patrick; Gottschalk, Kay-Eberhard; Frick, Manfred

    2015-01-01

    ABSTRACT In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion. PMID:25637593

  12. A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells.

    PubMed

    Kedar, Girish H; Munch, Anders S; van Weering, Jan R T; Malsam, Jörg; Scheutzow, Andrea; de Wit, Heidi; Houy, Sébastien; Tawfik, Bassam; Söllner, Thomas H; Sørensen, Jakob B; Verhage, Matthijs

    2015-10-21

    Synaptotagmin-1 (Syt1) is the principal Ca(2+) sensor for vesicle fusion and is also essential for vesicle docking in chromaffin cells. Docking depends on interactions of the Syt1-C2B domain with the t-SNARE SNAP25/Syntaxin1 complex and/or plasma membrane phospholipids. Here, we investigated the role of the positively charged "bottom" region of the C2B domain, proposed to help crosslink membranes, in vesicle docking and secretion in mouse chromaffin cells and in cell-free assays. We expressed a double mutation shown previously to interfere with lipid mixing between proteoliposomes and with synaptic transmission, Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2 and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQ-induced docking was strictly PI(4,5)P2-dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca(2+)-triggered SUV-GUV fusion was restored by the Syt1 mutants. Finally, overexpressing the RQ-mutant in wild type cells produced no effect on either docking or secretion. We conclude that the positively charged bottom region in the C2B domain--and, by inference, Syt1-mediated membrane crosslinking--is required for triggering fusion, but not for docking. Secretory vesicles dock by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. The R398/399 mutations selectively disrupt the latter and hereby help to discriminate protein regions involved in different aspects of Syt1 function in docking and fusion. This study provides new insights in how the two opposite sides of the C2B domain of Synaptotagmin-1 participate in secretory vesicle fusion, and in more upstream steps, especially vesicle docking. We show

  13. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    SciTech Connect

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  14. A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone

    PubMed Central

    Lübbert, Matthias; Goral, R Oliver; Satterfield, Rachel; Putzke, Travis; van den Maagdenberg, Arn MJM; Kamasawa, Naomi; Young, Samuel M

    2017-01-01

    In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses. DOI: http://dx.doi.org/10.7554/eLife.28412.001 PMID:28786379

  15. Temporal separation of insulin-stimulated GLUT4/IRAP vesicle plasma membrane docking and fusion in 3T3L1 adipocytes.

    PubMed

    Elmendorf, J S; Boeglin, D J; Pessin, J E

    1999-12-24

    Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.

  16. Secretory carrier membrane protein SCAMP2 and phosphatidylinositol 4,5-bisphosphate interactions in the regulation of dense core vesicle exocytosis.

    PubMed

    Liao, Haini; Ellena, Jeff; Liu, Lixia; Szabo, Gabor; Cafiso, David; Castle, David

    2007-09-25

    Secretory carrier membrane protein 2 (SCAMP2) functions in late steps of membrane fusion in calcium-dependent granule exocytosis. A basic/hydrophobic peptide segment within SCAMP2 (SCAMP2 E: CWYRPIYKAFR) has been implicated in this function and shown to bind and sequester phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] within membranes through an electrostatic mechanism. We now show that alanine substitution of tryptophan W2 within SCAMP2 E substantially weakens peptide binding to negatively charged liposomes; other substitutions for arginine R4 and lysine K8 have only limited effects on binding. Electron paramagnetic resonance analysis of liposomes containing spin-labeled PIP2 shows that R4 but not K8 is critical for SCAMP E binding to PIP2. The interfacial locations of SCAMP E and its structural variants within lipid bicelles measured by oxygen enhancement of nuclear relaxation are all similar. Corresponding point mutations within full-length SCAMP2 (SC2-R204A, SC2-K208A, and SC2-W202A) have been analyzed for biological effects on dense core vesicle exocytosis in neuroendocrine PC12 cells. With the same level of overexpression, SC2-R204A but not SC2-K208A inhibited secretion of cotransfected human growth hormone and of noradrenalin. Inhibition by SC2-R204A was the same as or greater than previously observed for SC2-W202A. Analysis of noradrenalin secretion by amperometry showed that inhibitory mutants of SCAMP2 decrease the probability of fusion pore opening and the stability of initially opened but not yet expanded fusion pores. The strong correlation between SCAMP2 E interactions with PIP2 and inhibition of exocytosis, particularly by SC2-R204A, led us to propose that SCAMP2 interaction with PIP2 within the membrane interface regulates fusion pore formation during exocytosis.

  17. PyroGlu-Aβ and Glutaminyl Cyclase are Co-Localized with Aβ in Secretory Vesicles and Undergo Activity-Dependent Secretion

    PubMed Central

    Cynis, Holger; Funkelstein, Lydiane; Toneff, Thomas; Mosier, Charles; Ziegler, Michael; Koch, Britta; Demuth, Hans-Ulrich; Hook, Vivian

    2014-01-01

    Background and Aims N-truncated pGlu-Aβ(3-40/42) peptides are key components that promote Aβ peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer’s disease. Because Aβ deposition in brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aβ(3-40/42) production by glutaminyl cyclase, and their localization with full-length Aβ(1-40/42) peptides for regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and glutaminyl cyclase (QC) are co-localized with Aβ in secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters. Methods Purified DCSV was assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitters. Neuronal-like chromaffin cells were analyzed for co-secretion of pGlu-Aβ, QC, Aβ, and neuropeptides. Cells were treated with a QC inhibitor and pGlu-Aβ was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC. Results Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ(3-40/42) and QC undergo activity-dependent co-secretion with Aβ(1-40/42) and enkephalin and galanin neurotransmitters. A QC inhibitor decreased levels of pGlu-Aβ. Human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that is co-localized with QC. Conclusions PyroGlu-Aβ and QC are present with Aβ in DCSV, and undergo activity-dependent, regulated co-secretion with neurotransmitters. PMID:24943989

  18. Fluconazole transport into Candida albicans secretory vesicles by the membrane proteins Cdr1p, Cdr2p, and Mdr1p.

    PubMed

    Basso, Luiz R; Gast, Charles E; Mao, Yuxin; Wong, Brian

    2010-06-01

    A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [(3)H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [(3)H]fluconazole than did PGVs from empty-vector controls. The K(m)s (expressed in micromolar concentrations) and V(max)s (expressed in picomoles per milligram of protein per minute), respectively, for [(3)H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [(3)H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [(3)H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [(3)H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC(50)s; expressed as proportions of the [(3)H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.

  19. Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p ▿

    PubMed Central

    Basso, Luiz R.; Gast, Charles E.; Mao, Yuxin; Wong, Brian

    2010-01-01

    A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [3H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [3H]fluconazole than did PGVs from empty-vector controls. The Kms (expressed in micromolar concentrations) and Vmaxs (expressed in picomoles per milligram of protein per minute), respectively, for [3H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [3H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [3H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [3H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC50s; expressed as proportions of the [3H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi. PMID:20348384

  20. Reconstitution of calcium-mediated exocytosis of dense-core vesicles

    PubMed Central

    Kreutzberger, Alex J. B.; Kiessling, Volker; Liang, Binyong; Seelheim, Patrick; Jakhanwal, Shrutee; Jahn, Reinhard; Castle, J. David; Tamm, Lukas K.

    2017-01-01

    Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)–dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction. PMID:28776026

  1. Two Distinct Modes of Exocytotic Fusion Pore Expansion in Large Astrocytic Vesicles*

    PubMed Central

    Peng, Hong; Kang, Ning; Xu, Jun; Stanton, Patric K.; Kang, Jian

    2013-01-01

    Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (2–7 μm) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and the movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. Shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101, stained the fusion-produced membrane fragment more brightly than FM 1-43. Sulforhodamine 101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism underlying fusion pore expansion and provide a new explanation for full and partial collapses of large secretory vesicles. PMID:23620588

  2. Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway

    PubMed Central

    Hariri, Hanaa; Bhattacharya, Nilakshee; Johnson, Kerri; Noble, Alex J; Stagg, Scott M.

    2014-01-01

    The small GTPase protein Sar1 is known to be involved in both the initiation of COPII coated vesicle formation and scission of the nascent vesicle from the ER. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission. PMID:25193674

  3. Secretory autophagy.

    PubMed

    Ponpuak, Marisa; Mandell, Michael A; Kimura, Tomonori; Chauhan, Santosh; Cleyrat, Cédric; Deretic, Vojo

    2015-08-01

    Autophagy, once viewed exclusively as a cytoplasmic auto-digestive process, has its less intuitive but biologically distinct non-degradative roles. One manifestation of these functions of the autophagic machinery is the process termed secretory autophagy. Secretory autophagy facilitates unconventional secretion of the cytosolic cargo such as leaderless cytosolic proteins, which unlike proteins endowed with the leader (N-terminal signal) peptides cannot enter the conventional secretory pathway normally operating via the endoplasmic reticulum and the Golgi apparatus. Secretory autophagy may also export more complex cytoplasmic cargo and help excrete particulate substrates. Autophagic machinery and autophagy as a process also affect conventional secretory pathways, including the constitutive and regulated secretion, as well as promote alternative routes for trafficking of integral membrane proteins to the plasma membrane. Thus, autophagy and autophagic factors are intimately intertwined at many levels with secretion and polarized sorting in eukaryotic cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Neuronal porosome - The secretory portal at the nerve terminal: Its structure-function, composition, and reconstitution

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu P.

    2014-09-01

    Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate secretion from cells. Membrane bound secretory vesicles transiently dock and fuse at the cytosolic compartment of the porosome base to expel intravesicular contents to the outside during cell secretion. In the past decade, the structure, isolation, composition, and functional reconstitution of the neuronal porosome complex has been accomplished providing a molecular understanding of its structure-function. Neuronal porosomes are 15 nm cup-shaped lipoprotein structures composed of nearly 40 proteins; compared to the 120 nm nuclear pore complex comprised of over 500 protein molecules composed of 30 different proteins. Being a membrane-associated supramolecular complex has precluded determination of the atomic structure of the porosome. However recent studies using small-angle X-ray solution scattering (SAXS), provide at sub-nanometer resolution, the native 3D structure of the neuronal porosome complex associated with docked synaptic vesicle at the nerve terminal. Additionally, results from the SAXS study and earlier studies using atomic force microscopy, provide the possible molecular mechanism involved in porosome-mediated neurotransmitter release at the nerve terminal.

  5. A biosynthetic regulated secretory pathway in constitutive secretory cells

    PubMed Central

    1996-01-01

    It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic- like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed. PMID:8682857

  6. Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells.

    PubMed

    Nili, U; de Wit, H; Gulyas-Kovacs, A; Toonen, R F; Sørensen, J B; Verhage, M; Ashery, U

    2006-12-01

    Activation of protein kinase C (PKC) after robust stimulation is necessary for vesicle pool replenishment in secretory cells. Here we studied the contribution of a prominent downstream PKC target, Munc18-1, to this process in bovine chromaffin cells. In these cells, both activation of endogenous PKC and overexpressing of Munc18-1 promote vesicle pool replenishment after an extensive stimulation. In order to study the physiological relevance of PKC-dependent Munc18-1 phosphorylation, we generated two Munc18-1 phospho-mutants; one that mimics a constitutively PKC-phosphorylated Munc18-1 (i.e. a phosphomimetic mutant; Munc18-1(S313D)) and a second that cannot be PKC-phosphorylated (Munc18-1(3A)). Overexpression of Munc18-1(3A) caused a significant decrease in vesicle pool replenishment following a depleting stimulation, while Munc18-1(S313D) caused a significant increase in vesicle pool replenishment. These findings suggested that the phosphorylation of Munc18-1 by PKC potentiates vesicle pool replenishment. This hypothesis was further strengthened by the finding that overexpression of wild type Munc18-1 in the presence of a PKC inhibitor caused a significant reduction in vesicle pool replenishment, similar to that observed with Munc18-1(3A). Moreover, overexpression of Munc18-1(S313D) in the presence of the PKC inhibitor partly alleviated this attenuation, elucidating Munc18-1's unique contribution to vesicle pool replenishment. Finally, we demonstrate that Munc18-1 promotes vesicle docking in a phosphorylation-independent manner. This is deduced from the findings that both the wild type and the two Munc18-1 phospho-mutants enhanced docking to the same extent in bovine chromaffin cells. We conclude that Munc18-1 facilitates docking in a PKC phosphorylation-independent manner, and that its phosphorylation by PKC potentiates vesicle pool replenishment following a depleting stimulation, at a post-docking stage.

  7. Vesicle Photonics

    SciTech Connect

    Vasdekis, Andreas E.; Scott, E. A.; Roke, Sylvie; Hubbell, J. A.; Psaltis, D.

    2013-04-03

    Thin membranes, under appropriate boundary conditions, can self-assemble into vesicles, nanoscale bubbles that encapsulate and hence protect or transport molecular payloads. In this paper, we review the types and applications of light fields interacting with vesicles. By encapsulating light-emitting molecules (e.g. dyes, fluorescent proteins, or quantum dots), vesicles can act as particles and imaging agents. Vesicle imaging can take place also under second harmonic generation from vesicle membrane, as well as employing mass spectrometry. Light fields can also be employed to transport vesicles using optical tweezers (photon momentum) or directly pertrurbe the stability of vesicles and hence trigger the delivery of the encapsulated payload (photon energy).

  8. Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

    PubMed Central

    MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

    2015-01-01

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

  9. Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

    PubMed

    MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

    2015-04-24

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

    PubMed Central

    Bäck, Nils; Rajagopal, Chitra; Mains, Richard E.; Eipper, Betty A.

    2010-01-01

    The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine α-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation demonstrated efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr946, Ser949) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100 kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins. PMID:20374556

  11. Involvement of vH+-ATPase in synaptic vesicle swelling

    PubMed Central

    Shin, Leah; Basi, Nirukti; Jeremic, Aleksandar; Lee, Jin-Sook; Cho, Won Jin; Chen, ZhiHui; Abu-Hamdah, Rania; Oupicky, David; Jena, Bhanu P

    2010-01-01

    Secretory vesicle swelling is central to cell secretion, however the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The Gαi3-PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas, and the Gαo-mediated AQP-6 involvement in synaptic vesicle swelling in neurons, has previously been reported. Furthermore, the role of vH+-ATPase in neurotransmitter transport into synaptic vesicles, has also been shown. Using nanometer scale precision measurements of isolated synaptic vesicles, the present study reports for the first time, the involvement of vH+-ATPase in GTP-Gαo-mediated synaptic vesicle swelling. Results from this study, demonstrate that the GTP-Gαo-mediated vesicle swelling is vH+-ATPase–dependent, and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate, a bafilomycin-sensitive vesicle acidification, following the GTP-Gαo-induced swelling stimulus. Since water channels are bidirectional, and the vH+-ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggests vH+-ATPase to be upstream of AQP-6, in the pathway leading from Gαo-stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6 mediated gating of water into synaptic vesicles. PMID:19610106

  12. Secretory granule biogenesis: rafting to the SNARE.

    PubMed

    Tooze, S A; Martens, G J; Huttner, W B

    2001-03-01

    Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.

  13. Toxoplasma PRP1 is an ortholog of parafusin (PFUS) in vesicle scaffold assembly in Ca(2+)-regulated exocytosis.

    PubMed

    Liu, Li; Tucker, Stephanie C; Satir, Birgit H

    2009-05-01

    The Paramecium tetraurelia protein parafusin (PFUS) and the Toxoplasma gondii protein parafusin-related protein 1 (PRP1) both have two covalent modifications (phosphorylation and phosphoglucosylation) and both are members of the phosphoglucomutase superfamily, associating with secretory vesicle scaffolds in their respective cells. This study tests the hypothesis that PRP1 is a functional ortholog of PFUS, functioning identically in Ca(2+)-regulated exocytosis. Electroporation of fluorescently labeled recombinant His-PRP1 into live Paramecium cells resulted in its localization to docked, dense-core secretory vesicles (DCSVs) in a pattern identical to endogenous PFUS. In tam8 mutants, defective in transport of DCSVs, the fluorescently labeled protein was restricted to the un-transported DCSVs. Specificity of PRP1 localization was demonstrated by electroporating labeled actin or pyruvate kinase, which both failed to localize to either docked or undocked vesicles. In wild-type Paramecium, electroporated His-PRP1 dissociated from DCSVs upon exocytosis, and re-associated as new organelles formed. Mutagenized His-PRP1 species (S146A or S146E) cannot be phosphorylated by P2 calcium-dependent kinase in vitro. Upon electroporation, these molecules remained cytoplasmic and un-associated with DCSVs, while mutation of another PRP1 serine residue (S560A) did neither affect the localization to the DCSVs nor the phosphorylation pattern. Therefore, in this heterologous system, localization, transport and dissociation/re-association of PRP1 substituted for PFUS, supporting the conclusion that the proteins are functional orthologs. The assay also identified a strategic residue S146 within the PFUS ortholog (S138 in PFUS by extrapolation) required for post-translational modification, DCSV scaffold association and for exocytosis.

  14. Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

    PubMed Central

    1992-01-01

    Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation. PMID:1572894

  15. Darwinian Docking

    NASA Astrophysics Data System (ADS)

    Kuntz, Irwin D.

    2012-01-01

    The Darwinian model of evolution is an optimization strategy that can be adapted to docking. It differs from the common use of genetic algorithms, primarily in its acceptance of diverse solutions over finding "global" optima. A related problem is selecting compounds using multiple criteria. I discuss these ideas and present the outlines of a protocol for selecting "hits" and "leads" in drug discovery.

  16. Gas vesicles.

    PubMed Central

    Walsby, A E

    1994-01-01

    The gas vesicle is a hollow structure made of protein. It usually has the form of a cylindrical tube closed by conical end caps. Gas vesicles occur in five phyla of the Bacteria and two groups of the Archaea, but they are mostly restricted to planktonic microorganisms, in which they provide buoyancy. By regulating their relative gas vesicle content aquatic microbes are able to perform vertical migrations. In slowly growing organisms such movements are made more efficiently than by swimming with flagella. The gas vesicle is impermeable to liquid water, but it is highly permeable to gases and is normally filled with air. It is a rigid structure of low compressibility, but it collapses flat under a certain critical pressure and buoyancy is then lost. Gas vesicles in different organisms vary in width, from 45 to > 200 nm; in accordance with engineering principles the narrower ones are stronger (have higher critical pressures) than wide ones, but they contain less gas space per wall volume and are therefore less efficient at providing buoyancy. A survey of gas-vacuolate cyanobacteria reveals that there has been natural selection for gas vesicles of the maximum width permitted by the pressure encountered in the natural environment, which is mainly determined by cell turgor pressure and water depth. Gas vesicle width is genetically determined, perhaps through the amino acid sequence of one of the constituent proteins. Up to 14 genes have been implicated in gas vesicle production, but so far the products of only two have been shown to be present in the gas vesicle: GvpA makes the ribs that form the structure, and GvpC binds to the outside of the ribs and stiffens the structure against collapse. The evolution of the gas vesicle is discussed in relation to the homologies of these proteins. Images PMID:8177173

  17. Signaling for Vesicle Mobilization and Synaptic Plasticity

    PubMed Central

    Levitan, Edwin S.

    2008-01-01

    The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs appears to be due to liberation of hindered vesicles to promote quicker diffusion. However, F-actin and synapsin, which have been featured in mobilization models, are not required for activity-dependent increases in the mobility of DCVs or SSVs. Most recently, the signaling required for sustained mobilization has been identified for Drosophila motoneuron DCVs and shown to increase synaptic transmission. Specifically, presynaptic endoplasmic reticulum ryanodine receptor (RyR)-mediated Ca2+ release activates Ca2+/calmodulin-dependent kinase II (CamKII) to mobilize DCVs and induce post-tetanic potentiation (PTP) of neuropeptide release in the Drosophila neuromuscular junction. The shared signaling for increasing vesicle mobility and PTP links vesicle mobilization and synaptic plasticity. PMID:18446451

  18. Hydrophobic contributions to the membrane docking of synaptotagmin 7 C2A domain: mechanistic contrast between isoforms 1 and 7.

    PubMed

    Brandt, Devin S; Coffman, Matthew D; Falke, Joseph J; Knight, Jefferson D

    2012-10-02

    Synaptotagmin (Syt) triggers Ca(2+)-dependent membrane fusion via its tandem C2 domains, C2A and C2B. The 17 known human isoforms are active in different secretory cell types, including neurons (Syt1 and others) and pancreatic β cells (Syt7 and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking mechanisms of Syt1 C2A and Syt7 C2A to vesicles comprised of physiological lipid mixtures. In agreement with previous studies, the Ca(2+) sensitivity of membrane binding is much higher for Syt7 C2A. We report here for the first time that this increased sensitivity is due to the slower target membrane dissociation of Syt7 C2A. Association and dissociation rate constants for Syt7 C2A are found to be ~2-fold and ~60-fold slower than Syt1 C2A, respectively. Furthermore, the membrane dissociation of Syt7 C2A but not Syt1 C2A is slowed by Na(2)SO(4) and trehalose, solutes that enhance the hydrophobic effect. Overall, the simplest model consistent with these findings proposes that Syt7 C2A first docks electrostatically to the target membrane surface and then inserts into the bilayer via a slow hydrophobic mechanism. In contrast, the membrane docking of Syt1 C2A is known to be predominantly electrostatic. Thus, these two highly homologous domains exhibit distinct mechanisms of membrane binding correlated with their known differences in function.

  19. Synaptic Vesicle Pools: An Update

    PubMed Central

    Denker, Annette; Rizzoli, Silvio O.

    2010-01-01

    During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or “pools”. We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are “fixed”. Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool. PMID:21423521

  20. The Cortical Acto-Myosin Network: From Diffusion Barrier to Functional Gateway in the Transport of Neurosecretory Vesicles to the Plasma Membrane

    PubMed Central

    Papadopulos, Andreas; Tomatis, Vanesa M.; Kasula, Ravikiran; Meunier, Frederic A.

    2013-01-01

    Dysregulation of regulated exocytosis is linked to an array of pathological conditions, including neurodegenerative disorders, asthma, and diabetes. Understanding the molecular mechanisms underpinning neuroexocytosis including the processes that allow neurosecretory vesicles to access and fuse with the plasma membrane and to recycle post-fusion, is therefore critical to the design of future therapeutic drugs that will efficiently tackle these diseases. Despite considerable efforts to determine the principles of vesicular fusion, the mechanisms controlling the approach of vesicles to the plasma membrane in order to undergo tethering, docking, priming, and fusion remain poorly understood. All these steps involve the cortical actin network, a dense mesh of actin filaments localized beneath the plasma membrane. Recent work overturned the long-held belief that the cortical actin network only plays a passive constraining role in neuroexocytosis functioning as a physical barrier that partly breaks down upon entry of Ca2+ to allow secretory vesicles to reach the plasma membrane. A multitude of new roles for the cortical actin network in regulated exocytosis have now emerged and point to highly dynamic novel functions of key myosin molecular motors. Myosins are not only believed to help bring about dynamic changes in the actin cytoskeleton, tethering and guiding vesicles to their fusion sites, but they also regulate the size and duration of the fusion pore, thereby directly contributing to the release of neurotransmitters and hormones. Here we discuss the functions of the cortical actin network, myosins, and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis. PMID:24155741

  1. Trafficking of astrocytic vesicles in hippocampal slices

    SciTech Connect

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  2. Expression of eosinophil target SNAREs as potential cognate receptors for vesicle-associated membrane protein-2 in exocytosis.

    PubMed

    Logan, Michael R; Lacy, Paige; Bablitz, Ben; Moqbel, Redwan

    2002-02-01

    Exocytosis of eosinophil granule-derived mediators is thought to be an important effector response contributing to allergic inflammation. Secretion from many cell types has been shown to be dependent on the formation of a docking complex composed of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on the vesicle (v-SNAREs) and the target membrane (t-SNAREs). The SNARE isoforms VAMP-2, SNAP-23, and syntaxin-4 have been described in secretory processes in myeloid cells. Previously, we have demonstrated that the v-SNARE VAMP-2 is a candidate v-SNARE involved in eosinophil exocytosis and is localized to a pool of RANTES-positive vesicles that translocate to the cell periphery after IFN-gamma-induced degranulation. We sought to determine whether eosinophils express the t-SNARE isoforms SNAP-23 and syntaxin-4 as potential binding targets for VAMP-2 during exocytosis. Human peripheral blood eosinophils (>97%) from atopic subjects were subjected to RT-PCR and sequence analysis by using specific primers for SNAP-23 and syntaxin-4. Protein expression and localization was determined by means of Western blot analysis of eosinophil subcellular fractions and confirmed with confocal laser scanning microscopy. Nucleotide sequences obtained from PCR products exhibited nearly identical (>95%) homology with reported sequences for human SNAP-23 and syntaxin-4. Both SNAP-23 and syntaxin-4 were present in plasma membranes, with some staining in endoplasmic reticulum and Golgi membranes. Negligible expression was detected in crystalloid and small secretory granules. The plasma membrane-associated t-SNAREs SNAP-23 and syntaxin-4 are expressed in human eosinophils and are likely candidates for association with VAMP-2 during docking, which is followed by exocytosis. These findings support a role for SNARE molecules in eosinophil mediator release.

  3. Studying Factors Involved in Biogenesis of Lysobacter sp. XL1 Outer Membrane Vesicles.

    PubMed

    Kudryakova, I V; Suzina, N E; Vinokurova, N G; Shishkova, N A; Vasilyeva, N V

    2017-04-01

    The Gram-negative bacterium Lysobacter sp. XL1 produces outer membrane vesicles that are heterogeneous in size, density, and protein composition. One of the subpopulations is secretory vesicles for lytic protease L5 of Lysobacter sp. XL1 (Kudryakova et al. (2015) FEMS Microbiol. Lett., 362, fnv137). Protein L5 was assumed to influence biogenesis of these secretory vesicles that contain it. Using a Pseudomonas fluorescens Q2-87/B expression system, it was shown that the recombinant L5 protein may act as a factor of vesicle biogenesis. This points to a possible involvement of L5 protein in Lysobacter sp. XL1 vesicle biogenesis. Furthermore, it was established that the main phospholipid of Lysobacter sp. XL1 vesicles is cardiolipin, and vesicles are formed predominantly of outer membrane regions enriched with this phospholipid. This indicates that cardiolipin participates in biogenesis of all vesicle subpopulations in Lysobacter sp. XL1.

  4. Human Platelet Vesicles Exhibit Distinct Size and Proteome.

    PubMed

    Jena, Bhanu P; Stemmer, Paul M; Wang, Sunxi; Mao, Guangzhao; Lewis, Kenneth T; Walz, Daniel A

    2017-07-07

    In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.

  5. An evolving paradigm for the secretory pathway?

    PubMed Central

    Lippincott-Schwartz, Jennifer

    2011-01-01

    The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes. PMID:22039065

  6. Cytoplasmic calcium stimulates exocytosis in a plant secretory cell

    PubMed Central

    Tester, Mark; Zorec, Robert

    1992-01-01

    Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (Cm), while dialyzing the cytosol with different Ca2+ containing solutions. Secretory activity increased with [Ca2+]i ∼ 1 μM. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca2+) for the control of exocytotic secretion. PMID:19431846

  7. Covalent attachment of lipid vesicles to a fluid supported bilayer allows observation of DNA-mediated vesicle interactions

    PubMed Central

    van Lengerich, Bettina; Rawle, Robert J.; Boxer, Steven G.

    2010-01-01

    Specific membrane interactions such as lipid vesicle docking and fusion can be mediated by synthetic DNA-lipid conjugates as a model for the protein-driven processes that are ubiquitous in biological systems. Here we present a method of tethering vesicles to a supported lipid bilayer that allows simultaneous deposition of cognate vesicle partners displaying complementary DNA, resulting in well-mixed populations of tethered vesicles that are laterally mobile. Vesicles are covalently attached to a supporting lipid bilayer using a DNA-templated click reaction; then DNA-mediated interactions between tethered vesicles are triggered by spiking the salt concentration. These interactions, such as docking and fusion, can then be observed for individual vesicles as they collide on the surface. The architecture of this new system also permits control over the number of lipid anchors that tether the vesicle to the supporting bilayer. The diffusion coefficient of tethered vesicles anchored by two lipids is approximately 1.6-fold slower than that of vesicles anchored only with a single lipid, consistent with a simple physical model. PMID:20180548

  8. Covalent attachment of lipid vesicles to a fluid-supported bilayer allows observation of DNA-mediated vesicle interactions.

    PubMed

    van Lengerich, Bettina; Rawle, Robert J; Boxer, Steven G

    2010-06-01

    Specific membrane interactions such as lipid vesicle docking and fusion can be mediated by synthetic DNA-lipid conjugates as a model for the protein-driven processes that are ubiquitous in biological systems. Here we present a method of tethering vesicles to a supported lipid bilayer that allows the simultaneous deposition of cognate vesicle partners displaying complementary DNA, resulting in well-mixed populations of tethered vesicles that are laterally mobile. Vesicles are covalently attached to a supporting lipid bilayer using a DNA-templated click reaction; then DNA-mediated interactions between tethered vesicles are triggered by spiking the salt concentration. These interactions, such as docking and fusion, can then be observed for individual vesicles as they collide on the surface. The architecture of this new system also permits control over the number of lipid anchors that tether the vesicle to the supporting bilayer. The diffusion coefficient of tethered vesicles anchored by two lipids is approximately 1.6-fold slower than that of vesicles anchored only with a single lipid, consistent with a simple physical model.

  9. Vesicle trafficking via the Spitzenkörper during hyphal tip growth in Rhizoctonia solani.

    PubMed

    Dijksterhuis, Jan; Molenaar, Douwe

    2013-04-01

    Growing hyphae of Rhizoctonia solani were stained with the endocytic marker dye FM4-64 and imaged by confocal microscopy. Staining of the plasma membrane was followed by labeling of organelles in the cytoplasm (after ~1 min) and of the Spitzenkörper (Spk; after ~2 min). Fluorescence recovery after photobleaching (FRAP) of the stained Spk demonstrated the vectorial flow of secretory vesicles from the apical cytoplasm to the Spk. This flux was modelled in a two-compartment model. The turnover time of the vesicles of the Spk was estimated to be 1.3-2.5 min. These results are roughly consistent with the expected flux of vesicles through the Spk based on the number of secretory vesicles within the Spk and the number of secretory vesicles that would be necessary to fuse with the apical plasma membrane to maintain hyphal extension rates. These results suggest that membrane retrieval via endocytosis is not as significant as previously suggested.

  10. Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells.

    PubMed

    Pangrsic, Tina; Lasarow, Livia; Reuter, Kirsten; Takago, Hideki; Schwander, Martin; Riedel, Dietmar; Frank, Thomas; Tarantino, Lisa M; Bailey, Janice S; Strenzke, Nicola; Brose, Nils; Müller, Ulrich; Reisinger, Ellen; Moser, Tobias

    2010-07-01

    Inner hair cell ribbon synapses indefatigably transmit acoustic information. The proteins mediating their fast vesicle replenishment (hundreds of vesicles per s) are unknown. We found that an aspartate to glycine substitution in the C(2)F domain of the synaptic vesicle protein otoferlin impaired hearing by reducing vesicle replenishment in the pachanga mouse model of human deafness DFNB9. In vitro estimates of vesicle docking, the readily releasable vesicle pool (RRP), Ca(2+) signaling and vesicle fusion were normal. Moreover, we observed postsynaptic excitatory currents of variable size and spike generation. However, mutant active zones replenished vesicles at lower rates than wild-type ones and sound-evoked spiking in auditory neurons was sparse and only partially improved during longer interstimulus intervals. We conclude that replenishment does not match the release of vesicles at mutant active zones in vivo and a sufficient standing RRP therefore cannot be maintained. We propose that otoferlin is involved in replenishing synaptic vesicles.

  11. Magnetic capture docking mechanism

    NASA Technical Reports Server (NTRS)

    Howard, Nathan (Inventor); Nguyen, Hai D. (Inventor)

    2010-01-01

    A mechanism uses a magnetic field to dock a satellite to a host vehicle. A docking component of the mechanism residing on the host vehicle has a magnet that is used to induce a coupled magnetic field with a docking component of the mechanism residing on the satellite. An alignment guide axially aligns the docking component of the satellite with the docking component of the host device dependent on the coupled magnetic field. Rotational alignment guides are used to rotationally align the docking component of the satellite with the docking component of the host device. A ball-lock mechanism is used to mechanically secure the docking component of the host vehicle and the docking component of the satellite.

  12. Distribution of secretory component in hepatocytes and its mode of transfer into bile

    PubMed Central

    Mullock, Barbara M.; Hinton, Richard H.; Dobrota, Miloslav; Peppard, Jane; Orlans, Eva

    1980-01-01

    Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound 125I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [14C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile. ImagesPLATE 1Fig. 5. PMID:7470082

  13. RNAi knockdown of parafusin inhibits the secretory pathway.

    PubMed

    Liu, Li; Wyroba, Elzbieta; Satir, Birgit H

    2011-10-01

    Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway. Copyright © 2011 Elsevier GmbH. All rights reserved.

  14. The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis*

    PubMed Central

    Petrie, Matt; Esquibel, Joseph; Maciuba, Stephanie; Takahashi, Hirohide

    2016-01-01

    Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming. PMID:27528604

  15. Proteomics of regulated secretory organelles.

    PubMed

    Brunner, Yannick; Schvartz, Domitille; Couté, Yohann; Sanchez, Jean-Charles

    2009-01-01

    Regulated secretory organelles are important subcellular structures of living cells that allow the release in the extracellular space of crucial compounds, such as hormones and neurotransmitters. Therefore, the regulation of biogenesis, trafficking, and exocytosis of regulated secretory organelles has been intensively studied during the last 30 years. However, due to the large number of different regulated secretory organelles, only a few of them have been specifically characterized. New insights into regulated secretory organelles open crucial perspectives for a better comprehension of the mechanisms that govern cell secretion. The combination of subcellular fractionation, protein separation, and mass spectrometry is also possible to study regulated secretory organelles at the proteome level. In this review, we present different strategies used to isolate regulated secretory organelles, separate their protein content, and identify the proteins by mass spectrometry. The biological significance of regulated secretory organelles-proteomic analysis is discussed as well. Copyright 2009 Wiley Periodicals, Inc.

  16. Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

    PubMed

    Tsuboi, Takashi; Kitaguchi, Tetsuya; Karasawa, Satoshi; Fukuda, Mitsunori; Miyawaki, Atsushi

    2010-01-01

    Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

  17. Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein

    PubMed Central

    Kitaguchi, Tetsuya; Karasawa, Satoshi; Fukuda, Mitsunori

    2010-01-01

    Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO–tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K+] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway. PMID:19889833

  18. Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

  19. Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

    PubMed Central

    Mulholland, J; Wesp, A; Riezman, H; Botstein, D

    1997-01-01

    Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized. Images PMID:9285820

  20. Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

    PubMed

    Mulholland, J; Wesp, A; Riezman, H; Botstein, D

    1997-08-01

    Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

  1. Mammary Analogue Secretory Carcinoma.

    PubMed

    Stevens, Todd M; Parekh, Vishwas

    2016-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined.

  2. Rendezvous Docking Simulator

    NASA Technical Reports Server (NTRS)

    1963-01-01

    Rendezvous Docking Simulator. The simulation demonstrated linear and gimbal motions of the capsule and a Gemini-Agena docking. [Entire movie available on DVD from CASI as Doc ID 20070030983. Contact help@sti.nasa.gov

  3. Kinetic regulation of coated vesicle secretion

    PubMed Central

    Foret, Lionel; Sens, Pierre

    2008-01-01

    The secretion of vesicles for intracellular transport often relies on the aggregation of specialized membrane-bound proteins into a coat able to curve cell membranes. The nucleation and growth of a protein coat is a kinetic process that competes with the energy-consuming turnover of coat components between the membrane and the cytosol. We propose a generic kinetic description of coat assembly and the formation of coated vesicles and discuss its implication to the dynamics of COP vesicles that traffic within the Golgi and with the endoplasmic reticulum. We show that stationary coats of fixed area emerge from the competition between coat growth and the recycling of coat components, in a fashion resembling the treadmilling of cytoskeletal filaments. We further show that the turnover of coat components allows for a highly sensitive switching mechanism between a quiescent and a vesicle producing membrane, upon a slowing down of the exchange kinetics. We claim that the existence of this switching behavior, also triggered by factors, such as the presence of cargo and variation of the membrane mechanical tension, allows for efficient regulation of vesicle secretion. We propose a model, supported by different experimental observations, in which vesiculation of secretory membranes is impaired by the energy-consuming desorption of coat proteins, until the presence of cargo or other factors triggers a dynamical switch into a vesicle producing state. PMID:18824695

  4. Involvement of water channels in synaptic vesicle swelling.

    PubMed

    Jeremic, Aleksandar; Cho, Won Jin; Jena, Bhanu P

    2005-10-01

    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.

  5. Poking Vesicles

    NASA Astrophysics Data System (ADS)

    Powers, Thomas R.; Huber, Greg; Goldstein, Raymond E.

    2000-03-01

    Lipid vesicles can exhibit a variety of interesting shapes when subject to point forcing, as has been demonstrated with laser-tweezed beads and with growing microtubules. Using numerical and analytic techniques, we study the force vs. extension in two regimes. At low forces, the resistance to deformation is primarily entropic, with a tension given by the fluctuating bending modes. At high forces, the thermal wrinkles are smoothed out, and there is bending and stretching which balance in a thin tether, which we treat using boundary-layer techniques. This work was supported in part by the Harvard MRSEC and Army Research Office Grant DAA655-97-1-014 (TRP) and NSF DMR9812526 (GH & REG).

  6. Recognition and tethering of transport vesicles at the Golgi apparatus.

    PubMed

    Witkos, Tomasz M; Lowe, Martin

    2017-02-23

    The Golgi apparatus occupies a central position within the secretory pathway where it is a hub for vesicle trafficking. Distinct classes of transport vesicles traffic diverse cargoes into and out of this organelle, as well as between the different Golgi subcompartments. A key feature of Golgi trafficking is the specific recognition of transport vesicles at the different regions of the Golgi apparatus, required for the correct cargo delivery. Specificity is ensured by coiled-coil golgins and multi-subunit tethering complexes (MTCs), which act together to capture vesicles and promote their subsequent fusion with the Golgi membrane. In this review we discuss our current understanding of how golgins and MTCs function together to mediate the specific recognition of vesicles at the Golgi apparatus.

  7. Spiperone: evidence for uptake into secretory granules.

    PubMed Central

    Dannies, P S; Rudnick, M S; Fishkes, H; Rudnick, G

    1984-01-01

    Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution. PMID:6584920

  8. Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo

    PubMed Central

    Lwin, Kyaw Myo; Li, Donghao; Bretscher, Anthony

    2016-01-01

    The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1—which binds Myo2—its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo. PMID:27307583

  9. In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts.

    PubMed

    Le Bihan, Marie-Catherine; Bigot, Anne; Jensen, Søren Skov; Dennis, Jayne L; Rogowska-Wrzesinska, Adelina; Lainé, Jeanne; Gache, Vincent; Furling, Denis; Jensen, Ole Nørregaard; Voit, Thomas; Mouly, Vincent; Coulton, Gary R; Butler-Browne, Gillian

    2012-12-21

    Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.

  10. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    PubMed Central

    Stevens, David R.; Schirra, Claudia; Becherer, Ute; Rettig, Jens

    2011-01-01

    The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP) and a slowly releasable (SRP) pool are followed by sustained release, due to maturation, and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin, and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles. PMID:21423410

  11. Coarse-Grained Model of SNARE-Mediated Docking

    PubMed Central

    Fortoul, Nicole; Singh, Pankaj; Hui, Chung-Yuen; Bykhovskaia, Maria; Jagota, Anand

    2015-01-01

    Synaptic transmission requires that vesicles filled with neurotransmitter molecules be docked to the plasma membrane by the SNARE protein complex. The SNARE complex applies attractive forces to overcome the long-range repulsion between the vesicle and membrane. To understand how the balance between the attractive and repulsive forces defines the equilibrium docked state we have developed a model that combines the mechanics of vesicle/membrane deformation with an apparently new coarse-grained model of the SNARE complex. The coarse-grained model of the SNARE complex is calibrated by comparison with all-atom molecular dynamics simulations as well as by force measurements in laser tweezer experiments. The model for vesicle/membrane interactions includes the forces produced by membrane deformation and hydration or electrostatic repulsion. Combining these two parts, the coarse-grained model of the SNARE complex with membrane mechanics, we study how the equilibrium docked state varies with the number of SNARE complexes. We find that a single SNARE complex is able to bring a typical synaptic vesicle to within a distance of ∼3 nm from the membrane. Further addition of SNARE complexes shortens this distance, but an overdocked state of >4–6 SNAREs actually increases the equilibrium distance. PMID:25954883

  12. What is Rate-Limiting during Sustained Synaptic Activity: Vesicle Supply or the Availability of Release Sites

    PubMed Central

    Neher, Erwin

    2010-01-01

    For some types of synapses the availability of release-ready vesicles is a limiting factor during ongoing activity. Synaptic strength in this case is determined both by the recruitment of such vesicles and the probability of their release during an action potential. Here it is argued that not the availability of vesicles is the limiting factor for recruitment, but rather the availability of specific sites to which vesicles can dock. PMID:21423530

  13. Evidence that vesicles undergo compound fusion on the synaptic ribbon

    PubMed Central

    Matthews, Gary; Sterling, Peter

    2008-01-01

    The ribbon synapse can release a stream of transmitter quanta at very high rates. Although the ribbon tethers numerous vesicles near the presynaptic membrane, most of the tethered vesicles are held at a considerable distance from the plasma membrane. Therefore, it remains unclear how their contents are released. We evoked prolonged bouts of exocytosis from a retinal bipolar cell, fixed within seconds, and then studied the ribbons by electron microscopy. Vesicle density on ribbons was reduced by ~50% compared with cells where exocytosis was blocked with intracellular ATP-γS. Large, irregularly shaped vesicles appeared on the ribbon in cells fixed during repetitive stimulation of exocytosis, and in some cases the large vesicles could be traced in adjacent sections to cisternae open to the medium. The large cisternal structures were attached to the ribbon by filaments similar to those that tether synaptic vesicles to the ribbon, and they occupied the base of the ribbon near the plasma membrane, where normal synaptic vesicles are found in resting cells. We suggest that the cisternae attached to ribbons represent synaptic vesicles that fused by compound exocytosis during strong repetitive stimulation, and thus that vesicles tethered to the ribbon can empty their contents by fusing to other vesicles docked at the presynaptic membrane. Such compound fusion could explain the extremely high release rates and the multivesicular release reported for auditory and visual ribbon synapses. PMID:18495874

  14. A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii.

    PubMed

    Breinich, Manuela S; Ferguson, David J P; Foth, Bernardo J; van Dooren, Giel G; Lebrun, Maryse; Quon, Doris V; Striepen, Boris; Bradley, Peter J; Frischknecht, Friedrich; Carruthers, Vern B; Meissner, Markus

    2009-02-24

    Apicomplexans contain only a core set of factors involved in vesicular traffic. Yet these obligate intracellular parasites evolved a set of unique secretory organelles (micronemes, rhoptries, and dense granules) that are required for invasion and modulation of the host cell. Apicomplexa replicate by budding from or within a single mother cell, and secretory organelles are synthesized de novo at the final stage of division. To date, the molecular basis for their biogenesis is unknown. We demonstrate that the apicomplexan dynamin-related protein B (DrpB) belongs to an alveolate specific family of dynamins that is expanded in ciliates. DrpB accumulates in a cytoplasmic region close to the Golgi that breaks up during replication and reforms after assembly of the daughter cells. Conditional ablation of DrpB function results in mature daughter parasites that are devoid of micronemes and rhoptries. In the absence of these organelles, invasion-related secretory proteins are mistargeted to the constitutive secretory pathway. Mutant parasites are able to replicate but are unable to escape from or invade into host cells. DrpB is the essential mechanoenzyme for the biogenesis of secretory organelles in Apicomplexa. We suggest that DrpB is required during replication to generate vesicles for the regulated secretory pathway that form the unique secretory organelles. Our study supports a role of an alveolate-specific dynamin that was required for the evolution of novel, secretory organelles. In the case of Apicomplexa, these organelles further evolved to enable a parasitic lifestyle.

  15. Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

    PubMed Central

    Rohrbough, Jeffrey; Rushton, Emma; Palanker, Laura; Woodruff, Elvin; Matthies, Heinrich J. G.; Acharya, Usha; Acharya, Jairaj K.; Broadie, Kendal

    2009-01-01

    A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50–70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50–70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission. PMID:15356190

  16. An inside job: subversion of the host secretory pathway by intestinal pathogens.

    PubMed

    Sharp, Tyler M; Estes, Mary K

    2010-10-01

    The cellular secretory pathway, composed of the endoplasmic reticulum, Golgi apparatus, and cellular vesicles, mediates the intracellular trafficking of proteins and lipids. Gastrointestinal pathogens frequently affect the functions of enterocytes, the differentiated cells involved in secretion and absorption of extracellular molecules. Microbial pathogenesis can be enhanced by altering the trafficking of key molecules such as brush border enzymes, soluble immune mediators such as cytokines and chemokines, and MHC Class I molecules, all of which rely on the secretory pathway for their appropriate cellular localization. This review focuses on our current understanding of the distinct mechanisms employed by enteric pathogens to antagonize the secretory pathway. Many pathogens encode individual or multiple proteins to antagonize the secretory pathway, including disrupting the trafficking of vesicles between the endoplasmic reticulum, Golgi, and plasma membrane. This antagonism allows for increased pathogenesis and can assist, directly or indirectly, in microbial replication. Virtually all arms of the secretory pathway are targeted by intestinal pathogens, supporting the pathogenic significance of shutting this pathway down. This review summarizes the mechanisms utilized by gut pathogens to disrupt the cellular secretory pathway and addresses potential therapeutic targets to combat these highly prevalent and burdensome microbes.

  17. International Docking Standardization NASA

    NASA Technical Reports Server (NTRS)

    Donahoe, Stanley; Lewis, J.; Carroll, M.; Le, T.

    2009-01-01

    This slide presentation reviews the different types of docking types. The objective is the pressurized vehicle connection and crew transfer. Androgynous Docking is defined as the joining or coming together of two free flying space vehicles with alike interfaces. Androgynous mating allows for collaboration between any two vehicles. The subsytems of an androgynous mating system are reviewed, including: Hard docking subsystems: latch system, tunnel housing, alignment system and seal.

  18. A Critical Role for Toxoplasma gondii Vacuolar Protein Sorting VPS9 in Secretory Organelle Biogenesis and Host Infection

    PubMed Central

    Sakura, Takaya; Sindikubwabo, Fabien; Oesterlin, Lena K.; Bousquet, Hugo; Slomianny, Christian; Hakimi, Mohamed-Ali; Langsley, Gordon; Tomavo, Stanislas

    2016-01-01

    Accurate sorting of proteins to the three types of parasite-specific secretory organelles namely rhoptry, microneme and dense granule in Toxoplasma gondii is crucial for successful host cell invasion by this obligate intracellular parasite. Despite its tiny body architecture and limited trafficking machinery, T. gondii relies heavily on transport of vesicles containing proteins, lipids and important virulence-like factors that are delivered to these secretory organelles. However, our understanding on how trafficking of vesicles operates in the parasite is still limited. Here, we show that the T. gondii vacuolar protein sorting 9 (TgVps9), has guanine nucleotide exchange factor (GEF) activity towards Rab5a and is crucial for sorting of proteins destined to secretory organelles. Our results illuminate features of TgVps9 protein as a key trafficking facilitator that regulates protein maturation, secretory organelle formation and secretion, thereby ensuring a primary role in host infection by T. gondii. PMID:27966671

  19. Docking and retrieval mechanism

    NASA Technical Reports Server (NTRS)

    Tewell, J. R.; Spencer, R. A.

    1977-01-01

    An engineering prototype docking and retrieval mechanism (DRM) which enables two spacecraft to dock and be structurally joined on-orbit is described. The joining of two spacecraft or payloads on-orbit supports future planned space activities such as payload servicing, deployment and retrieval, and assembly or large space systems. Advantages of the DRM include: it is a nonimpact docking mechanism; does not require impact absorbing mechanisms or attitude stabilization on the target spacecraft; is capable of docking to a spinning spacecraft; and can spin up and deploy a spinning spacecraft or payload.

  20. Docking and retrieval mechanism

    NASA Technical Reports Server (NTRS)

    Tewell, J. R.; Spencer, R. A.

    1977-01-01

    An engineering prototype docking and retrieval mechanism (DRM) is described which enables two spacecraft to dock and be structurally joined on-orbit. The joining of two spacecraft or payloads on-orbit supports future planned space activities such as payload servicing, deployment and retrieval, and assembly of large space systems. The DRM, as developed, provides advantages over prior approaches because it is a nonimpact docking mechanism, does not require impact absorbing mechanisms or attitude stabilization on the target spacecraft, is capable of docking to a spinning spacecraft, and can spin up and deploy a spinning spacecraft or payload.

  1. Docking system for spacecraft

    NASA Technical Reports Server (NTRS)

    Kahn, Jon B. (Inventor)

    1988-01-01

    A mechanism is disclosed for the docking of a spacecraft to a space station where a connection for transfer of personnel and equipment is desired. The invention comprises an active docking structure on a spacecraft and a passive docking structure on the station. The passive structure includes a docking ring mounted on a tunnel structure fixed to the space station. The active structure includes a docking ring carried by an actuator-attenuator devices, each attached at one end to the ring and at its other end in the spacecraft payload bay. The devices respond to command signals for moving the docking ring between a stowed position in the spacecraft to a deployed position suitable for engagement with the docking ring. The devices comprise means responsive to signals of sensed loadings to absorb impact energy and retraction means for drawing the coupled spacecraft and station into final docked configuration and moving the tunnel structure to a berthed position in the spacecraft. Latches couple the spacecraft and space station upon contact of the docking rings and latches establish a structural tie between the spacecraft when retracted.

  2. Docking structure for spacecraft

    NASA Technical Reports Server (NTRS)

    Belew, R. R. (Inventor)

    1973-01-01

    A docking structure for a pair of spacecraft is described comprising a conical receptacle on the docking end of a first spacecraft that receives a mating conical projection on the docking end of the second spacecraft. The conical receptacle of the first spacecraft constitutes an exterior portion of a sealed gas-tight compartment. Pressurization of the sealed compartment causes the conical receptacle to extend toward the incoming conical projection of the second spacecraft. When the mating conical portions are latched together, the docking energy is absorbed by the compressed gas in the sealed compartment. Rebound forces are countered by a plurality of actuator cylinders supporting the conical receptacle.

  3. Docking system for spacecraft

    NASA Technical Reports Server (NTRS)

    Kahn, Jon B. (Inventor)

    1990-01-01

    A mechanism for the docking of a space vehicle to a space station where a connection for transfer of personnel and equipment is desired. The invention comprises an active docking structure on a space vehicle 10 and a passive docking structure on a station 11. The passive structure includes a docking ring 50 mounted on a tunnel structure 35 fixed to the space station. The active structure including a docking ring 18 carried by actuator-attenuator devices 20, each attached at one end to the ring 18 and at its other end in the vehicle's payload bay 12. The devices 20 respond to command signals for moving the docking ring 18 between a stowed position in the space vehicle to a deployed position suitable for engagement with the docking ring 50. The devices 20 comprise means responsive to signals of sensed loadings to absorb impact energy and retraction means for drawing the coupled space vehicle and station into final docked configuration and moving the tunnel structure to a berthed position in the space vehicle 10. Latches 60 couple the space vehicle and space station upon contact of docking rings 18 and 50 and latches 41-48 establish a structural tie between the spacecraft when retracted.

  4. HOPS interacts with Apl5 at the vacuole membrane and is required for consumption of AP-3 transport vesicles.

    PubMed

    Angers, Cortney G; Merz, Alexey J

    2009-11-01

    Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 delta subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.

  5. The puzzle of chloroplast vesicle transport – involvement of GTPases

    PubMed Central

    Karim, Sazzad; Aronsson, Henrik

    2014-01-01

    In the cytosol of plant cells vesicle transport occurs via secretory pathways among the endoplasmic reticulum network, Golgi bodies, secretory granules, endosome, and plasma membrane. Three systems transfer lipids, proteins and other important molecules through aqueous spaces to membrane-enclosed compartments, via vesicles that bud from donor membranes, being coated and uncoated before tethered and fused with acceptor membranes. In addition, molecular, biochemical and ultrastructural evidence indicates presence of a vesicle transport system in chloroplasts. Little is known about the protein components of this system. However, as chloroplasts harbor the photosynthetic apparatus that ultimately supports most organisms on the planet, close attention to their pathways is warranted. This may also reveal novel diversification and/or distinct solutions to the problems posed by the targeted intra-cellular trafficking of important molecules. To date two homologs to well-known yeast cytosolic vesicle transport proteins, CPSAR1 and CPRabA5e (CP, chloroplast localized), have been shown to have roles in chloroplast vesicle transport, both being GTPases. Bioinformatic data indicate that several homologs of cytosolic vesicle transport system components are putatively chloroplast-localized and in addition other proteins have been implicated to participate in chloroplast vesicle transport, including vesicle-inducing protein in plastids 1, thylakoid formation 1, snowy cotyledon 2/cotyledon chloroplast biogenesis factor, curvature thylakoid 1 proteins, and a dynamin like GTPase FZO-like protein. Several putative potential cargo proteins have also been identified, including building blocks of the photosynthetic apparatus. Here we discuss details of the largely unknown putative chloroplast vesicle transport system, focusing on GTPase-related components. PMID:25295043

  6. The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

    PubMed Central

    Capoccia, Benjamin J.; Jin, Ramon U.; Kong, Young-Yun; Peek, Richard M.; Fassan, Matteo; Rugge, Massimo; Mills, Jason C.

    2013-01-01

    After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease. PMID:23478405

  7. Signaling from the secretory granule to the nucleus

    PubMed Central

    Rajagopal, Chitra; Mains, Richard E.; Eipper, Betty A.

    2014-01-01

    Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40–60 nm vesicles carry conventional or small molecule neurotransmitters, and 200–400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, since the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by Cadherins, Notch and ErbB4. PMID:22681236

  8. Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

    PubMed

    Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A

    2008-04-01

    Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

  9. Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion.

    PubMed

    Verkhratsky, Alexei; Matteoli, Michela; Parpura, Vladimir; Mothet, Jean-Pierre; Zorec, Robert

    2016-02-01

    Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes.

  10. Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Mecheri, Salahedine; Peronet, Roger; Bonnerot, Christian; Desaymard, Catherine

    1997-01-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles. PMID:9398681

  11. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

    PubMed

    Raposo, G; Tenza, D; Mecheri, S; Peronet, R; Bonnerot, C; Desaymard, C

    1997-12-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

  12. Trichocysts-Paramecium's Projectile-like Secretory Organelles: Reappraisal of their Biogenesis, Composition, Intracellular Transport, and Possible Functions.

    PubMed

    Plattner, Helmut

    2017-01-01

    This review summarizes biogenesis, composition, intracellular transport, and possible functions of trichocysts. Trichocyst release by Paramecium is the fastest dense core-secretory vesicle exocytosis known. This is enabled by the crystalline nature of the trichocyst "body" whose matrix proteins (tmp), upon contact with extracellular Ca(2+) , undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated trichocyst exocytosis involves Ca(2+) mobilization from alveolar sacs and tightly coupled store-operated Ca(2+) -influx, initiated by activation of ryanodine receptor-like Ca(2+) -release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of trichocysts, i.e. defense against predators, by vigorous, local trichocyst discharge. The tmp's contained in the main "body" of a trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a trichocyst, the "tip", contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of trichocysts. We consider ciliary reversal accompanying stimulated trichocyst exocytosis (also in mutants devoid of depolarization-activated Ca(2+) channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a trichocyst with its membrane; these may signal the filling state, full or

  13. Multiple Docking Adapter Illustration

    NASA Technical Reports Server (NTRS)

    1972-01-01

    This cutaway drawing details the major characteristics of the Skylab Multiple Docking Adapter (MDA). The MDA, built under the direction of the Marshall Space Flight Center, housed the control units for the Apollo Telescope Mount (ATM), Earth Resources Experiment Package (EREP), and Zero-Gravity Materials Processing Facility, and provided a docking port for the Apollo Command Module (CM).

  14. The development and differentiation of human seminal vesicles.

    PubMed Central

    Brewster, S F

    1985-01-01

    The development and differentiation of the human seminal vesicles has been studied using 14 human male fetuses and 4 postnatal specimens. Earlier accounts of the morphogenesis of the seminal vesicle have been reviewed and commented upon in the light of the present findings. Previously undocumented observations include the movements of the mesenchyme, the formation of epithelial folds and the development of the lamina propria and tunica muscularis. Epithelial differentiation is also described. The function of the basal cells and the possible prepubertal secretory activity of the seminal vesicle are discussed. More work is required on the differentiation of the vesicle between birth and puberty. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:3870731

  15. Secretory Carcinoma of the Breast

    PubMed Central

    Aktepe, Fatma; Sarsenov, Dauren; Özmen, Vahit

    2016-01-01

    Secretory carcinoma is a very rare subtype of breast carcinoma. These tumors are generally associated with a favorable prognosis, although having triple-negative phenotype (estrogen receptor (ER), progesterone receptor (PR) negative and c-erbB2 (HER2) negative). In this presentation, a rare secretory carcinoma of the breast in a woman aged 24 years is discussed and the literature is reviewed. PMID:28331758

  16. Docking mechanism for spacecraft

    NASA Technical Reports Server (NTRS)

    Lange, Gregory A. (Inventor); Mcmanamen, John P. (Inventor); Schliesing, John A. (Inventor)

    1989-01-01

    A system is presented for docking a space vehicle to a space station where a connecting tunnel for in-flight transfer of personnel is required. Cooperable coupling mechanisms include docking rings on the space vehicle and space station. The space station is provided with a tunnel structure, a retraction mechanism, and a docking ring. The vehicle coupling mechanism is designed to capture the station coupling mechanism, arrest relative spacecraft motions while limiting loads to acceptable levels, and then realign the spacecraft for final docking and tunnel interconnection. The docking ring of the space vehicle coupling mechanism is supported by linear attentuator actuator devices, each of which is controlled by a control system which receives loading information signals and attenuator stroke information signals from each device and supplies output signals for controlling its linear actuation to attenuate impact loading or to realign the spacecraft for final docking and tunnel interconnection. The retraction mechanism is used to draw the spacecraft together after initial contact and coupling. Tunnel trunnions, cooperative with the latches on the space vehicle constitute the primary structural tie between the spacecraft in final docked configuration.

  17. Extracellular Vesicles as New Players in Cellular Senescence

    PubMed Central

    Urbanelli, Lorena; Buratta, Sandra; Sagini, Krizia; Tancini, Brunella; Emiliani, Carla

    2016-01-01

    Cell senescence is associated with the secretion of many factors, the so-called “senescence-associated secretory phenotype”, which may alter tissue microenvironment, stimulating the organism to clean up senescent cells and replace them with newly divided ones. Therefore, although no longer dividing, these cells are still metabolically active and influence the surrounding tissue. Much attention has been recently focused not only on soluble factors released by senescent cells, but also on extracellular vesicles as conveyors of senescence signals outside the cell. Here, we give an overview of the role of extracellular vesicles in biological processes and signaling pathways related to senescence and aging. PMID:27571072

  18. The COPII cage: unifying principles of vesicle coat assembly.

    PubMed

    Gürkan, Cemal; Stagg, Scott M; Lapointe, Paul; Balch, William E

    2006-10-01

    Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers - vesicles and tubules - that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.

  19. Spacecraft Docking System

    NASA Technical Reports Server (NTRS)

    Ghofranian, Siamak (Inventor); Chuang, Li-Ping Christopher (Inventor); Motaghedi, Pejmun (Inventor)

    2016-01-01

    A method and apparatus for docking a spacecraft. The apparatus comprises elongate members, movement systems, and force management systems. The elongate members are associated with a docking structure for a spacecraft. The movement systems are configured to move the elongate members axially such that the docking structure for the spacecraft moves. Each of the elongate members is configured to move independently. The force management systems connect the movement systems to the elongate members and are configured to limit a force applied by the each of the elongate members to a desired threshold during movement of the elongate members.

  20. Color fingerprinting of proteins by calixarenes embedded in lipid/polydiacetylene vesicles.

    PubMed

    Kolusheva, Sofiya; Zadmard, Reza; Schrader, Thomas; Jelinek, Raz

    2006-10-18

    "Naked eye" color detection of proteins was achieved by embedding calixarene receptors within vesicles comprising phospholipids and the chromatic polymer polydiacetylene. Dramatic visible absorbance changes were induced through electrostatic interactions between the protein surface and the vesicle-incorporated hosts. The colorimetric responses could be induced by micromolar protein concentrations, and furthermore, specific protein fingerprints could be obtained by incorporating different receptors within the vesicles. Fluorescence and circular dichroism experiments confirmed the relationship between the colorimetric phenomena and protein docking on the surface of the chromatic vesicles. The colorimetric assay constitutes a generic platform for high-sensitivity detection of soluble proteins and for evaluation of protein surface charge distribution.

  1. DOCK8 Deficiency

    MedlinePlus

    ... care at NIAID, visit the NIAID PIDD site . Credit: NIAID Scientist at microscope. Causes DOCK8 deficiency is ... The End of an Era Acknowledgments References Photo Credits Dr. Joseph Kinyoun: Selected Bibliography NIAID 60th Anniversary ...

  2. Glucocorticoid affects dendritic transport of BDNF-containing vesicles.

    PubMed

    Adachi, Naoki; Numakawa, Tadahiro; Nakajima, Shingo; Fukuoka, Masashi; Odaka, Haruki; Katanuma, Yusuke; Ooshima, Yoshiko; Hohjoh, Hirohiko; Kunugi, Hiroshi

    2015-08-04

    Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and functions in the central nervous system (CNS). Because BDNF protein is sorted into secretory vesicles at the trans-Golgi network in the cell body after translation, transport of BDNF-containing vesicles to the secretion sites is an important process for its function. Here we examined the effect of dexamethasone (DEX), a synthetic glucocorticoid, on BDNF-containing vesicle transport and found that DEX decreased the proportion of stationary vesicles and increased velocity of the microtubule-based vesicle transport in dendrites of cortical neurons. Furthermore, DEX increased huntingtin (Htt) protein levels via glucocorticoid receptor (GR) activation, and reduction in the amount of Htt by a specific shRNA reversed the action of DEX on BDNF vesicle transport. Given that Htt protein is a positive regulator for the microtubule-dependent vesicular transport in neurons, our data suggest that glucocorticoid stimulates BDNF vesicle transport through upregulation of Htt protein levels.

  3. Glucocorticoid affects dendritic transport of BDNF-containing vesicles

    PubMed Central

    Adachi, Naoki; Numakawa, Tadahiro; Nakajima, Shingo; Fukuoka, Masashi; Odaka, Haruki; Katanuma, Yusuke; Ooshima, Yoshiko; Hohjoh, Hirohiko; Kunugi, Hiroshi

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and functions in the central nervous system (CNS). Because BDNF protein is sorted into secretory vesicles at the trans-Golgi network in the cell body after translation, transport of BDNF-containing vesicles to the secretion sites is an important process for its function. Here we examined the effect of dexamethasone (DEX), a synthetic glucocorticoid, on BDNF-containing vesicle transport and found that DEX decreased the proportion of stationary vesicles and increased velocity of the microtubule-based vesicle transport in dendrites of cortical neurons. Furthermore, DEX increased huntingtin (Htt) protein levels via glucocorticoid receptor (GR) activation, and reduction in the amount of Htt by a specific shRNA reversed the action of DEX on BDNF vesicle transport. Given that Htt protein is a positive regulator for the microtubule-dependent vesicular transport in neurons, our data suggest that glucocorticoid stimulates BDNF vesicle transport through upregulation of Htt protein levels. PMID:26239075

  4. Expedition 27 Docking

    NASA Image and Video Library

    2011-04-06

    Top officials from the Russian Federal Space Agency and NASA hold a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Thursday, April 7, 2011. The Soyuz TMA-21 docked to the International Space Station carrying Expedition 27 Soyuz Commander Alexander Samokutyaev, NASA Flight Engineer Ron Garan and Russian Flight Engineer Andrey Borisenko. Photo Credit: (NASA/Carla Cioffi)

  5. Expedition 28 Docking

    NASA Image and Video Library

    2011-06-10

    William Gerstenmaier, Associate Administrator for Space Operations, is interviewed by Russian Federal Space Agency (ROSCOSMOS) TV following a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Friday, June 10, 2011. The Soyuz TMA-02M docked to the International Space Station carrying Expedition 28 Soyuz Commander Sergei Volkov, NASA Flight Engineer Mike Fossum and JAXA (Japanase Aerospace Exploration Agency) Flight Engineer Satoshi Furukawa. Photo Credit: (NASA/Carla Cioffi)

  6. Expedition 28 Docking

    NASA Image and Video Library

    2011-06-10

    Top officials from the Russian Federal Space Agency and NASA hold a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Friday, June 10, 2011. The Soyuz TMA-02M docked to the International Space Station carrying Expedition 28 Soyuz Commander Sergei Volkov, NASA Flight Engineer Mike Fossum and JAXA (Japanase Aerospace Exploration Agency) Flight Engineer Satoshi Furukawa. Photo Credit: (NASA/Carla Cioffi)

  7. Expedition 26 Docking

    NASA Image and Video Library

    2010-12-18

    Vitaly Davyidov, second from right, Deputy Head of the Russian Federal Space Agency, answers reporter’s questions during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Saturday, Dec. 18, 2010. The Soyuz TMA-20 docked to the International Space Station carrying Expedition 26 Soyuz Commander Dmitry Kondratyev, Flight Engineer Catherine Coleman and European Space Agency Flight Engineer Paolo Nespoli. Photo Credit: (NASA/Carla Cioffi)

  8. Expedition 28 Docking

    NASA Image and Video Library

    2011-06-10

    Vladimir Popovkin, Head of the Russian Federal Space Agency (ROSCOSMOS) answers a reporter’s question during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Friday, June 10, 2011. The Soyuz TMA-02M docked to the International Space Station carrying Expedition 28 Soyuz Commander Sergei Volkov, NASA Flight Engineer Mike Fossum and JAXA (Japanase Aerospace Exploration Agency) Flight Engineer Satoshi Furukawa. Photo Credit: (NASA/Carla Cioffi)

  9. Expedition 26 Docking

    NASA Image and Video Library

    2010-12-18

    Kirk Shireman, third from right, NASA's deputy ISS program manager, answers reporter’s questions during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Saturday, Dec. 18, 2010. The Soyuz TMA-20 docked to the International Space Station carrying Expedition 26 Soyuz Commander Dmitry Kondratyev, Flight Engineer Catherine Coleman and European Space Agency Flight Engineer Paolo Nespoli. Photo Credit: (NASA/Carla Cioffi)

  10. Expedition 23 Docking

    NASA Image and Video Library

    2010-04-03

    Kirk Shireman, right, NASA's deputy ISS program manager, answers reporter’s questions during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Sunday, April 4, 2010. The Soyuz TMA-18 docked to the International Space Station carrying Expedition 23 Soyuz Commander Alexander Skvortsov, Flight Engineer Mikhail Kornienko and NASA Flight Engineer Tracy Caldwell Dyson. Photo Credit: (NASA/Carla Cioffi)

  11. Expedition 27 Docking

    NASA Image and Video Library

    2011-04-06

    Russian Federal Space Agency Director of Human Space Flight, Alexey Krasnov, third from right, answers reporter’s questions during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Thursday, April 7, 2011. The Soyuz TMA-21 docked to the International Space Station carrying Expedition 27 Soyuz Commander Alexander Samokutyaev, NASA Flight Engineer Ron Garan and Russian Flight Engineer Andrey Borisenko. Photo Credit: (NASA/Carla Cioffi)

  12. Expedition 28 Docking

    NASA Image and Video Library

    2011-06-10

    Kuniaki Shiraki, President of the Japan Aerospace Exploration Agency (JAXA), center, answers a reporter’s question during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Friday, June 10, 2011. The Soyuz TMA-02M docked to the International Space Station carrying Expedition 28 Soyuz Commander Sergei Volkov, NASA Flight Engineer Mike Fossum and JAXA (Japanase Aerospace Exploration Agency) Flight Engineer Satoshi Furukawa. Photo Credit: (NASA/Carla Cioffi)

  13. Ca{sup 2+}-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

    SciTech Connect

    Stenovec, Matjaz Kreft, Marko Grilc, Sonja Potokar, Maja Kreft, Mateja Erdani Pangrsic, Tina Zorec, Robert

    2007-11-01

    Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca{sup 2+}-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca{sup 2+} level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca{sup 2+}-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.

  14. Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.

    PubMed

    Lefèbvre, Fabien; Prouzet-Mauléon, Valérie; Hugues, Michel; Crouzet, Marc; Vieillemard, Aurélie; McCusker, Derek; Thoraval, Didier; Doignon, François

    2012-05-01

    Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

  15. Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

    PubMed Central

    Lefèbvre, Fabien; Prouzet-Mauléon, Valérie; Hugues, Michel; Crouzet, Marc; Vieillemard, Aurélie; McCusker, Derek; Thoraval, Didier

    2012-01-01

    Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck. PMID:22447923

  16. Adhesion of Polymer Vesicles

    NASA Astrophysics Data System (ADS)

    Lin, John J.; Bates, Frank S.; Hammer, Daniel A.; Silas, James A.

    2005-07-01

    The adhesion and bending modulus of polybutadiene-poly(ethylene oxide) block copolymer vesicles made from a bidisperse mixture of polymers is measured using micropipette aspiration. The adhesion energy between biotinylated vesicles and avidin beads is modeled by incorporating the extension of the adhesive ligands above the surface brush of the vesicle according to the blob model of bidisperse polymer mixtures of Komura and Safran assuming the polymer brush at the surface of the vesicle is compact. The same model accurately reproduces the scaling of the bending modulus with polymer composition.

  17. HID-1 is required for homotypic fusion of immature secretory granules during maturation

    PubMed Central

    Du, Wen; Zhou, Maoge; Zhao, Wei; Cheng, Dongwan; Wang, Lifen; Lu, Jingze; Song, Eli; Feng, Wei; Xue, Yanhong; Xu, Pingyong; Xu, Tao

    2016-01-01

    Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules. DOI: http://dx.doi.org/10.7554/eLife.18134.001 PMID:27751232

  18. The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis

    PubMed Central

    Villanueva, José; Torregrosa-Hetland, Cristina J.; Gil, Amparo; González-Vélez, Virginia; Segura, Javier; Viniegra, Salvador; Gutiérrez, Luis M.

    2010-01-01

    The organization of cytoplasm in excitable cells was a largely ignored factor when mathematical models were developed to understand intracellular calcium and secretory behavior. Here we employed a combination of fluorescent evanescent and transmitted light microscopy to explore the F-actin cytoskeletal organization in the vicinity of secretory sites in cultured bovine chromaffin cells. This technique and confocal fluorescent microscopy show chromaffin granules associated with the borders of cortical cytoskeletal cages forming an intricate tridimensional network. Furthermore, the overexpression of SNAP-25 in these cells also reveals the association of secretory machinery clusters with the borders of these cytoskeletal cages. The importance of these F-actin cage borders is stressed when granules appear to interact and remain associated during exocytosis visualized in acridin orange loaded vesicles. These results will prompt us to propose a model of cytoskeletal cages, where the secretory machinery is associated with its borders. Both the calcium level and the secretory response are enhanced in this geometrical arrangement when compared with a random distribution of the secretory machinery that is not restricted to the borders of the cage. PMID:20885775

  19. Characteristics of endoplasmic reticulum-derived transport vesicles

    PubMed Central

    1994-01-01

    We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules. PMID:8063853

  20. Smart tunnel: Docking mechanism

    NASA Technical Reports Server (NTRS)

    Schliesing, John A. (Inventor); Edenborough, Kevin L. (Inventor)

    1989-01-01

    A docking mechanism is presented for the docking of a space vehicle to a space station comprising a flexible tunnel frame structure which is deployable from the space station. The tunnel structure comprises a plurality of series connected frame sections, one end section of which is attached to the space station and the other end attached to a docking module of a configuration adapted for docking in the payload bay of the space vehicle. The docking module is provided with trunnions, adapted for latching engagement with latches installed in the vehicle payload bay and with hatch means connectable to a hatch of the crew cabin of the space vehicle. Each frame section comprises a pair of spaced ring members, interconnected by actuator-attenuator devices which are individually controllable by an automatic control means to impart relative movement of one ring member to the other in six degrees of freedom of motion. The control means includes computer logic responsive to sensor signals of range and attitude information, capture latch condition, structural loads, and actuator stroke for generating commands to the onboard flight control system and the individual actuator-attenuators to deploy the tunnel to effect a coupling with the space vehicle and space station after coupling. A tubular fluid-impervious liner, preferably fabric, is disposed through the frame sections of a size sufficient to accommodate the passage of personnel and cargo.

  1. The key target of neuroprotection after the onset of ischemic stroke: secretory pathway Ca2+-ATPase 1

    PubMed Central

    Li, Li-hua; Tian, Xiang-rong; Hu, Zhi-ping

    2015-01-01

    The regulatory mechanisms of cytoplasmic Ca2+ after myocardial infarction-induced Ca2+ overload involve secretory pathway Ca2+-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi apparatus on Ca2+ overload after cerebral ischemia and reperfusion remains unclear. Four-vessel occlusion rats were used as animal models of cerebral ischemia. The expression of secretory pathway Ca2+-ATPase 1 in the cortex and hippocampus was detected by immunoblotting, and Ca2+ concentrations in the cytoplasm and Golgi vesicles were determined. Results showed an overload of cytoplasmic Ca2+ during ischemia and reperfusion that reached a peak after reperfusion. Levels of Golgi Ca2+ showed an opposite effect. The expression of Golgi-specific secretory pathway Ca2+-ATPase 1 in the cortex and hippocampus decreased before ischemia and reperfusion, and increased after reperfusion for 6 hours. This variation was similar to the alteration of calcium in separated Golgi vesicles. These results indicate that the Golgi apparatus participates in the formation and alleviation of calcium overload, and that secretory pathway Ca2+-ATPase 1 tightly responds to ischemia and reperfusion in nerve cells. Thus, we concluded that secretory pathway Ca2+-ATPase 1 plays an essential role in cytosolic calcium regulation and its expression can be used as a marker of Golgi stress, responding to cerebral ischemia and reperfusion. The secretory pathway Ca2+-ATPase 1 can be an important neuroprotective target of ischemic stroke. PMID:26487855

  2. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  3. Proteolysis in the secretory pathway

    SciTech Connect

    Guzowski, D.E.; Bienkowski, R.S.

    1987-05-01

    Many secretory proteins are degraded intracellularly rather than secreted, however the location of this catabolic process is not known. The authors have tested the hypothesis that the degradation occurs in the organelles of the secretory pathway. Slices of rat liver were incubated with (/sup 14/C)leucine for 3 h and then incubated under chase conditions for 30 min. The tissue was homogenized and the Golgi apparatus, smooth endoplasmic reticulum (sER) and rough endoplasmic reticulum (rER) were isolated by ultracentrifugation on a discontinuous sucrose gradient. The organelles were incubated in 0.3M sucrose-50 mM citrate (pH 4) for 8-12 h at 37 C; control samples were incubated at 4 C. Percent degradation was calculated as the amount of acid soluble radioactivity released relative to total radioactivity in the sample. Proteolysis in the organelles incubated at 37 C was as follows: Golgi: 15-25%; sER: 10-20%; rER: 10-20%. Proteolysis at 4 C was negligible in all cases. These results support the hypothesis that the compartments of the secretory pathway are capable of degrading newly synthesized secretory proteins.

  4. The Secretory System of Arabidopsis

    PubMed Central

    Bassham, Diane C.; Brandizzi, Federica; Otegui, Marisa S.; Sanderfoot, Anton A.

    2008-01-01

    Over the past few years, a vast amount of research has illuminated the workings of the secretory system of eukaryotic cells. The bulk of this work has been focused on the yeast Saccharomyces cerevisiae, or on mammalian cells. At a superficial level, plants are typical eukaryotes with respect to the operation of the secretory system; however, important differences emerge in the function and appearance of endomembrane organelles. In particular, the plant secretory system has specialized in several ways to support the synthesis of many components of the complex cell wall, and specialized kinds of vacuole have taken on a protein storage role—a role that is intended to support the growing seedling, but has been co-opted to support human life in the seeds of many crop plants. In the past, most research on the plant secretory system has been guided by results in mammalian or fungal systems but recently plants have begun to stand on their own as models for understanding complex trafficking events within the eukaryotic endomembrane system. PMID:22303241

  5. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    SciTech Connect

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-07-12

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP/sub 1/ synaptic vesicles banding at 1.055 g/mL actively transported (/sup 3/H)acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more (/sup 3/H)AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP/sub 1/ vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP/sub 2/ synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of (/sup 3/H)vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP/sub 1/ vesicles the VP/sub 2/ vesicles had a ratio of (/sup 3/H)AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients ..cap alpha../sub H/ and equilibrium dissociation constants K for vesamicol binding to VP/sub 1/ and VP/sub 2/ vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP/sub 1/ and VP/sub 2/ synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.

  6. Autonomous docking ground demonstration

    NASA Technical Reports Server (NTRS)

    Lamkin, Steve L.; Le, Thomas Quan; Othon, L. T.; Prather, Joseph L.; Eick, Richard E.; Baxter, Jim M.; Boyd, M. G.; Clark, Fred D.; Spehar, Peter T.; Teters, Rebecca T.

    1991-01-01

    The Autonomous Docking Ground Demonstration is an evaluation of the laser sensor system to support the docking phase (12 ft to contact) when operated in conjunction with the guidance, navigation, and control (GN&C) software. The docking mechanism being used was developed for the Apollo/Soyuz Test Program. This demonstration will be conducted using the 6-DOF Dynamic Test System (DTS). The DTS simulates the Space Station Freedom as the stationary or target vehicle and the Orbiter as the active or chase vehicle. For this demonstration, the laser sensor will be mounted on the target vehicle and the retroflectors will be on the chase vehicle. This arrangement was chosen to prevent potential damage to the laser. The laser sensor system, GN&C, and 6-DOF DTS will be operated closed-loop. Initial conditions to simulate vehicle misalignments, translational and rotational, will be introduced within the constraints of the systems involved.

  7. Gemini Rendezvous Docking Simulator

    NASA Technical Reports Server (NTRS)

    1964-01-01

    Gemini Rendezvous Docking Simulator suspended from the roof of the Langley Research Center's aircraft hanger. Francis B. Smith wrote: 'The rendezvous and docking operation of the Gemini spacecraft with the Agena and of the Apollo Command Module with the Lunar Excursion Module have been the subject of simulator studies for several years. [This figure] illustrates the Gemini-Agena rendezvous docking simulator at Langley. The Gemini spacecraft was supported in a gimbal system by an overhead crane and gantry arrangement which provided 6 degrees of freedom - roll, pitch, yaw, and translation in any direction - all controllable by the astronaut in the spacecraft. Here again the controls fed into a computer which in turn provided an input to the servos driving the spacecraft so that it responded to control motions in a manner which accurately simulated the Gemini spacecraft.'

  8. The native structure of cytoplasmic dynein at work translocating vesicles in Paramecium.

    PubMed

    Ishida, Masaki; Aihara, Marilynn S; Allen, Richard D; Fok, Agnes K

    2011-01-01

    In Paramecium multimicronucleatum, the discoidal vesicles, the acidosomes and the 100-nm carrier vesicles are involved in phagosome formation, phagosome acidification and endosomal processing, respectively. Numerous cross bridges link these vesicles to the kinetic side of the microtubules of a cytopharyngeal microtubular ribbon. Vesicles are translocated along these ribbons in a minus-end direction towards the cytopharynx. A monoclonal antibody specific for the light vanadate-photocleaved fragment of the heavy chain of cytoplasmic dynein was used to show that this dynein is located between the discoidal vesicles and the ribbons as well as on the cytosolic surface of the acidosomes and the 100-nm carrier vesicles. This antibody inhibited the docking of the vesicles to the microtubular ribbons so that the transport of discoidal vesicles and acidosomes were reduced by 60% and 70%, respectively. It had little effect on the dynein's velocity of translocation. These results show that cytoplasmic dynein is the motor for vesicle translocation and its location, between the vesicles and the ribbons, indicates that the cross bridges seen at this location in thin sections and in quick-frozen, deep-etched replicas are apparently the working dyneins. Such a working dynein cross bridge, as preserved by ultra-rapid freezing, is 54 nm long and has two legs arising from a globular head that appears to be firmly bound to its cargo vesicle and each leg consists of ≥3 beaded subunits with the last subunit making contact with the microtubular ribbon.

  9. Suitlock Docking Mechanism

    NASA Technical Reports Server (NTRS)

    Culbertson, Philip, Jr. (Inventor)

    1997-01-01

    An environmental protective suit used for hazardous clean-up or space applications includes a suitlock docking mechanism that allows for easy egress and ingress of a crew member between a sealed vessel and a possibly contaminated environment. The suitlock docking mechanism comprises a single actuator that controls latches which, in turn, respectfully control rack and pinion assemblies that allow for easy removal and attachment of a life support equipment enclosure shell to the environmental protective suit or to the vehicle from which the operator performs his/her duties.

  10. last ATV docking OBT

    NASA Image and Video Library

    2014-08-07

    ISS040-E-089627 (7 Aug. 2014) --- In the International Space Station?s Zvezda Service Module, European Space Agency astronaut Alexander Gerst (foreground) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, participate in a training session in preparation for the rendezvous and docking of ESA?s fifth and final Automated Transfer Vehicle (ATV-5). Nicknamed the ?Georges Lemaitre? in honor of the Belgian physicist and astronomer who first proposed the Big Bang theory, ATV-5 will deliver more than seven tons of scientific experiments, food and other supplies when it docks to the aft end of Zvezda on Aug. 12.

  11. last ATV docking OBT

    NASA Image and Video Library

    2014-08-07

    ISS040-E-089629 (7 Aug. 2014) --- In the International Space Station?s Zvezda Service Module, European Space Agency astronaut Alexander Gerst (foreground) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, participate in a training session in preparation for the rendezvous and docking of ESA?s fifth and final Automated Transfer Vehicle (ATV-5). Nicknamed the ?Georges Lemaitre? in honor of the Belgian physicist and astronomer who first proposed the Big Bang theory, ATV-5 will deliver more than seven tons of scientific experiments, food and other supplies when it docks to the aft end of Zvezda on Aug. 12.

  12. 9. Southeast end, dock no. 492. Dock no. 493 in ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Southeast end, dock no. 492. Dock no. 493 in background. View to west. - Offutt Air Force Base, Looking Glass Airborne Command Post, Nose Docks, On either side of Hangar Access Apron at Northwest end of Project Looking Glass Historic District, Bellevue, Sarpy County, NE

  13. Highly Flexible Protein-Peptide Docking Using CABS-Dock.

    PubMed

    Ciemny, Maciej Paweł; Kurcinski, Mateusz; Kozak, Konrad Jakub; Kolinski, Andrzej; Kmiecik, Sebastian

    2017-01-01

    Protein-peptide molecular docking is a difficult modeling problem. It is even more challenging when significant conformational changes that may occur during the binding process need to be predicted. In this chapter, we demonstrate the capabilities and features of the CABS-dock server for flexible protein-peptide docking. CABS-dock allows highly efficient modeling of full peptide flexibility and significant flexibility of a protein receptor. During CABS-dock docking, the peptide folding and binding process is explicitly simulated and no information about the peptide binding site or its structure is used. This chapter presents a successful CABS-dock use for docking a potentially therapeutic peptide to a protein target. Moreover, simulation contact maps, a new CABS-dock feature, are described and applied to the docking test case. Finally, a tutorial for running CABS-dock from the command line or command line scripts is provided. The CABS-dock web server is available from http://biocomp.chem.uw.edu.pl/CABSdock/ .

  14. Dry dock no. 4. Service Building between dry docks 4 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Dry dock no. 4. Service Building between dry docks 4 and 5. Floor plans (Navy Yard Public Works Office 1941). In files of Cushman & Wakefield, building 501. Philadelphia Naval Business Center. - Naval Base Philadelphia-Philadelphia Naval Shipyard, Service Building, Dry Docks No. 4 & 5, League Island, Philadelphia, Philadelphia County, PA

  15. Synaptic Vesicle Endocytosis

    PubMed Central

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

  16. Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii.

    PubMed

    Stewart, Rebecca J; Ferguson, David J P; Whitehead, Lachlan; Bradin, Clare H; Wu, Hong J; Tonkin, Christopher J

    2016-02-01

    Upon infection, apicomplexan parasites quickly invade host cells and begin a replicative cycle rapidly increasing in number over a short period of time, leading to tissue lysis and disease. The secretory pathway of these highly polarized protozoan parasites tightly controls, in time and space, the biogenesis of specialized structures and organelles required for invasion and intracellular survival. In other systems, regulation of protein trafficking can occur by phosphorylation of vesicle fusion machinery. Previously, we have shown that Toxoplasma gondii αSNAP - a protein that controls the disassembly of cis-SNARE complexes--is phosphorylated. Here, we show that this post-translational modification is required for the correct function of αSNAP in controlling secretory traffic. We demonstrate that during intracellular development conditional expression of a non-phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, we show that the vestigial plastid (termed apicoplast), although reported not to be reliant on Golgi trafficking for biogenesis, is also affected upon overexpression of αSNAP and is much more sensitive to the levels of this protein than targeting to other organelles. This work highlights the importance of αSNAP and its phosphorylation in Toxoplasma organelle biogenesis and exposes a hereto fore-unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Understanding Plastid Vesicle Transport - Could it Provide Benefit for Human Medicine?

    PubMed

    Khan, Nadir Zaman; Lindquist, Emelie; Alezzawi, Mohamed; Aronsson, Henrik

    2017-01-01

    In plants, vesicle transport occurs in the secretory pathway in the cytosol, between the membranes of different compartments. Several protein components have been identified to be involved in the process and their functions were characterized. Both cargos and other molecules (such as hormones) have been shown to use vesicle transport, although the major constituents of vesicles are lipids which are transferred from donor to acceptor membranes. In humans, malfunction of the cytosolic vesicle transport system leads to different diseases. To better understand and ultimately cure these human diseases, studying other model systems such as yeast can be beneficial. Plants with their cytosolic vesicle transport system could serve as another model system. However, this review focuses on plant vesicles not present in the cytosol but in the chloroplasts, where lipids produced in the surrounding envelope are transported through the aqueous stroma to the thylakoid membranes. Although chloroplast vesicles have found both biochemical and ultrastructural support, only two proteins have been characterized as components of the pathway. However, using bioinformatics a number of other proteins have been suggested as homologs to the cytosolic system. Based on these findings vesicles of chloroplasts are likely most similar to the vesicles trafficking from ER to Golgi, or may even be unique, but important experimental support is yet lacking. In this review, proposed vesicle transport components in chloroplasts are presented, and their possible future implementation for human medicine is discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps

    PubMed Central

    Harris, Mark C.; Cislo, Dillon; Lenz, Joan S.; Umbach, Christopher

    2017-01-01

    Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link. PMID:28323853

  19. Expedition 27 Docking

    NASA Image and Video Library

    2011-04-06

    Russian Mission Control Center is seen on Thursday, April 7, 2011 in Korolev, Russia. The Soyuz TMA-21 docked to the International Space Station carrying Expedition 27 Soyuz Commander Alexander Samokutyaev, NASA Flight Engineer Ron Garan and Russian Flight Engineer Andrey Borisenko. Photo Credit: (NASA/Carla Cioffi)

  20. Expedition 27 Docking

    NASA Image and Video Library

    2011-04-06

    View from the balcony of the Russian Mission Control Center in Korolev, Russia as the Soyuz TMA-21 nears the International Space Station on Thursday, April 7, 2011. The Soyuz TMA-21 docked to the International Space Station carrying Expedition 27 Soyuz Commander Alexander Samokutyaev, NASA Flight Engineer Ron Garan and Russian Flight Engineer Andrey Borisenko. Photo Credit: (NASA/Carla Cioffi)

  1. Soyuz docked on ISS

    NASA Image and Video Library

    2014-05-29

    ISS040-E-005997 (29 May 2014) --- Featured near the center of this night photograph, taken by one of the Expedition 40 crew members aboard the International Space Station, is Seoul, South Korea. A Russian Soyuz spacecraft, docked to the orbital outpost, takes up the top part of the frame.

  2. Expedition 25 Docking

    NASA Image and Video Library

    2010-10-09

    Alexei Krasnov, center, Director of Manned Space Programs Department, Roscosmos, answers a reporter’s question during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Sunday, Oct. 10, 2010. Photo Credit: (NASA/Carla Cioffi)

  3. Expedition 25 Docking

    NASA Image and Video Library

    2010-10-09

    William Gerstenmaier, third from right, Associate Administrator for Space Operations answers a reporter’s question during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Sunday, Oct. 10, 2010. Gerstenmaier is flanked by NASA and ROSCOSMOS senior officials. Photo Credit: (NASA/Carla Cioffi)

  4. Expedition 25 Docking

    NASA Image and Video Library

    2010-10-09

    The Soyuz TMA-01M nears its docking with the International Space Station as seen in the video monitor at Russian Mission Control Center in Korolev, Russia on Sunday, Oct. 10, 2010. The TMA-01M delivered the crew of Expedition 25 Soyuz Commander Alexander Kaleri, Flight Engineer Scott Kelly and Flight Engineer Oleg Skripochka to the ISS. Photo Credit: (NASA/Carla Cioffi)

  5. Expedition 25 Docking

    NASA Image and Video Library

    2010-10-09

    The crews of Expedition 24 and Expedition 25 are seen on a video monitor at Russian Mission Control in Korolev, Russia shortly after crew members of Expedition 25 docked to and entered the International Space Station on Sunday, Oct. 10, 2010. Photo Credit: (NASA/Carla Cioffi)

  6. Expedition 28 Docking

    NASA Image and Video Library

    2011-06-10

    Guests at the Russian Mission Control Center in Korolove, Russia watch on a large screen TV as the Soyuz TMA-02M nears its docking to the International Space Station on Friday, June 10, 2011. Photo Credit: (NASA/Carla Cioffi)

  7. ATV docking ops

    NASA Image and Video Library

    2014-08-12

    ISS040-E-091673 (12 Aug. 2014) --- In the Zvezda Service Module, European Space Agency astronaut Alexander Gerst (right) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, take a brief moment for a photo during the approach and docking operations of ESA's "Georges Lemaitre" Automated Transfer Vehicle-5 (ATV-5) to the International Space Station.

  8. ATV docking ops

    NASA Image and Video Library

    2014-08-12

    ISS040-E-091688 (12 Aug. 2014) --- In the Zvezda Service Module, European Space Agency astronaut Alexander Gerst (left) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, monitor the approach and docking of ESA?s "Georges Lemaitre" Automated Transfer Vehicle-5 (ATV-5) to the International Space Station.

  9. ATV docking ops

    NASA Image and Video Library

    2014-08-12

    ISS040-E-091635 (12 Aug. 2014) --- In the Zvezda Service Module, European Space Agency astronaut Alexander Gerst (left) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, monitor the approach and docking of ESA's "Georges Lemaitre" Automated Transfer Vehicle-5 (ATV-5) to the International Space Station.

  10. ATV docking ops

    NASA Image and Video Library

    2014-08-12

    ISS040-E-091638 (12 Aug. 2014) --- In the Zvezda Service Module, European Space Agency astronaut Alexander Gerst (left) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, monitor the approach and docking of ESA's "Georges Lemaitre" Automated Transfer Vehicle-5 (ATV-5) to the International Space Station.

  11. ATV docking ops

    NASA Image and Video Library

    2014-08-12

    ISS040-E-091634 (12 Aug. 2014) --- In the Zvezda Service Module, European Space Agency astronaut Alexander Gerst (left) and Russian cosmonaut Alexander Skvortsov, both Expedition 40 flight engineers, monitor the approach and docking of ESA's "Georges Lemaitre" Automated Transfer Vehicle-5 (ATV-5) to the International Space Station.

  12. Vesicle Pool Size at the Salamander Cone Ribbon Synapse

    PubMed Central

    Bartoletti, Theodore M.; Babai, Norbert

    2010-01-01

    Cone light responses are transmitted to postsynaptic neurons by changes in the rate of synaptic vesicle release. Vesicle pool size at the cone synapse constrains the amount of release and can thus shape contrast detection. We measured the number of vesicles in the rapidly releasable and reserve pools at cone ribbon synapses by performing simultaneous whole cell recording from cones and horizontal or off bipolar cells in the salamander retinal slice preparation. We found that properties of spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs) are representative of mEPSCs evoked by depolarizing presynaptic stimulation. Strong, brief depolarization of the cone stimulated release of the entire rapidly releasable pool (RRP) of vesicles. Comparing charge transfer of the EPSC with mEPSC charge transfer, we determined that the fast component of the EPSC reflects release of ∼40 vesicles. Comparing EPSCs with simultaneous presynaptic capacitance measurements, we found that horizontal cell EPSCs constitute 14% of the total number of vesicles released from a cone terminal. Using a fluorescent ribeye-binding peptide, we counted ∼13 ribbons per cone. Together, these results suggest each cone contacts a single horizontal cell at ∼2 ribbons. The size of discrete components in the EPSC amplitude histogram also suggested ∼2 ribbon contacts per cell pair. We therefore conclude there are ∼20 vesicles per ribbon in the RRP, similar to the number of vesicles contacting the plasma membrane at the ribbon base. EPSCs evoked by lengthy depolarization suggest a reserve pool of ∼90 vesicles per ribbon, similar to the number of additional docking sites further up the ribbon. PMID:19923246

  13. PharmDock: a pharmacophore-based docking program

    PubMed Central

    2014-01-01

    Background Protein-based pharmacophore models are enriched with the information of potential interactions between ligands and the protein target. We have shown in a previous study that protein-based pharmacophore models can be applied for ligand pose prediction and pose ranking. In this publication, we present a new pharmacophore-based docking program PharmDock that combines pose sampling and ranking based on optimized protein-based pharmacophore models with local optimization using an empirical scoring function. Results Tests of PharmDock on ligand pose prediction, binding affinity estimation, compound ranking and virtual screening yielded comparable or better performance to existing and widely used docking programs. The docking program comes with an easy-to-use GUI within PyMOL. Two features have been incorporated in the program suite that allow for user-defined guidance of the docking process based on previous experimental data. Docking with those features demonstrated superior performance compared to unbiased docking. Conclusion A protein pharmacophore-based docking program, PharmDock, has been made available with a PyMOL plugin. PharmDock and the PyMOL plugin are freely available from http://people.pharmacy.purdue.edu/~mlill/software/pharmdock. PMID:24739488

  14. Excitatory and Inhibitory Neurons in the Hippocampus Exhibit Molecularly Distinct Large Dense Core Vesicles

    PubMed Central

    Ramírez-Franco, José J.; Munoz-Cuevas, Francisco J.; Luján, Rafael; Jurado, Sandra

    2016-01-01

    Hippocampal interneurons comprise a diverse family of inhibitory neurons that are critical for detailed information processing. Along with gamma-aminobutyric acid (GABA), interneurons secrete a myriad of neuroactive substances via secretory vesicles but the molecular composition and regulatory mechanisms remain largely unknown. In this study, we have carried out an immunohistofluorescence analysis to describe the molecular content of vesicles in distinct populations of hippocampal neurons. Our results indicate that phogrin, an integral protein of secretory vesicles in neuroendocrine cells, is highly enriched in parvalbumin-positive interneurons. Consistently, immunoelectron microscopy revealed phogrin staining in axon terminals of symmetrical synapses establishing inhibitory contacts with cell bodies of CA1 pyramidal neurons. Furthermore, phogrin is highly expressed in CA3 and dentate gyrus (DG) interneurons which are both positive for PV and neuropeptide Y. Surprisingly, chromogranin B a canonical large dense core vesicle marker, is excluded from inhibitory cells in the hippocampus but highly expressed in excitatory CA3 pyramidal neurons and DG granule cells. Our results provide the first evidence of phogrin expression in hippocampal interneurons and suggest the existence of molecularly distinct populations of secretory vesicles in different types of inhibitory neurons. PMID:27630542

  15. Ca(2+)-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

    PubMed

    Vashi, Nimi; Andrabi, Syed Bilal Ahmad; Ghanwat, Swapnil; Suar, Mrutyunjay; Kumar, Dhiraj

    2017-03-31

    The role of Golgi apparatus during phagocytic uptake by macrophages has been ruled out in the past. Notably, all such reports were limited to Fcγ receptor-mediated phagocytosis. Here, we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos, except the IgG-coated ones. We report recruitment of mannosidase-II-positive Golgi-derived vesicles during uptake of diverse targets, including latex beads, Escherichia coli, Salmonella typhimurium, and Mycobacterium tuberculosis in human and mouse macrophages. The recruitment of mannosidase-II vesicles was an early event mediated by focal exocytosis and coincided with the recruitment of transferrin receptor, VAMP3, and dynamin-2. Brefeldin A treatment inhibited mannosidase-II recruitment and phagocytic uptake of serum-coated or -uncoated latex beads and E. coli However, consistent with previous studies, brefeldin A treatment did not affect uptake of IgG-coated latex beads. Mechanistically, recruitment of mannosidase-II vesicles during phagocytic uptake required Ca(2+) from both extra- and intracellular sources apart from PI3K, microtubules, and dynamin-2. Extracellular Ca(2+) via voltage-gated Ca(2+) channels established a Ca(2+)-dependent local phosphatidylinositol 1,4,5-trisphosphate gradient, which guides the focal movement of Golgi-derived vesicles to the site of uptake. We confirmed Golgi-derived vesicles recruited during phagocytosis were secretory vesicles as their recruitment was sensitive to depletion of VAMP2 or NCS1, whereas recruitment of the recycling endosome marker VAMP3 was unaffected. Depletion of both VAMP2 and NCS1 individually resulted in the reduced uptake by macrophages. Together, the study provides a previously unprecedented role of Golgi-derived secretory vesicles in phagocytic uptake, the key innate defense function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Secretory adenocarcinoma of the endometrium.

    PubMed

    Tobon, H; Watkins, G J

    1985-01-01

    Secretory adenocarcinomas of the endometrium are uncommon tumors distinct from clear cell carcinomas. We reviewed nine cases that included the original endometrial curettings and the specimens of uteri with both adnexa [total abdominal hysterectomy-bilateral salpingo-oophorectomy (TAH-BSO)]. The patients' ages ranged from 36 to 79 years (with an average of 55 years). Six women were postmenopausal, and most complained of vaginal bleeding. Two patients were nulliparous and the others had one to four children. Four patients were obese, of whom one was diabetic and hypertensive. Eight tumors were of grade I and one was grade II. The histologic pattern was comparable to that of secretory endometrium, days 17 to 22, and the glands were positive with periodic acid-Schiff (whether predigested or not); they were partly positive with alcian blue and negative with Best Carmine. The carcinoma in one case was positive for carcinoembryonic antigen; all cases were negative with alpha-fetoprotein. Six patients were staged as IA and three as IB. One 47-year-old patient had a concurrent endometrioid adenocarcinoma (grade II) of the right ovary with squamous, clear cell, and mucinous components. Three cases had no tumour penetration of the myometrium while in the others penetration varied from 5 to 70%. One patient received intracavitary radium prior to TAH-BSO and two had postoperative radiation. All patients are alive 11 to 113 months following surgery. Secretory adenocarcinoma of the endometrium should be separated from clear cell carcinoma, as it has the pattern of secretory endometrium day 17 to 22, is very well differentiated, and has a relatively good prognosis.

  17. Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

    PubMed

    Vomund, Anthony N; Zinselmeyer, Bernd H; Hughes, Jing; Calderon, Boris; Valderrama, Carolina; Ferris, Stephen T; Wan, Xiaoxiao; Kanekura, Kohsuke; Carrero, Javier A; Urano, Fumihiko; Unanue, Emil R

    2015-10-06

    Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

  18. Bacterial Vesicle Secretion and the Evolutionary Origin of the Eukaryotic Endomembrane System.

    PubMed

    Gould, Sven B; Garg, Sriram G; Martin, William F

    2016-07-01

    Eukaryotes possess an elaborate endomembrane system with endoplasmic reticulum, nucleus, Golgi, lysosomes, peroxisomes, autophagosomes, and dynamic vesicle traffic. Theories addressing the evolutionary origin of eukaryotic endomembranes have overlooked the outer membrane vesicles (OMVs) that bacteria, archaea, and mitochondria secrete into their surroundings. We propose that the eukaryotic endomembrane system originated from bacterial OMVs released by the mitochondrial ancestor within the cytosol of its archaeal host at eukaryote origin. Confined within the host's cytosol, OMVs accumulated naturally, fusing either with each other or with the host's plasma membrane. This matched the host's archaeal secretory pathway for cotranslational protein insertion with outward bound mitochondrial-derived vesicles consisting of bacterial lipids, forging a primordial, secretory endoplasmic reticulum as the cornerstone of the eukaryotic endomembrane system. VIDEO ABSTRACT. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. PLURIVESICULAR SECRETORY PROCESSES AND NERVE ENDINGS IN THE PINEAL GLAND OF THE RAT

    PubMed Central

    De Robertis, Eduardo; de Iraldi, Amanda Pellegrino

    1961-01-01

    The pineal body of white normal rats, 1.5 to 3 months old, was studied under the electron microscope. A single type of parenchymal cell—the pinealocyte—is recognized as the main component of the tissue, and some of the structural characteristics of the nucleus and cytoplasm are described. The main morphological characteristic of the pinealocytes is represented by club-shaped perivascular expansions connected to the cell by thin pedicles. They are found lying in a large, clear space surrounding the blood capillaries. The name plurivesicular secretory processes is proposed, to emphasize the main structural feature and the probable function of these cellular expansions. A tubulofibrillar component is mainly found in the pedicle, and within the expansion there are numerous small mitochondria and densily packed vesicles of about 425 A. Two types of vesicles, one with a homogeneous content and another with a very dense osmium deposit, are described. Between the two types there are intermediary forms. In these processes, mitochondria show profound changes which may lead to complete vacuolization. The significance of this plurivesicular secretory component is discussed in the light of recent work on the biogenic amines of the pineal body and preliminary experiments showing the release of the vesicles containing dense granules after treatment with reserpine. These vesicles are interpreted as the site of storage of some of the biogenic amines. Bundles of unmyelinated nerve fibers and endings on large blood vessels which also contain a plurivesicular content are described and tentatively interpreted as adrenergic nerve terminals. PMID:13720811

  20. Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles.

    PubMed

    Boguslavsky, Shlomit; Chiu, Tim; Foley, Kevin P; Osorio-Fuentealba, Cesar; Antonescu, Costin N; Bayer, K Ulrich; Bilan, Philip J; Klip, Amira

    2012-10-01

    GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding-deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.

  1. Vesicle extrusion in nanopores

    NASA Astrophysics Data System (ADS)

    Joóos, B. Éla; Bertrand, Martin; Ouellet, S. Ébastien

    2010-03-01

    Monodisperse vesicles of nearly circular shape or liposomes are used as drug delivery systems. Their fabrication involves repeated passage of large vesicles through small pores. At each passage the vesicle ruptures and the fragments reform into smaller vesicles. We report on the last stages of the process where small liposomes are pushed by pressure differences into nano-sized pores, and we study the stress distribution along the lipid bilayer to determine the rupture lines. This is done by performing coarse grained Molecular Dynamics simulations. We have developed a technique to measure the stress in the membrane based on a tessellation of the surface which allows us to monitor the local area per lipid fluctuations. The results show subtle and complex flow phenomena. We can predict the final size distribution after many passages. Comparisons will be made with existing experimental data.

  2. Synaptic vesicle populations in saccular hair cells reconstructed by electron tomography.

    PubMed

    Lenzi, D; Runyeon, J W; Crum, J; Ellisman, M H; Roberts, W M

    1999-01-01

    We used electron tomography to map the three-dimensional architecture of the ribbon-class afferent synapses in frog saccular hair cells. The synaptic body (SB) at each synapse was nearly spherical (468 +/- 65 nm diameter; mean +/- SD) and was covered by a monolayer of synaptic vesicles (34.3 nm diameter; 8.8% coefficient of variation), many of them tethered to it by approximately 20-nm-long filaments, at an average density of 55% of close-packed (376 +/- 133 vesicles per SB). These vesicles could support approximately 900 msec of exocytosis at the reported maximal rate, which the cells can sustain for at least 2 sec, suggesting that replenishment of vesicles on the SB is not rate limiting. Consistent with this interpretation, prolonged K+ depolarization did not deplete vesicles on the SB. The monolayer of SB-associated vesicles remained after cell lysis in the presence of 4 mM Ca2+, indicating that the association is tight and Ca2+-resistant. The space between the SB and the plasma membrane contained numerous vesicles, many of which ( approximately 32 per synapse) were in contact with the plasma membrane. This number of docked vesicles could support maximal exocytosis for at most approximately 70 msec. Additional docked vesicles were seen within a few hundred nanometers of the synapse and occasionally at greater distances. The presence of omega profiles on the plasma membrane around active zones, in the same locations as coated pits and coated vesicles labeled with an extracellular marker, suggests that local membrane recycling may contribute to the three- to 14-fold greater abundance of vesicles in the cytoplasm (not associated with the SB) near synapses than in nonsynaptic regions.

  3. Pulling on adhered vesicles

    NASA Astrophysics Data System (ADS)

    Smith, Ana-Suncana; Goennenwein, Stefanie; Lorz, Barbara; Seifert, Udo; Sackmann, Erich

    2004-03-01

    A theoretical model describing pulling of vesicles adhered in a contact potential has been developed. Two different regimes have been recognized. For weak to middle-strength adhesive potentials, locally stable shapes are found in a range of applied forces, separated from the free shape by an energy barrier. The phase diagram contains regions with either a unique bound shape or an additional meta-stable shape. Upon pulling, these shapes unbind discontinuously since the vesicle disengage from the surface while still possessing a finite adhesion area (Smith 2003a). In a strong adhesion regime, a competition between adhesion and tether formation is observed. A critical onset force is identified where a tether spontaneously appears as a part of a second order shape transition. Further growth of a tether is followed by a detachment process which terminates at a finite force when a vesicle continuously unbinds from the substrate (Smith 2003b). Both critical forces, as well as all shape parameters, are calculated as a function of the reduced volume and the strength of adhesive potential. Analogous experimental study has been performed where a vertical magnetic tweezers are used in combination with micro-interferometric and confocal techniques to reproduce the same symmetry as in the theoretical investigation. Giant vesicles are bound to the substrate by numerous specific bonds formed between ligands and receptors incorporated into the vesicle and the substrate, respectively. Application of a constant force is inducing a new thermodynamic equilibrium of the system where the vesicle is partially unbound from the substrate (Goennenwein 2003). The shapes of vesicles are compared prior and during application of the force. Very good agreement is obtained, particularly in the middle-strength adhesion regime (Smith 2003c). References: 1. A.-S. Smith, E. Sackmann, U. Seifert: Effects of a pulling force on the shape of a bound vesicle, Europhys. Lett., 64, 2 (2003). 2. A.-S. Smith

  4. Pro-hormone secretogranin II regulates dense core secretory granule biogenesis in catecholaminergic cells.

    PubMed

    Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L; Mahata, Sushil K; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J; O'Connor, Daniel T; Taupenot, Laurent

    2010-03-26

    Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

  5. Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells*

    PubMed Central

    Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L.; Mahata, Sushil K.; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J.; O'Connor, Daniel T.; Taupenot, Laurent

    2010-01-01

    Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H+-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network. PMID:20061385

  6. F2Dock: Fast Fourier Protein-Protein Docking

    PubMed Central

    Bajaj, Chandrajit; Chowdhury, Rezaul; Siddavanahalli, Vinay

    2009-01-01

    The functions of proteins is often realized through their mutual interactions. Determining a relative transformation for a pair of proteins and their conformations which form a stable complex, reproducible in nature, is known as docking. It is an important step in drug design, structure determination and understanding function and structure relationships. In this paper we extend our non-uniform fast Fourier transform docking algorithm to include an adaptive search phase (both translational and rotational) and thereby speed up its execution. We have also implemented a multithreaded version of the adaptive docking algorithm for even faster execution on multicore machines. We call this protein-protein docking code F2Dock (F2 = Fast Fourier). We have calibrated F2Dock based on an extensive experimental study on a list of benchmark complexes and conclude that F2Dock works very well in practice. Though all docking results reported in this paper use shape complementarity and Coulombic potential based scores only, F2Dock is structured to incorporate Lennard-Jones potential and re-ranking docking solutions based on desolvation energy. PMID:21071796

  7. Magnetic docking aid for orbiter to ISS docking

    NASA Technical Reports Server (NTRS)

    Schneider, William C.; Nagy, Kornel; Schliesing, John A.

    1996-01-01

    The present docking system for the Orbiter uses mechanical capture latches that are actuated by contact forces. The forces are generated when the two approaching masses collide at the docking mechanism. There is always a trade-off between having high enough momentum to effect capture and low enough momentum to avoid structural overload or unacceptable angular displacements. The use of the present docking system includes a contact thrusting maneuver that causes high docking loads to be included into Space Station. A magnetic docking aid has been developed to reduce the load s during docking. The magnetic docking aid is comprised of two extendible booms that are attached adjacent to the docking structure with electromagnets attached on the end of the boom. On the mating vehicle, two steel plates are attached. As the Orbiter approaches Space Station, the booms are extended, and the magnets attach to the actuated (without thrusting), by slowly driving the extendible booms to the stowed position, thus reacting the load into the booms. This results in a docking event that has lower loads induced into Space Station structure. This method also greatly simplifies the Station berthing tasks, since the Shuttle Remote Manipulation System (SRMS) arm need only place the element to be berthed on the magnets (no load required), rather than firing the Reaction Control System (RCS) jets to provide the required force for capture latch actuation. The Magnetic Docking Aid was development testing on a six degree-of-freedom (6 DOF) system at JSC.

  8. A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta.

    PubMed

    Tzelos, Thomas; Matthews, Jacqueline B; Buck, Amy H; Simbari, Fabio; Frew, David; Inglis, Neil F; McLean, Kevin; Nisbet, Alasdair J; Whitelaw, C Bruce A; Knox, David P; McNeilly, Tom N

    2016-05-15

    Teladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Excretory/secretory products from the gastrointestinal nematode Trichuris muris.

    PubMed

    Tritten, Lucienne; Tam, Mifong; Vargas, Mireille; Jardim, Armando; Stevenson, Mary M; Keiser, Jennifer; Geary, Timothy G

    2017-07-01

    To better control gastrointestinal nematode infections in humans and animals, it is important to understand the strategies used by these parasites to modulate the host immune system. In this regard, molecules released by parasites have been attributed crucially important roles in host-parasite negotiations. We characterized the excretory/secretory (E/S) microRNA (miRNA) and protein profiles from the mouse gastrointestinal nematode parasite Trichuris muris. Released miRNAs were subjected to miRNA sequencing and E/S proteins were analysed by mass spectrometry. Fourteen miRNAs were identified in T. muris exosome-like vesicles, as well as 73 proteins of nematode origin, 11 of which were unique to this study. Comparison with published nematode protein secretomes revealed high conservation at the functional level. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Expedition 27 Docking

    NASA Image and Video Library

    2011-04-06

    The Soyuz TMA-21 is seen as it approaches the International Space Station on a large screen TV at the Russian Mission Control Center in Korolev, Russia on Thursday, April 7, 2011. The Soyuz TMA-21 docked to the International Space Station carrying Expedition 27 Soyuz Commander Alexander Samokutyaev, NASA Flight Engineer Ron Garan and Russian Flight Engineer Andrey Borisenko. Photo Credit: (NASA/Carla Cioffi)

  11. Expedition 21 Docking

    NASA Image and Video Library

    2009-10-01

    Anna-Marie Williams talks on the phone to her husband Expedition 21 with Flight Engineer Jeffrey N. Williams who is onboard the International Space Station (ISS) from the Mission Control Center Moscow in Korolev, Russia shortly after the successful docking of the Soyuz TMA-16 spacecraft with the International Space Station marking the start of Expedition 21 with Williams, Expedition 21 Flight Engineer Maxim Suraev, and Spaceflight Participant Guy Laliberté, Friday, Oct. 2, 2009. Photo Credit: (NASA/Bill Ingalls)

  12. Rendezvous Docking Simulator

    NASA Technical Reports Server (NTRS)

    1964-01-01

    Multiple exposure of Rendezvous Docking Simulator. The Gemini spacecraft was supported in a gimbal system by an overhead crane and gantry arrangement which provided 6 degrees of freedom - roll, pitch, yaw, and translation in any direction - all controllable by the astronaut in the spacecraft. The controls fed into a computer which in turn provided an input to the servos driving the spacecraft so that it responded to control motions in a manner which accurately simulated the Gemini spacecraft.

  13. Cell-Dock: high-performance protein-protein docking.

    PubMed

    Pons, Carles; Jiménez-González, Daniel; González-Álvarez, Cecilia; Servat, Harald; Cabrera-Benítez, Daniel; Aguilar, Xavier; Fernández-Recio, Juan

    2012-09-15

    The application of docking to large-scale experiments or the explicit treatment of protein flexibility are part of the new challenges in structural bioinformatics that will require large computer resources and more efficient algorithms. Highly optimized fast Fourier transform (FFT) approaches are broadly used in docking programs but their optimal code implementation leaves hardware acceleration as the only option to significantly reduce the computational cost of these tools. In this work we present Cell-Dock, an FFT-based docking algorithm adapted to the Cell BE processor. We show that Cell-Dock runs faster than FTDock with maximum speedups of above 200×, while achieving results of similar quality. The source code is released under GNU General Public License version 2 and can be downloaded from http://mmb.pcb.ub.es/~cpons/Cell-Dock. djimenez@ac.upc.edu or juanf@bsc.es Supplementary data are available at Bioinformatics online.

  14. Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

    PubMed

    Santodomingo, Jaime; Vay, Laura; Camacho, Marcial; Hernández-Sanmiguel, Esther; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2008-10-01

    The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

  15. Fusion of Endosomes Involved in Synaptic Vesicle Recycling

    PubMed Central

    Holroyd, Claudia; Kistner, Ute; Annaert, Wim; Jahn, Reinhard

    1999-01-01

    Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 × 10−6 M. Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca2+. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior. PMID:10473644

  16. Involvement of AQP6 in the Mercury-sensitive osmotic lysis of rat parotid secretory granules.

    PubMed

    Matsuki-Fukushima, Miwako; Fujita-Yoshigaki, Junko; Murakami, Masataka; Katsumata-Kato, Osamu; Yokoyama, Megumi; Sugiya, Hiroshi

    2013-03-01

    In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br⁻ > I⁻ > Cl⁻ and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid

  17. Quantitative Analysis of Ribbons, Vesicles, and Cisterns at the Cat Inner Hair Cell Synapse: Correlations with Spontaneous Rate

    PubMed Central

    Kantardzhieva, Albena; Liberman, M. Charles; Sewell, William F.

    2015-01-01

    Cochlear hair cells form ribbon synapses with terminals of the cochlear nerve. To test the hypothesis that one function of the ribbon is to create synaptic vesicles from the cisternal structures that are abundant at the base of hair cells, we analyzed the distribution of vesicles and cisterns around ribbons from serial sections of inner hair cells in the cat, and compared data from low and high spontaneous rate (SR) synapses. Consistent with the hypothesis, we identified a “sphere of influence” of 350 nm around the ribbon, with fewer cisterns and many more synaptic vesicles. Although high- and low-SR ribbons tended to be longer and thinner than high-SR ribbons, the total volume of the two ribbon types was similar. There were almost as many vesicles docked at the active zone as attached to the ribbon. The major SR-related difference was that low-SR ribbons had more synaptic vesicles intimately associated with them. Our data suggest a trend in which low-SR synapses had more vesicles attached to the ribbon (51.3 vs. 42.8), more docked between the ribbon and the membrane (12 vs. 8.2), more docked at the active zone (56.9 vs. 44.2), and more vesicles within the “sphere of influence” (218 vs. 166). These data suggest that the structural differences between high-and low-SR synapses may be more a consequence, than a determinant, of the physiological differences. PMID:23787810

  18. Quantitative analysis of ribbons, vesicles, and cisterns at the cat inner hair cell synapse: correlations with spontaneous rate.

    PubMed

    Kantardzhieva, Albena; Liberman, M Charles; Sewell, William F

    2013-10-01

    Cochlear hair cells form ribbon synapses with terminals of the cochlear nerve. To test the hypothesis that one function of the ribbon is to create synaptic vesicles from the cisternal structures that are abundant at the base of hair cells, we analyzed the distribution of vesicles and cisterns around ribbons from serial sections of inner hair cells in the cat, and compared data from low and high spontaneous rate (SR) synapses. Consistent with the hypothesis, we identified a "sphere of influence" of 350 nm around the ribbon, with fewer cisterns and many more synaptic vesicles. Although high- and low-SR ribbons tended to be longer and thinner than high-SR ribbons, the total volume of the two ribbon types was similar. There were almost as many vesicles docked at the active zone as attached to the ribbon. The major SR-related difference was that low-SR ribbons had more synaptic vesicles intimately associated with them. Our data suggest a trend in which low-SR synapses had more vesicles attached to the ribbon (51.3 vs. 42.8), more docked between the ribbon and the membrane (12 vs. 8.2), more docked at the active zone (56.9 vs. 44.2), and more vesicles within the "sphere of influence" (218 vs. 166). These data suggest that the structural differences between high- and low-SR synapses may be more a consequence, than a determinant, of the physiological differences.

  19. A new efficient method for synaptic vesicle quantification reveals differences between medial prefrontal cortex perforated and nonperforated synapses.

    PubMed

    Nava, Nicoletta; Chen, Fenghua; Wegener, Gregers; Popoli, Maurizio; Nyengaard, Jens Randel

    2014-02-01

    Communication between neurons is mediated by the release of neurotransmitter-containing vesicles from presynaptic terminals. Quantitative characterization of synaptic vesicles can be highly valuable for understanding mechanisms underlying synaptic function and plasticity. We performed a quantitative ultrastructural analysis of cortical excitatory synapses by mean of a new, efficient method, as an alternative to three-dimensional (3D) reconstruction. Based on a hierarchical sampling strategy and unequivocal identification of the region of interest, serial sections from excitatory synapses of medial prefrontal cortex (mPFC) of six Sprague-Dawley rats were acquired with a transmission electron microscope. Unbiased estimates of total 3D volume of synaptic terminals were obtained through the Cavalieri estimator, and adequate correction factors for vesicle profile number estimation were applied for final vesicle quantification. Our analysis was based on 79 excitatory synapses, nonperforated (NPSs) and perforated (PSs) subtypes. We found that total number of docked and reserve-pool vesicles in PSs significantly exceeded that in NPSs (by, respectively, 77% and 78%). These differences were found to be related to changes in size between the two subtypes (active zone area by 86%; bouton volume by 105%) rather than to postsynaptic density shape. Positive significant correlations were found between number of docked and reserve-pool vesicles, active zone area and docked vesicles, and bouton volume and reserve pool vesicles. Our method confirmed the large size of mPFC PSs and a linear correlation between presynaptic features of typical hippocampal synapses. Moreover, a greater number of docked vesicles in PSs may promote a high synaptic strength of these synapses.

  20. Origin and characterization of small membranous vesicles present in the venom of Crotalus durissus terrificus.

    PubMed

    Souza-Imberg, Andréia; Carneiro, Sylvia Mendes; Giannotti, Karina Cristina; Sant'Anna, Sávio Stefanini; Yamanouye, Norma

    2017-09-15

    Small membranous vesicles are small closed fragments of membrane. They are released from multivesicular bodies (exosomes) or shed from the surface membrane (microvesicles). They contains various bioactive molecules and their molecular composition varies depending on their cellular origin. Small membranous vesicles have been identified in snake venoms, but the origin of these small membranous vesicles in the venom is controversial. The aim of this study was to verify the origin of the small membranous vesicles in venom of Crotalus durissus terrificus by morphological analyses using electron microscopy. In addition, the protein composition of the vesicles was analyzed by using a proteome approach. The small membranous vesicles present in the venom were microvesicles, since they originated from microvilli on the apical membrane of secretory cells. They contained cytoplasmic proteins, and proteins from the plasma membrane, endoplasmic reticulum (ER), and Golgi membrane. The release of microvesicles may be a mechanism to control the size of the cell membrane of the secretory cells after intense exocytosis. Microvesicle components that may have a role in envenoming include ecto-5'-nucleotidase, a cell membrane protein that releases adenosine, and aminopeptidase N, a cell membrane protein that may modulate the action of many peptides. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Using enrichment index for quality control of secretory protein sample and identification of secretory proteins.

    PubMed

    Chen, Yong; Gu, Bei; Wu, Shuzhen; Sun, Wei; Ma, Sucan; Liu, Yuqin; Gao, Youhe

    2009-03-01

    Analysis of secretory proteins is an important area in proteomic research. We propose that a good secretory protein sample should be enriched with known secretory proteins, and a secretory protein should be enriched in the secretory protein sample compared with its corresponding soluble cell lysate. Positive identifications of proteins were subjected to quantitation of spectral counts, which reflect relative protein abundance. Enrichment index of the sample (EIS) and the enrichment index for protein (EIP) were obtained by comparing proteins identified in the secretory protein sample and those in the soluble cell lysate sample. The quality of the secretory protein sample can be represented by EIS. EIP was used to identify the secretory proteins.The secretory proteins from mouse dendritic cell sarcoma (DCS) were analyzed by MS. The EISs of two samples were 75.4 and 84.65, respectively. 72 proteins were significantly enriched in secretory protein samples, of which 42 proteins were either annotated in Swiss-Prot and/or predicted by signal peptides to be secretory. In the remaining 30 proteins, 12 and 15 proteins were positively predicted by SecretomeP and ProP, respectively, and 5 proteins were positive by both methods. Furthermore, 11 proteins were found to be present in exosome in other studies that involved mice dendritic cell lines. We suggest that this assessment method is helpful for systemic research of secretory proteins and biomarker discovery for diseases such as cancer. Copyright (c) 2009 John Wiley & Sons, Ltd.

  2. How the stimulus defines the dynamics of vesicle pool recruitment, fusion mode, and vesicle recycling in neuroendocrine cells.

    PubMed

    Cárdenas, Ana María; Marengo, Fernando D

    2016-06-01

    The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval, and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more intense stimulation will provoke a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location. The pattern of stimulation, and therefore the characteristics of the Ca(2+) signal generated by the stimulus also influence the mode of exocytosis and the type of endocytosis. Indeed, low-frequency stimulation favors kiss-and-run exocytosis and clathrin-independent fast endocytosis, whereas higher frequencies promote full fusion and clathrin-dependent endocytosis. This latter type of endocytosis is accelerated at high-frequency stimulation. Synaptotagmins, calcineurin, dynamin, complexin, and actin remodeling, appear to be involved in the mechanisms that determine the response of these processes to Ca(2+) . In chromaffin cells, a brief stimulus induces the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels (A), whereas longer or high-frequency stimulation provokes a global Ca(2+) increase, promoting exocytosis irrespective of

  3. Multipose binding in molecular docking.

    PubMed

    Atkovska, Kalina; Samsonov, Sergey A; Paszkowski-Rogacz, Maciej; Pisabarro, M Teresa

    2014-02-14

    Molecular docking has been extensively applied in virtual screening of small molecule libraries for lead identification and optimization. A necessary prerequisite for successful differentiation between active and non-active ligands is the accurate prediction of their binding affinities in the complex by use of docking scoring functions. However, many studies have shown rather poor correlations between docking scores and experimental binding affinities. Our work aimed to improve this correlation by implementing a multipose binding concept in the docking scoring scheme. Multipose binding, i.e., the property of certain protein-ligand complexes to exhibit different ligand binding modes, has been shown to occur in nature for a variety of molecules. We conducted a high-throughput docking study and implemented multipose binding in the scoring procedure by considering multiple docking solutions in binding affinity prediction. In general, improvement of the agreement between docking scores and experimental data was observed, and this was most pronounced in complexes with large and flexible ligands and high binding affinities. Further developments of the selection criteria for docking solutions for each individual complex are still necessary for a general utilization of the multipose binding concept for accurate binding affinity prediction by molecular docking.

  4. Spacecraft capture and docking system

    NASA Technical Reports Server (NTRS)

    Kong, Kinyuen (Inventor); Rafeek, Shaheed (Inventor); Myrick, Thomas (Inventor)

    2001-01-01

    A system for capturing and docking an active craft to a passive craft has a first docking assembly on the active craft with a first contact member and a spike projecting outwardly, a second docking assembly on the passive craft having a second contact member and a flexible net deployed over a target area with an open mesh for capturing the end of the spike of the active craft, and a motorized net drive for reeling in the net and active craft to mate with the passive craft's docking assembly. The spike has extendable tabs to allow it to become engaged with the net. The net's center is coupled to a net spool for reeling in. An alignment funnel has inclined walls to guide the net and captured spike towards the net spool. The passive craft's docking assembly includes circumferentially spaced preload wedges which are driven to lock the wedges against the contact member of the active craft. The active craft's docking assembly includes a rotary table and drive for rotating it to a predetermined angular alignment position, and mating connectors are then engaged with each other. The system may be used for docking spacecraft in zero or low-gravity environments, as well as for docking underwater vehicles, docking of ancillary craft to a mother craft in subsonic flight, in-flight refueling systems, etc.

  5. Evolution of apicomplexan secretory organelles

    PubMed Central

    Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2013-01-01

    The alveolate superphylum includes many free-living and parasitic organisms, which are united by the presence of alveolar sacs lying proximal to the plasma membrane, providing cell structure. All species comprising the apicomplexan group of alveolates are parasites and have adapted to the unique requirements of the parasitic lifestyle. Here the evolution of apicomplexan secretory organelles that are involved in the critical process of egress from one cell and invasion of another is explored. The variations within the Apicomplexa and how these relate to species-specific biology will be discussed. In addition, recent studies have identified specific calcium-sensitive molecules that coordinate the various events and regulate the release of these secretory organelles within apicomplexan parasites. Some aspects of this machinery are conserved outside the Apicomplexa, and are beginning to elucidate the conserved nature of the machinery. Briefly, the relationship of this secretion machinery within the Apicomplexa will be discussed, compared with free-living and predatory alveolates, and how these might have evolved from a common ancestor. PMID:23068912

  6. Hyperviscous diblock copolymer vesicles

    NASA Astrophysics Data System (ADS)

    Dimova, R.; Seifert, U.; Pouligny, B.; Förster, S.; Döbereiner, H.-G.

    2002-03-01

    Giant vesicles prepared from the diblock copolymer polybutadien-b-polyethyleneoxide (PB-PEO) exhibit a shear surface viscosity, which is about 500 times higher than those found in common phospholipid bilayers. Our result constitutes the first direct measurement of the shear surface viscosity of such polymersomes. At the same time, we measure bending and stretching elastic constants, which fall in the range of values typical for lipid membranes. Pulling out a tether from an immobilized polymersome and following its relaxation back to the vesicle body provides an estimate of the viscous coupling between the two monolayers composing the polymer membrane. The detected intermonolayer friction is about an order of magnitude higher than the characteristic one for phospholipid membranes. Polymersomes are tough vesicles with a high lysis tension. This, together with their robust rheological properties, makes them interesting candidates for a number of technological applications.

  7. Expedition 23 Docking

    NASA Image and Video Library

    2010-04-03

    The crew of Expedition 23 are seen on a large TV screen in the Russian Mission Control Center in Korolev, Russia, Sunday, April 4, 2010, shortly after the Soyuz TMA-18 spacecraft docked to the International Space Station and delivered Expedition 23 Flight Engineers Alexander Skvortsov, Mikhail Kornienko and Tracy Caldwell Dyson. Clockwise from top right are NASA astronaut TJ Creamer, NASA astronaut Tracy Caldwell Dyson, Russian cosmonaut Alexander Skvortsov, Russian cosmonaut Mikhail Kornienko, JAXA (Japan Aerospace Exploration Agency) astronaut Soichi Noguchi and Expedition 23 commander Russian cosmonaut Oleg Kotov . Photo Credit: (NASA/Carla Cioffi)

  8. Expedition 21 Docking

    NASA Image and Video Library

    2009-10-01

    NASA Administrator Charles Bolden, left, Head of the Russian Federal Space Agency, Anatoly Perminov, center, and Deputy Head of the Russian Federal Space Agency, Vitaly A. Davyidov listen to reporters questions during a press conference at Mission Control Center Moscow in Korolev, Russia shortly after the successful docking of the Soyuz TMA-16 spacecraft with the International Space Station (ISS) marking the start of Expedition 21 with Flight Engineer Jeffrey N. Williams, Expedition 21 Flight Engineer Maxim Suraev, and Spaceflight Participant Guy Laliberté, Friday, Oct. 2, 2009. The entire crew onboard the ISS can be seen in the monitor below. Photo Credit: (NASA/Bill Ingalls)

  9. Expedition 21 Docking

    NASA Image and Video Library

    2009-10-01

    NASA Administrator Charles Bolden, Left, and Head of the Russian Federal Space Agency, Anatoly Perminov listen to reporter's questions during a press conference at Mission Control Center Moscow in Korolev, Russia shortly after the successful docking of the Soyuz TMA-16 spacecraft with the International Space Station (ISS) marking the start of Expedition 21 with Flight Engineer Jeffrey N. Williams, Expedition 21 Flight Engineer Maxim Suraev, and Spaceflight Participant Guy Laliberté, Friday, Oct. 2, 2009. Laliberté will return to Earth with the Expedition 20 crew on October 11, 2009. Photo Credit: (NASA/Bill Ingalls)

  10. Vesicles in Poiseuille flow.

    PubMed

    Danker, Gerrit; Vlahovska, Petia M; Misbah, Chaouqi

    2009-04-10

    Blood microcirculation critically depends on the migration of red cells towards the flow centerline. We identify theoretically the ratio of the inner over the outer fluid viscosities lambda as a key parameter. At low lambda, the vesicle deforms into a tank-treading ellipsoid shape far away from the flow centerline. The migration is always towards the flow centerline, unlike drops. Above a critical lambda, the vesicle tumbles or breaths and migration is suppressed. A surprising coexistence of two types of shapes at the centerline, a bulletlike and a parachutelike shape, is predicted.

  11. 12. Ore unloading dock, looking south. Dock, built in 1908, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. Ore unloading dock, looking south. Dock, built in 1908, featured two 10-ton-capacity Hulett unloaders (shown here) built by the Wellman-Seaver-Morgan Co. of Cleveland. - Central Furnaces, 2650 Broadway, east bank of Cuyahoga River, Cleveland, Cuyahoga County, OH

  12. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

    PubMed Central

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2015-01-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  13. [Study of human secretory immunoglobulin A. I. Obtaining monospecific antiserum to human secretory immunoglobulin A].

    PubMed

    German, G P; Chernokhvostova, E V; Gol'derman, S Ia

    1975-10-01

    A method of obtaining monospecific antiserum to the human secretory IgA is described. Immunochemically pure secretory IgA (isolated from human colostrum by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200) was used for immunization of rabbits or sheep. Heterologous antibodies were removed by adsorption with commercial gamma globulin, normal serum, the serum of a patient suffering from A-myeloma with the IgA polymere and purified lactoferrin. Monospecific antiserum to the secretory IgA gave a reaction of complete immunological identity with the secretory IgA and a free secretory component.

  14. DockingShop: A Tool for Interactive Molecular Docking

    SciTech Connect

    Lu, Ting-Cheng; Max, Nelson L.; Ding, Jinhui; Bethel, E. Wes; Crivelli, Silvia N.

    2005-04-24

    Given two independently determined molecular structures, the molecular docking problem predicts the bound association, or best fit between them, while allowing for conformational changes of the individual molecules during construction of a molecular complex. Docking Shop is an integrated environment that permits interactive molecular docking by navigating a ligand or protein to an estimated binding site of a receptor with real-time graphical feedback of scoring factors as visual guides. Our program can be used to create initial configurations for a protein docking prediction process. Its output--the structure of aprotein-ligand or protein-protein complex--may serve as an input for aprotein docking algorithm, or an optimization process. This tool provides molecular graphics interfaces for structure modeling, interactive manipulation, navigation, optimization, and dynamic visualization to aid users steer the prediction process using their biological knowledge.

  15. Phospholipid flippases: building asymmetric membranes and transport vesicles.

    PubMed

    Sebastian, Tessy T; Baldridge, Ryan D; Xu, Peng; Graham, Todd R

    2012-08-01

    Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal membranes, thereby impacting the sorting and localization of many different proteins in the secretory and endocytic pathways. At the organismal level, P4-ATPase deficiencies are linked to liver disease, obesity, diabetes, hearing loss, neurological deficits, immune deficiency and reduced fertility. Here, we review the biochemical, cellular and physiological functions of P4-ATPases, with an emphasis on their roles in vesicle-mediated protein transport. This article is part of a Special Issue entitled Lipids and Vesicular Transport. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Phospholipid flippases: building asymmetric membranes and transport vesicles

    PubMed Central

    Sebastian, Tessy T.; Baldridge, Ryan D.; Xu, Peng; Graham, Todd R.

    2012-01-01

    Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal membranes, thereby impacting the sorting and localization of many different proteins in the secretory and endocytic pathways. At the organismal level, P4-ATPase deficiencies are linked to liver disease, obesity, diabetes, hearing loss, neurological deficits, immune deficiency and reduced fertility. Here, we review the biochemical, cellular and physiological functions of P4-ATPases, with an emphasis on their roles in vesicle-mediated protein transport. PMID:22234261

  17. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Russian Orthodox Priest, Vladyka Feofan speaks during a Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  18. Complexity of vesicle microcirculation

    NASA Astrophysics Data System (ADS)

    Kaoui, B.; Tahiri, N.; Biben, T.; Ez-Zahraouy, H.; Benyoussef, A.; Biros, G.; Misbah, C.

    2011-10-01

    This study focuses numerically on dynamics in two dimensions of vesicles in microcirculation. The method used is based on boundary integral formulation. This study is inspired by the behavior of red blood cells (RBCs) in the microvasculature. Red RBCs carry oxygen from the lungs and deliver it through the microvasculature. The shape adopted by RBCs can affect blood flow and influence oxygen delivery. Our simulation using vesicles (a simple model for RBC) reveals unexpected complexity as compared to the case where a purely unbounded Poiseuille flow is considered [Kaoui, Biros, and Misbah, Phys. Rev. Lett.10.1103/PhysRevLett.103.188101 103, 188101 (2009)]. In sufficiently large channels (in the range of 100μm; the vesicle size and its reduced volume are taken in the range of those of a human RBC), such as arterioles, a slipperlike (asymmetric) shape prevails. A parachutelike (symmetric) shape is adopted in smaller channels (in the range of 20μm, as in venules), but this shape loses stability and again changes to a pronounced slipperlike morphology in channels having a size typical of capillaries (5-10 μm). Stiff membranes, mimicking malaria infection, for example, adopt a centered or off-centered snakelike locomotion instead (the denomination snaking is used for this regime). A general scenario of how and why vesicles adopt their morphologies and dynamics among several distinct possibilities is provided. This finding potentially points to nontrivial RBCs dynamics in the microvasculature.

  19. Stress modulates intestinal secretory immunoglobulin A

    PubMed Central

    Campos-Rodríguez, Rafael; Godínez-Victoria, Marycarmen; Abarca-Rojano, Edgar; Pacheco-Yépez, Judith; Reyna-Garfias, Humberto; Barbosa-Cabrera, Reyna Elizabeth; Drago-Serrano, Maria Elisa

    2013-01-01

    Stress is a response of the central nervous system to environmental stimuli perceived as a threat to homeostasis. The stress response triggers the generation of neurotransmitters and hormones from the hypothalamus pituitary adrenal axis, sympathetic axis and brain gut axis, and in this way modulates the intestinal immune system. The effects of psychological stress on intestinal immunity have been investigated mostly with the restraint/immobilization rodent model, resulting in an up or down modulation of SIgA levels depending on the intensity and time of exposure to stress. SIgA is a protein complex formed by dimeric (dIgA) or polymeric IgA (pIgA) and the secretory component (SC), a peptide derived from the polymeric immunoglobulin receptor (pIgR). The latter receptor is a transmembrane protein expressed on the basolateral side of gut epithelial cells, where it uptakes dIgA or pIgA released by plasma cells in the lamina propria. As a result, the IgA-pIgR complex is formed and transported by vesicles to the apical side of epithelial cells. pIgR is then cleaved to release SIgA into the luminal secretions of gut. Down modulation of SIgA associated with stress can have negative repercussions on intestinal function and integrity. This can take the form of increased adhesion of pathogenic agents to the intestinal epithelium and/or an altered balance of inflammation leading to greater intestinal permeability. Most studies on the molecular and biochemical mechanisms involved in the stress response have focused on systemic immunity. The present review analyzes the impact of stress (mostly by restraint/immobilization, but also with mention of other models) on the generation of SIgA, pIgR and other humoral and cellular components involved in the intestinal immune response. Insights into these mechanisms could lead to better therapies for protecting against pathogenic agents and avoiding epithelial tissue damage by modulating intestinal inflammation. PMID:24348350

  20. Muscle as a secretory organ.

    PubMed

    Pedersen, Bente K

    2013-07-01

    Skeletal muscle is the largest organ in the body. Skeletal muscles are primarily characterized by their mechanical activity required for posture, movement, and breathing, which depends on muscle fiber contractions. However, skeletal muscle is not just a component in our locomotor system. Recent evidence has identified skeletal muscle as a secretory organ. We have suggested that cytokines and other peptides that are produced, expressed, and released by muscle fibers and exert either autocrine, paracrine, or endocrine effects should be classified as "myokines." The muscle secretome consists of several hundred secreted peptides. This finding provides a conceptual basis and a whole new paradigm for understanding how muscles communicate with other organs such as adipose tissue, liver, pancreas, bones, and brain. In addition, several myokines exert their effects within the muscle itself. Many proteins produced by skeletal muscle are dependent upon contraction. Therefore, it is likely that myokines may contribute in the mediation of the health benefits of exercise.

  1. Morphology and histology of secretory setae in terrestrial larvae of biting midges of the genus Forcipomyia (Diptera: Ceratopogonidae).

    PubMed

    Urbanek, Aleksandra; Richert, Malwina; Giłka, Wojciech; Szadziewski, Ryszard

    2011-11-01

    Apneustic larvae of the genus Forcipomyia possess unique secretory setae located on the dorsal surface along the body in two rows, one pair on each thoracic and abdominal segment and two pairs on the head. Morphological and histological studies of secretory setae in fourth instar larvae of Forcipomyia nigra (Winnertz) and Forcipomyia nigrans Remm indicate they are modified mechanoreceptors (sensilla trichodea) in which the trichogen cell is a glandular cell producing a hygroscopic secretion. The cytoplasm of the glandular trichogen cell fills the lumen of a secretory seta, which shows one or more pores on the apex. The cytoplasm contains numerous microtubules responsible for transportation of proteinaceous vesicles, and an extremely large polyploid nucleus typical of gland cells. The main role of the hygroscopic secretion is to moist the body and thus facilitate cuticular respiration.

  2. Surflex-Dock: Docking benchmarks and real-world application

    NASA Astrophysics Data System (ADS)

    Spitzer, Russell; Jain, Ajay N.

    2012-06-01

    Benchmarks for molecular docking have historically focused on re-docking the cognate ligand of a well-determined protein-ligand complex to measure geometric pose prediction accuracy, and measurement of virtual screening performance has been focused on increasingly large and diverse sets of target protein structures, cognate ligands, and various types of decoy sets. Here, pose prediction is reported on the Astex Diverse set of 85 protein ligand complexes, and virtual screening performance is reported on the DUD set of 40 protein targets. In both cases, prepared structures of targets and ligands were provided by symposium organizers. The re-prepared data sets yielded results not significantly different than previous reports of Surflex-Dock on the two benchmarks. Minor changes to protein coordinates resulting from complex pre-optimization had large effects on observed performance, highlighting the limitations of cognate ligand re-docking for pose prediction assessment. Docking protocols developed for cross-docking, which address protein flexibility and produce discrete families of predicted poses, produced substantially better performance for pose prediction. Performance on virtual screening performance was shown to benefit by employing and combining multiple screening methods: docking, 2D molecular similarity, and 3D molecular similarity. In addition, use of multiple protein conformations significantly improved screening enrichment.

  3. Surflex-Dock: Docking benchmarks and real-world application.

    PubMed

    Spitzer, Russell; Jain, Ajay N

    2012-06-01

    Benchmarks for molecular docking have historically focused on re-docking the cognate ligand of a well-determined protein-ligand complex to measure geometric pose prediction accuracy, and measurement of virtual screening performance has been focused on increasingly large and diverse sets of target protein structures, cognate ligands, and various types of decoy sets. Here, pose prediction is reported on the Astex Diverse set of 85 protein ligand complexes, and virtual screening performance is reported on the DUD set of 40 protein targets. In both cases, prepared structures of targets and ligands were provided by symposium organizers. The re-prepared data sets yielded results not significantly different than previous reports of Surflex-Dock on the two benchmarks. Minor changes to protein coordinates resulting from complex pre-optimization had large effects on observed performance, highlighting the limitations of cognate ligand re-docking for pose prediction assessment. Docking protocols developed for cross-docking, which address protein flexibility and produce discrete families of predicted poses, produced substantially better performance for pose prediction. Performance on virtual screening performance was shown to benefit by employing and combining multiple screening methods: docking, 2D molecular similarity, and 3D molecular similarity. In addition, use of multiple protein conformations significantly improved screening enrichment.

  4. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  5. A role for Yip1p in COPII vesicle biogenesis

    PubMed Central

    Heidtman, Matthew; Chen, Catherine Z.; Collins, Ruth N.; Barlowe, Charles

    2003-01-01

    Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis. PMID:14557247

  6. A role for Yip1p in COPII vesicle biogenesis.

    PubMed

    Heidtman, Matthew; Chen, Catherine Z; Collins, Ruth N; Barlowe, Charles

    2003-10-13

    Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

  7. DockingApp: a user friendly interface for facilitated docking simulations with AutoDock Vina

    NASA Astrophysics Data System (ADS)

    Di Muzio, Elena; Toti, Daniele; Polticelli, Fabio

    2017-02-01

    Molecular docking is a powerful technique that helps uncover the structural and energetic bases of the interaction between macromolecules and substrates, endogenous and exogenous ligands, and inhibitors. Moreover, this technique plays a pivotal role in accelerating the screening of large libraries of compounds for drug development purposes. The need to promote community-driven drug development efforts, especially as far as neglected diseases are concerned, calls for user-friendly tools to allow non-expert users to exploit the full potential of molecular docking. Along this path, here is described the implementation of DockingApp, a freely available, extremely user-friendly, platform-independent application for performing docking simulations and virtual screening tasks using AutoDock Vina. DockingApp sports an intuitive graphical user interface which greatly facilitates both the input phase and the analysis of the results, which can be visualized in graphical form using the embedded JMol applet. The application comes with the DrugBank set of more than 1400 ready-to-dock, FDA-approved drugs, to facilitate virtual screening and drug repurposing initiatives. Furthermore, other databases of compounds such as ZINC, available also in AutoDock format, can be readily and easily plugged in.

  8. DockingApp: a user friendly interface for facilitated docking simulations with AutoDock Vina

    NASA Astrophysics Data System (ADS)

    Di Muzio, Elena; Toti, Daniele; Polticelli, Fabio

    2017-01-01

    Molecular docking is a powerful technique that helps uncover the structural and energetic bases of the interaction between macromolecules and substrates, endogenous and exogenous ligands, and inhibitors. Moreover, this technique plays a pivotal role in accelerating the screening of large libraries of compounds for drug development purposes. The need to promote community-driven drug development efforts, especially as far as neglected diseases are concerned, calls for user-friendly tools to allow non-expert users to exploit the full potential of molecular docking. Along this path, here is described the implementation of DockingApp, a freely available, extremely user-friendly, platform-independent application for performing docking simulations and virtual screening tasks using AutoDock Vina. DockingApp sports an intuitive graphical user interface which greatly facilitates both the input phase and the analysis of the results, which can be visualized in graphical form using the embedded JMol applet. The application comes with the DrugBank set of more than 1400 ready-to-dock, FDA-approved drugs, to facilitate virtual screening and drug repurposing initiatives. Furthermore, other databases of compounds such as ZINC, available also in AutoDock format, can be readily and easily plugged in.

  9. DockingApp: a user friendly interface for facilitated docking simulations with AutoDock Vina.

    PubMed

    Di Muzio, Elena; Toti, Daniele; Polticelli, Fabio

    2017-02-01

    Molecular docking is a powerful technique that helps uncover the structural and energetic bases of the interaction between macromolecules and substrates, endogenous and exogenous ligands, and inhibitors. Moreover, this technique plays a pivotal role in accelerating the screening of large libraries of compounds for drug development purposes. The need to promote community-driven drug development efforts, especially as far as neglected diseases are concerned, calls for user-friendly tools to allow non-expert users to exploit the full potential of molecular docking. Along this path, here is described the implementation of DockingApp, a freely available, extremely user-friendly, platform-independent application for performing docking simulations and virtual screening tasks using AutoDock Vina. DockingApp sports an intuitive graphical user interface which greatly facilitates both the input phase and the analysis of the results, which can be visualized in graphical form using the embedded JMol applet. The application comes with the DrugBank set of more than 1400 ready-to-dock, FDA-approved drugs, to facilitate virtual screening and drug repurposing initiatives. Furthermore, other databases of compounds such as ZINC, available also in AutoDock format, can be readily and easily plugged in.

  10. Cryo-electron microscopy of extracellular vesicles in fresh plasma.

    PubMed

    Yuana, Yuana; Koning, Roman I; Kuil, Maxim E; Rensen, Patrick C N; Koster, Abraham J; Bertina, Rogier M; Osanto, Susanne

    2013-12-31

    Extracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited. To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM). Fresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM. EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25-260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules. Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

  11. Analysis of COPII vesicles indicates a role for the Emp47-Ssp120 complex in transport of cell surface glycoproteins

    PubMed Central

    Margulis, Neil G.; Wilson, Joshua D.; Bentivoglio, Christine M.; Dhungel, Nripesh; Gitler, Aaron D.; Barlowe, Charles

    2015-01-01

    Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium-binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47-Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway. PMID:26650540

  12. OMV docking simulator

    NASA Technical Reports Server (NTRS)

    Teoh, W.; Hawkins, J.

    1988-01-01

    The Boeing Orbital Maneuvering Vehicle (OMV) Docking and Proximity Operation System (DAPOS) was completed. The system constructed involves the use of four separate processors. Appropriate software was developed that drives each of these four processors. The hand controller logic coordinates all the activities in the control station, and communicates with the OMV mathematical model. The state vector generated by the model is in turn transmitted to the control station as well as the POLY 2000 (via the ALCYON host computer) for real time graphics generation. The OMV characteristics are stored in a data file which may be easily updated and modified without disturbing the software, thereby making the system very flexible. The current system supports two types of hand controllers. The system was flown by several volunteers, some of whom are airplane pilots. A user manual is also enclosed.

  13. Identification and staining of distinct populations of secretory organelles in astrocytes.

    PubMed

    Bezzi, Paola; Volterra, Andrea

    2014-05-01

    Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

  14. Identification of a Munc13-sensitive step in chromaffin cell large dense-core vesicle exocytosis

    PubMed Central

    Man, Kwun Nok M; Imig, Cordelia; Walter, Alexander M; Pinheiro, Paulo S; Stevens, David R; Rettig, Jens; Sørensen, Jakob B; Cooper, Benjamin H; Brose, Nils; Wojcik, Sonja M

    2015-01-01

    It is currently unknown whether the molecular steps of large dense-core vesicle (LDCV) docking and priming are identical to the corresponding reactions in synaptic vesicle (SV) exocytosis. Munc13s are essential for SV docking and priming, and we systematically analyzed their role in LDCV exocytosis using chromaffin cells lacking individual isoforms. We show that particularly Munc13-2 plays a fundamental role in LDCV exocytosis, but in contrast to synapses lacking Munc13s, the corresponding chromaffin cells do not exhibit a vesicle docking defect. We further demonstrate that ubMunc13-2 and Munc13-1 confer Ca2+-dependent LDCV priming with similar affinities, but distinct kinetics. Using a mathematical model, we identify an early LDCV priming step that is strongly dependent upon Munc13s. Our data demonstrate that the molecular steps of SV and LDCV priming are very similar while SV and LDCV docking mechanisms are distinct. DOI: http://dx.doi.org/10.7554/eLife.10635.001 PMID:26575293

  15. Low Impact Docking System (LIDS)

    NASA Technical Reports Server (NTRS)

    LaBauve, Tobie E.

    2009-01-01

    Since 1996, NASA has been developing a docking system that will simplify operations and reduce risks associated with mating spacecraft. This effort has focused on developing and testing an original, reconfigurable, active, closed-loop, force-feedback controlled docking system using modern technologies. The primary objective of this effort has been to design a docking interface that is tunable to the unique performance requirements for all types of mating operations (i.e. docking and berthing, autonomous and piloted rendezvous, and in-space assembly of vehicles, modules and structures). The docking system must also support the transfer of crew, cargo, power, fluid, and data. As a result of the past 10 years of docking system advancement, the Low Impact Docking System or LIDS was developed. The current LIDS design incorporates the lessons learned and development experiences from both previous and existing docking systems. LIDS feasibility was established through multiple iterations of prototype hardware development and testing. Benefits of LIDS include safe, low impact mating operations, more effective and flexible mission implementation with an anytime/anywhere mating capability, system level redundancy, and a more affordable and sustainable mission architecture with reduced mission and life cycle costs. In 1996 the LIDS project, then known as the Advanced Docking Berthing System (ADBS) project, launched a four year developmental period. At the end of the four years, the team had built a prototype of the soft-capture hardware and verified the control system that will be used to control the soft-capture system. In 2001, the LIDS team was tasked to work with the X- 38 Crew Return Vehicle (CRV) project and build its first Engineering Development Unit (EDU).

  16. Limited intermixing of synaptic vesicle components upon vesicle recycling.

    PubMed

    Opazo, Felipe; Punge, Annedore; Bückers, Johanna; Hoopmann, Peer; Kastrup, Lars; Hell, Stefan W; Rizzoli, Silvio O

    2010-06-01

    Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing approximately 19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH-sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin-tagged proteins. While the native proteins had a clustered distribution, the GFP-tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin-tagged vesicle proteins are less well integrated in clusters.

  17. Equilibrium of nematic vesicles

    NASA Astrophysics Data System (ADS)

    Napoli, Gaetano; Vergori, Luigi

    2010-11-01

    A variational scheme is proposed which allows the derivation of a concise and elegant formulation of the equilibrium equations for closed fluid membranes, endowed with a nematic microstructure. The nematic order is described by an in-plane nematic director and a degree of orientation, as customary in the theory of uniaxial nematics. The only constitutive ingredient in this scheme is a free-energy density which depends on the vesicle geometry and order parameters. The stress and the couple stress tensors related to this free-energy density are provided. As an application of the proposed scheme, a certain number of special theories are deduced: soap bubbles, lipid vesicles, chiral and achiral nematic membranes, and nematics on curved substrates.

  18. After Progress Resupply Vehicle Docking

    NASA Image and Video Library

    2012-01-28

    ISS030-E-050949 (27 Jan. 2012) --- NASA astronaut Dan Burbank (right), Expedition 30 commander; Russian cosmonauts Anton Shkaplerov (bottom), Oleg Kononenko (center) and Anatoly Ivanishin (left background); and European Space Agency astronaut Andre Kuipers, all flight engineers, take a moment for a photo in the Zvezda Service Module of the International Space Station following the successful docking of the unpiloted ISS Progress 46 resupply vehicle. Progress 46 docked automatically to the Pirs Docking Compartment via the Kurs automated rendezvous system at 7:00 p.m. (EST) on Jan. 27, 2012.

  19. After Progress Resupply Vehicle Docking

    NASA Image and Video Library

    2012-01-28

    ISS030-E-050946 (27 Jan. 2012) --- NASA astronaut Dan Burbank (right), Expedition 30 commander; Russian cosmonauts Anton Shkaplerov (bottom), Oleg Kononenko (center) and Anatoly Ivanishin (left background); and European Space Agency astronaut Andre Kuipers, all flight engineers, take a moment for a photo in the Zvezda Service Module of the International Space Station following the successful docking of the unpiloted ISS Progress 46 resupply vehicle. Progress 46 docked automatically to the Pirs Docking Compartment via the Kurs automated rendezvous system at 7:00 p.m. (EST) on Jan. 27, 2012.

  20. Poking vesicles in silico

    NASA Astrophysics Data System (ADS)

    Barlow, Ben; Bertrand, Martin; Joos, Bela

    2014-03-01

    The Atomic Force Microscope (AFM) is used to poke cells and study their mechanical properties. Using Coarse-Grained Molecular Dynamics simulations, we study the deformation and relaxation of lipid bilayer vesicles, when poked with a constant force. The relaxation time, equilibrium area expansion, and surface tension of the vesicle membrane are studied over a range of applied forces. The relaxation time exhibits a strong force-dependence. Our force-compression curves show a strong similarity with results from a recent experiment by Schafer et al. (Langmuir, 2013). They used an AFM to ``poke'' adherent giant liposomes with constant nanonewton forces and observed the resulting deformation with a Laser Scanning Confocal Microscope. Results of such experiments, whether on vesicles or cells, are often interpreted in terms of dashpots and springs. This simple approach used to describe the response of a whole cell --complete with cytoskeleton, organelles etc.-- can be problematic when trying to measure the contribution of a single cell component. Our modeling is a first step in a ``bottom-up'' approach where we investigate the viscoelastic properties of an in silico cell prototype with constituents added step by step. Supported by NSERC (Canada).

  1. Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry.

    PubMed

    Lewis, Kenneth T; Maddipati, Krishna R; Naik, Akshata R; Jena, Bhanu P

    2017-03-02

    Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.

  2. Studying calcium triggered vesicle fusion in a single vesicle-vesicle content/lipid mixing system

    PubMed Central

    Kyoung, Minjoung; Zhang, Yunxiang; Diao, Jiajie; Chu, Steven; Brunger, Axel T.

    2013-01-01

    This Protocol describes a single vesicle-vesicle microscopy system to study Ca2+-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and SNAP-25, and are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at zero Ca2+-concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca2+-injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-hundred millisecond timescale. Other factors, such as complexin, can be easily added. Our system is unique by monitoring both content and lipid mixing, and by starting from a metastable state of interacting vesicle pairs prior to Ca2+-injection. PMID:23222454

  3. Evaluation of docking calculations on X-ray structures using CONSENSUS-DOCK.

    PubMed

    Okamoto, Masako; Masuda, Yoshiaki; Muroya, Ayumu; Yasuno, Kazuhiro; Takahashi, Osamu; Furuya, Toshio

    2010-12-01

    We are participating in the challenge of identifying active compounds for target proteins using structure-based virtual screening (SBVS). We use an in-house customized docking program, CONSENSUS-DOCK, which is a customized version of the DOCK4 program in which three scoring functions (DOCK4, FlexX and PMF) and consensus scoring have been implemented. This paper compares the docking calculation results obtained using CONSENSUS-DOCK and DOCK4, and demonstrates that CONSENSUS-DOCK produces better results than DOCK4 for major X-ray structures obtained from the Protein Data Bank (PDB).

  4. Plasmadesmatal frequency, apoplast-symplast ratio, and photosynthetic transfer in grapefruit juice vesicles. [Citrus paradisi Macf

    SciTech Connect

    Koch, K.E.; Lowell, C.A.; Avigne, W.T.

    1986-04-01

    Structure and function were examined in phloem-free vesicles and vesicle stalks of grapefruit (Citrus paradisi Macf.) by light and electron microscopy and /sup 14/C-photosynthate transport in intact and dissected tissues. Plasmodesmatal frequencies were approximately 0.3 to 0.5 ..mu..m/sup -1/ cell wall interface (3 to 5 ..mu..m/sup -2/), less than that of known secretory structures but similar to root parenchyma. Cell wall or apoplast comprised 18 to 24% of the total cross-sectional area of the vesicle stalk. The mass of total photosynthate transfer through individual vesicle stalks was ca. 0.5 ..mu..g C h/sup -1/ and rate of /sup 14/C-movement 0.1 to 0.4 mm h/sup -1/. Transport continued in rows of vesicles dissected in association with a vascular bundle. If isolated from fully-expanded fruit, translocation was similar for systems with frozen vs. non-frozen vesicle stalks. Similar freezing treatment decreased transport in vesicles from younger fruit. Symplastic and apoplastic pathways may therefore both operate in this system.

  5. Two distinct populations of synaptic-like vesicles from rat brain

    PubMed Central

    Thoidis, Galini; Chen, Peng; Pushkin, Alexander V.; Vallega, Gino; Leeman, Susan E.; Fine, Richard E.; Kandror, Konstantin V.

    1998-01-01

    In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain. PMID:9419350

  6. Secretory diarrhoea: mechanisms and emerging therapies.

    PubMed

    Thiagarajah, Jay R; Donowitz, Mark; Verkman, Alan S

    2015-08-01

    Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca(2+) signalling pathways. In many secretory diarrhoeas, activation of Cl(-) channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, increases fluid secretion, while inhibition of Na(+) transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics.

  7. Secretory diarrhoea: mechanisms and emerging therapies

    PubMed Central

    Thiagarajah, Jay R.; Donowitz, Mark; Verkman, Alan S.

    2016-01-01

    Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca2+ signalling pathways. In many secretory diarrhoeas, activation of Cl− channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl− channels, increases fluid secretion, while inhibition of Na+ transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics. PMID:26122478

  8. [Overweight and secretory male infertility].

    PubMed

    Oshakbaev, K P; Abylaĭuly, Zh; Dukenbaeva, B A

    2009-01-01

    We have performed a trial with participation of 60 males aged 23-52. Of them, 30 had secretory male iufertility (SMI) and obesity. The control 30 patients were healthy volunteers. The protocol was performed by two stages. Stage 1 included: investigation of a clinico-laboratory status, of correlation between a sorption function of erythrocytes, endogenic metabolic intoxication (EMI) and spermogram parameters, concentration of serum testosterone in SMI patients. Stage 2 consisted in treatment of the intoxication by reducing body mass. All the infertile men were obese; 30% of them had low glucose tolerance, 46.7% had stage 2 hypertension, 23.3%--seasonal allergic symptoms. The level of organic substances on the surface of erythrocytes in infertile men was higher than in the controls (p < 0.01). A negative correlation was seen between spermogram parameters and organic substances content on erythrocytic surface (p < 0/05), concentration of serum testosterone and the above substances (p < 0.01). The loss of fat tissue by 7-14 kg by infertile men resulted in a positive trend in spermogram parameters and the level of serum testosterone (p < 0.01).

  9. Individual Vesicle Fusion Events Mediated by Lipid-Anchored DNA

    PubMed Central

    van Lengerich, Bettina; Rawle, Robert J.; Bendix, Poul Martin; Boxer, Steven G.

    2013-01-01

    Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed. PMID:23870262

  10. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

    PubMed Central

    Topalidou, Irini; Cattin-Ortolá, Jérôme; MacCoss, Michael J.

    2016-01-01

    The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment. PMID:27191843

  11. Histochemical Analysis of Plant Secretory Structures.

    PubMed

    Demarco, Diego

    2017-01-01

    Histochemical analysis is essential for the study of plant secretory structures whose classification is based, at least partially, on the composition of their secretion. As each gland may produce one or more types of substances, a correct analysis of its secretion should be done using various histochemical tests to detect metabolites of different chemical classes. Here I describe some of the most used methods to detect carbohydrates, proteins, lipids, phenolic compounds, and alkaloids in the secretory structures.

  12. Giant Polymersome Protocells Dock with Virus Particle Mimics via Multivalent Glycan-Lectin Interactions

    PubMed Central

    Kubilis, Artur; Abdulkarim, Ali; Eissa, Ahmed M.; Cameron, Neil R.

    2016-01-01

    Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the de novo creation of synthetic cell mimics in the form of giant polymeric vesicles (polymersomes), which are capable of behavior approaching that of living cells. These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction. PMID:27576579

  13. Giant Polymersome Protocells Dock with Virus Particle Mimics via Multivalent Glycan-Lectin Interactions.

    PubMed

    Kubilis, Artur; Abdulkarim, Ali; Eissa, Ahmed M; Cameron, Neil R

    2016-08-31

    Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the de novo creation of synthetic cell mimics in the form of giant polymeric vesicles (polymersomes), which are capable of behavior approaching that of living cells. These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction.

  14. Giant Polymersome Protocells Dock with Virus Particle Mimics via Multivalent Glycan-Lectin Interactions

    NASA Astrophysics Data System (ADS)

    Kubilis, Artur; Abdulkarim, Ali; Eissa, Ahmed M.; Cameron, Neil R.

    2016-08-01

    Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the de novo creation of synthetic cell mimics in the form of giant polymeric vesicles (polymersomes), which are capable of behavior approaching that of living cells. These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction.

  15. Autonomous spacecraft rendezvous and docking

    NASA Technical Reports Server (NTRS)

    Tietz, J. C.; Almand, B. J.

    1985-01-01

    A storyboard display is presented which summarizes work done recently in design and simulation of autonomous video rendezvous and docking systems for spacecraft. This display includes: photographs of the simulation hardware, plots of chase vehicle trajectories from simulations, pictures of the docking aid including image processing interpretations, and drawings of the control system strategy. Viewgraph-style sheets on the display bulletin board summarize the simulation objectives, benefits, special considerations, approach, and results.

  16. Autonomous spacecraft rendezvous and docking

    NASA Astrophysics Data System (ADS)

    Tietz, J. C.; Almand, B. J.

    A storyboard display is presented which summarizes work done recently in design and simulation of autonomous video rendezvous and docking systems for spacecraft. This display includes: photographs of the simulation hardware, plots of chase vehicle trajectories from simulations, pictures of the docking aid including image processing interpretations, and drawings of the control system strategy. Viewgraph-style sheets on the display bulletin board summarize the simulation objectives, benefits, special considerations, approach, and results.

  17. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Vladimir Solovyov, Chief Flight Director, MCC-M, answers reporters questions during a Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  18. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Mike Hawes, NASA's Acting Associate Administrator, left, looks on as Kirk Shireman, NASA's deputy ISS program manager, answers reporters questions during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  19. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Alexei Krasnov, Director of Manned Space Programs Department, Roscosmos, answers reporters questions during a Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  20. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Managers from NASA, Roscosmos, RSC-Energia, TsNIIMash and other related agencies answer reporters questions during a Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  1. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Vitaly Lopota, President, General Designer, RSC-Energia, answers reporters questions during a Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  2. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Managers from NASA, Roscosmos, RSC-Energia and other related agencies answer reporters questions during a Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  3. Progress 28 supply vehicle docking

    NASA Image and Video Library

    2008-02-07

    ISS016-E-027827 (7 Feb. 2008) --- An unpiloted Progress supply vehicle approaches the International Space Station. Progress 28 resupply craft launched at 7:03 a.m. (CST) on Feb. 5, 2008 from the Baikonur Cosmodrome in Kazakhstan to deliver more than 2.5 tons of food, fuel, oxygen and other supplies to the Expedition 16 crewmembers onboard the station. Progress automatically docked to the Pirs Docking Compartment at 8:30 a.m. (CST) on Feb. 7.

  4. Optical Docking Aid Containing Fresnel Lenses

    NASA Technical Reports Server (NTRS)

    Pierce, Cole J.

    1995-01-01

    Proposed device provides self-contained visual cues to aid in docking. Similar to devices used to guide pilots in landing on aircraft carriers. Positions and directions of beams of light give observer visual cues of position relative to docking target point. Optical assemblies generate directed, diverging beams of light that, together, mark approach path to docking point. Conceived for use in docking spacecraft at Space Station Freedom, device adapted to numerous industrial docking and alignment applications.

  5. Optical Docking Aid Containing Fresnel Lenses

    NASA Technical Reports Server (NTRS)

    Pierce, Cole J.

    1995-01-01

    Proposed device provides self-contained visual cues to aid in docking. Similar to devices used to guide pilots in landing on aircraft carriers. Positions and directions of beams of light give observer visual cues of position relative to docking target point. Optical assemblies generate directed, diverging beams of light that, together, mark approach path to docking point. Conceived for use in docking spacecraft at Space Station Freedom, device adapted to numerous industrial docking and alignment applications.

  6. Swelling of the vesicle is prerequisite for PTH secretion.

    PubMed

    Lim, S K; Kwon, Y H; Song, Y D; Lee, H C; Ryu, K J; Huh, K B; Park, C S

    1996-02-01

    Unlike most secretory cells, high extra cellular calcium inhibits rather than stimulates hormonal secretion in several cells such as parathyroid cells, Juxtaglomerular cells and osteoclast. To gain further insight into the common but unique stimulus-secretion coupling mechanism in these cells, bovine parathyroid slices were incubated in various conditions of Krebs-Ringer (KR) solution containing essential amino acids. Parathyroid cells showed the inverse dependency of secretion on extra cellular calcium concentration as we expected. Ammonium acetate overcame the inhibitory effect of 2.5 mM of calcium and the maximum effect was as much as the five times of the basal value, while there was a little additive effect under 0 mM CaCl2. PTH secretion was biphasic according to the change of extra cellular osmolarity and the lowest response was observed at 300 mOsm/l. In Na-rich KR solution, high concentration of nigericin (> 10(-4)M) completely overcame the inhibitory effect of 2.5 mM CaCl2 and the maximum stimulatory effect was 8 times greater whereas it was only 2 times greater without CaCl2. In K-rich KR solution that abolished the K-gradient between the extra cellular solution and the cytoplasm, the rate of PTH secretion increased, and furthermore the addition of nigericin increased the rate of secretion significantly. The results above suggested that the osmotic swelling of the secretory vesicle in parathyroid cells might promote exocytosis as in Juxtaglomerular cells. We propose that the swelling of the vesicle is also prerequisite for secretion in several cells inhibited paradoxically by Ca++, whatever the signal transduction pathway for swelling of the secretory granules induced by the lowering of Ca++ in cytoplasm are.

  7. Russian Docking Module is lowered

    NASA Technical Reports Server (NTRS)

    1995-01-01

    The Russian-built Docking Module (DM) is lowered for installation into the payload bay of the Space Shuttle Orbiter Atlantis while the spaceplane is in Orbiter Processing Facility bay 2. The module will fly as a primary payload on the second Space Shuttle/Mir space station docking mission, STS-74, which is now scheduled for liftoff in the fall of 1995. During the mission, the module will first be attached with the orbiter's robot arm to the Orbiter Docking System (ODS) in the payload bay of the orbiter Atlantis and then be docked with the Mir. When Atlantis undocks from the Mir, it will leave the new docking module permanently attached to the space station for use during future Shuttle Mir docking missions. The new module will simplify future Shuttle linkups with Mir by improving orbiter clearances when it serves as a bridge between the two space vehicles. The white structures attached to the module's sides are solar panels that will be attached to the Mir after the conclusion of the STS-74 mission.

  8. Enabling Exploration Through Docking Standards

    NASA Technical Reports Server (NTRS)

    Hatfield, Caris A.

    2012-01-01

    Human exploration missions beyond low earth orbit will likely require international cooperation in order to leverage limited resources. International standards can help enable cooperative missions by providing well understood, predefined interfaces allowing compatibility between unique spacecraft and systems. The International Space Station (ISS) partnership has developed a publicly available International Docking System Standard (IDSS) that provides a solution to one of these key interfaces by defining a common docking interface. The docking interface provides a way for even dissimilar spacecraft to dock for exchange of crew and cargo, as well as enabling the assembly of large space systems. This paper provides an overview of the key attributes of the IDSS, an overview of the NASA Docking System (NDS), and the plans for updating the ISS with IDSS compatible interfaces. The NDS provides a state of the art, low impact docking system that will initially be made available to commercial crew and cargo providers. The ISS will be used to demonstrate the operational utility of the IDSS interface as a foundational technology for cooperative exploration.

  9. Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons.

    PubMed

    Arthur, C P; Dean, C; Pagratis, M; Chapman, E R; Stowell, M H B

    2010-04-28

    Fusion of synaptic vesicles with the plasma membrane is mediated by the SNARE (soluble NSF attachment receptor) proteins and is regulated by synaptotagmin (syt). There are at least 17 syt isoforms that have the potential to act as modulators of membrane fusion events. Synaptotagmin IV (syt IV) is particularly interesting; it is an immediate early gene that is regulated by seizures and certain classes of drugs, and, in humans, syt IV maps to a region of chromosome 18 associated with schizophrenia and bipolar disease. Syt IV has recently been found to localize to dense core vesicles in hippocampal neurons, where it regulates neurotrophin release. Here we have examined the ultrastructure of cultured hippocampal neurons from wild-type and syt IV -/- mice using electron tomography. Perhaps surprisingly, we observed a potential synaptic vesicle transport defect in syt IV -/- neurons, with the accumulation of large numbers of small clear vesicles (putative axonal transport vesicles) near the trans-Golgi network. We also found an interaction between syt IV and KIF1A, a kinesin known to be involved in vesicle trafficking to the synapse. Finally, we found that syt IV -/- synapses exhibited reduced numbers of synaptic vesicles and a twofold reduction in the proportion of docked vesicles compared to wild-type. The proportion of docked vesicles in syt IV -/- boutons was further reduced, 5-fold, following depolarization. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. DOCK 6: Impact of new features and current docking performance.

    PubMed

    Allen, William J; Balius, Trent E; Mukherjee, Sudipto; Brozell, Scott R; Moustakas, Demetri T; Lang, P Therese; Case, David A; Kuntz, Irwin D; Rizzo, Robert C

    2015-06-05

    This manuscript presents the latest algorithmic and methodological developments to the structure-based design program DOCK 6.7 focused on an updated internal energy function, new anchor selection control, enhanced minimization options, a footprint similarity scoring function, a symmetry-corrected root-mean-square deviation algorithm, a database filter, and docking forensic tools. An important strategy during development involved use of three orthogonal metrics for assessment and validation: pose reproduction over a large database of 1043 protein-ligand complexes (SB2012 test set), cross-docking to 24 drug-target protein families, and database enrichment using large active and decoy datasets (Directory of Useful Decoys [DUD]-E test set) for five important proteins including HIV protease and IGF-1R. Relative to earlier versions, a key outcome of the work is a significant increase in pose reproduction success in going from DOCK 4.0.2 (51.4%) → 5.4 (65.2%) → 6.7 (73.3%) as a result of significant decreases in failure arising from both sampling 24.1% → 13.6% → 9.1% and scoring 24.4% → 21.1% → 17.5%. Companion cross-docking and enrichment studies with the new version highlight other strengths and remaining areas for improvement, especially for systems containing metal ions. The source code for DOCK 6.7 is available for download and free for academic users at http://dock.compbio.ucsf.edu/. © 2015 Wiley Periodicals, Inc.

  11. Shapes of Mixed Phospholipid Vesicles

    PubMed Central

    Aranda-Espinoza, Helim; Maldonado, Amir

    2006-01-01

    We studied the shape of phospholipid vesicles prepared by hydration of a mixture of phosphatidylcholine (SOPC) and phosphatidylserine (SOPS) in different proportions. The aim of the work is to obtain some insight into the influence of the chemical composition of a biomembrane on its shape. The optical microscopy results show that the shape of the vesicles depend on the SOPC:SOPS composition. For low SOPS contents, coiled cylindrical vesicles are observed. The results suggest that specific compositions of the SOPC:SOPS vesicles produce some spontaneous curvature on the membrane and then a coiling instability. PMID:19669461

  12. Gαo Represses Insulin Secretion by Reducing Vesicular Docking in Pancreatic β-Cells

    PubMed Central

    Zhao, Aizhen; Ohara-Imaizumi, Mica; Brissova, Marcella; Benninger, Richard K.P.; Xu, Yanwen; Hao, Yuhan; Abramowitz, Joel; Boulay, Guylain; Powers, Alvin C.; Piston, David; Jiang, Meisheng; Nagamatsu, Shinya; Birnbaumer, Lutz; Gu, Guoqiang

    2010-01-01

    OBJECTIVE Pertussis toxin uncoupling–based studies have shown that Gαi and Gαo can inhibit insulin secretion in pancreatic β-cells. Yet it is unclear whether Gαi and Gαo operate through identical mechanisms and how these G-protein–mediated signals inhibit insulin secretion in vivo. Our objective is to examine whether/how Gαo regulates islet development and insulin secretion in β-cells. RESEARCH DESIGN AND METHODS Immunoassays were used to analyze the Gαo expression in mouse pancreatic cells. Gαo was specifically inactivated in pancreatic progenitor cells by pancreatic cell–specific gene deletion. Hormone expression and insulin secretion in response to different stimuli were assayed in vivo and in vitro. Electron microscope and total internal reflection fluorescence–based assays were used to evaluate how Gαo regulates insulin vesicle docking and secretion in response to glucose stimulation. RESULTS Islet cells differentiate properly in Gαo−/− mutant mice. Gαo inactivation significantly enhances insulin secretion both in vivo and in isolation. Gαo nullizygous β-cells contain an increased number of insulin granules docked on the cell plasma membrane, although the total number of vesicles per β-cell remains unchanged. CONCLUSIONS Gαo is not required for endocrine islet cell differentiation, but it regulates the number of insulin vesicles docked on the β-cell membrane. PMID:20622165

  13. Monte Carlo docking with ubiquitin.

    PubMed Central

    Cummings, M. D.; Hart, T. N.; Read, R. J.

    1995-01-01

    The development of general strategies for the performance of docking simulations is prerequisite to the exploitation of this powerful computational method. Comprehensive strategies can only be derived from docking experiences with a diverse array of biological systems, and we have chosen the ubiquitin/diubiquitin system as a learning tool for this process. Using our multiple-start Monte Carlo docking method, we have reconstructed the known structure of diubiquitin from its two halves as well as from two copies of the uncomplexed monomer. For both of these cases, our relatively simple potential function ranked the correct solution among the lowest energy configurations. In the experiments involving the ubiquitin monomer, various structural modifications were made to compensate for the lack of flexibility and for the lack of a covalent bond in the modeled interaction. Potentially flexible regions could be identified using available biochemical and structural information. A systematic conformational search ruled out the possibility that the required covalent bond could be formed in one family of low-energy configurations, which was distant from the observed dimer configuration. A variety of analyses was performed on the low-energy dockings obtained in the experiment involving structurally modified ubiquitin. Characterization of the size and chemical nature of the interface surfaces was a powerful adjunct to our potential function, enabling us to distinguish more accurately between correct and incorrect dockings. Calculations with the structure of tetraubiquitin indicated that the dimer configuration in this molecule is much less favorable than that observed in the diubiquitin structure, for a simple monomer-monomer pair. Based on the analysis of our results, we draw conclusions regarding some of the approximations involved in our simulations, the use of diverse chemical and biochemical information in experimental design and the analysis of docking results, as well as

  14. Docking system of androgynous and peripheral type

    NASA Technical Reports Server (NTRS)

    Syromyatnikov, V. S.

    1972-01-01

    Soviet and American space engineers have proceeded with creating compatible means for closing and docking spacecraft. It was decided to make a new advanced docking system of a peripheral and androgynous type. Because of a more complex design of the new-type docking mechanism, a number of technical problems arose. To a great extent, the solution of these problems depends on a chosen concept of the docking mechanism. The report deals with the docking system concept accepted by the Soviet engineers as the basis for further development. The description and structural arrangement of the docking system as a whole, its basic assemblies, and a kinematic scheme of the docking mechanism using a system of differentials are given. It should be noted that the experience that was gained from the development of previous docking systems was used to create a new type of docking system. The main problems to be solved in the course of designing and developing the advanced system are noted.

  15. Segregation of the Qb-SNAREs GS27 and GS28 into Golgi vesicles regulates intra-Golgi transport.

    PubMed

    Fusella, Aurora; Micaroni, Massimo; Di Giandomenico, Daniele; Mironov, Alexandre A; Beznoussenko, Galina V

    2013-05-01

    The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgi-resident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb-SNAREs GS27 (membrin) and GS28 (GOS-28), and depleted of nucleotide sugar transporters. A block of intra-Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra-Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP-galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra-Golgi transport; re-addition of isolated Golgi vesicles devoid of UDP-galactose translocase obtained from normal cells restores intra-Golgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter-cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of ϵCOP accelerates the cis-to-trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intra-Golgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter-cisternal connections. © 2013 John Wiley & Sons A/S.

  16. Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes.

    PubMed

    Pellett, Patrina A; Dietrich, Felix; Bewersdorf, Jörg; Rothman, James E; Lavieu, Grégory

    2013-10-01

    A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI:http://dx.doi.org/10.7554/eLife.01296.001.

  17. Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes

    PubMed Central

    Pellett, Patrina A; Dietrich, Felix; Bewersdorf, Jörg; Rothman, James E; Lavieu, Grégory

    2013-01-01

    A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI: http://dx.doi.org/10.7554/eLife.01296.001 PMID:24137546

  18. Secretory function of adipose tissue.

    PubMed

    Kuryszko, J; Sławuta, P; Sapikowski, G

    2016-01-01

    There are two kinds of adipose tissue in mammals: white adipose tissue - WAT and brown adipose tissue - BAT. The main function of WAT is accumulation of triacylglycerols whereas the function of BAT is heat generation. At present, WAT is also considered to be an endocrine gland that produces bioactive adipokines, which take part in glucose and lipid metabolism. Considering its endocrine function, the adipose tissue is not a homogeneous gland but a group of a few glands which act differently. Studies on the secretory function of WAT began in 1994 after discovery of leptin known as the satiation hormone, which regulates body energy homeostasis and maintainence of body mass. Apart from leptin, the following belong to adipokines: adiponectin, resistin, apelin, visfatin and cytokines: TNF and IL 6. Adiponectin is a polypeptide hormone of antidiabetic, anti-inflammatory and anti-atherogenic activity. It plays a key role in carbohydrate and fat metabolism. Resistin exerts a counter effect compared to adiponectin and its physiological role is to maintain fasting glycaemia. Visfatin stimulates insulin secretion and increases insulin sensitivity and glucose uptake by muscle cells and adipocytes. Apelin probably increases the insulin sensitivity of tissues. TNF evokes insulin resistance by blocking insulin receptors and inhibits insulin secretion. Approximately 30% of circulating IL 6 comes from adipose tissue. It causes insulin resistance by decreasing the expression of insulin receptors, decreases adipogenesis and adiponectin and visfatin secretion, and stimulates hepatic gluconeogenesis. In 2004, Bays introduced the notion of adiposopathy, defined as dysfunction of the adipose tissue, whose main feature is insulin and leptin resistance as well as the production of inflammatory cytokines: TNF and IL 6 and monocyte chemoattractant protein. This means that excess of adipose tissue, especially visceral adipose tissue, leads to the development of a chronic subclinical

  19. Protein Mobility within Secretory Granules

    PubMed Central

    Weiss, Annita Ngatchou; Bittner, Mary A.; Holz, Ronald W.; Axelrod, Daniel

    2014-01-01

    We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10−10 cm2/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the

  20. POSE Algorithms for Automated Docking

    NASA Technical Reports Server (NTRS)

    Heaton, Andrew F.; Howard, Richard T.

    2011-01-01

    POSE (relative position and attitude) can be computed in many different ways. Given a sensor that measures bearing to a finite number of spots corresponding to known features (such as a target) of a spacecraft, a number of different algorithms can be used to compute the POSE. NASA has sponsored the development of a flash LIDAR proximity sensor called the Vision Navigation Sensor (VNS) for use by the Orion capsule in future docking missions. This sensor generates data that can be used by a variety of algorithms to compute POSE solutions inside of 15 meters, including at the critical docking range of approximately 1-2 meters. Previously NASA participated in a DARPA program called Orbital Express that achieved the first automated docking for the American space program. During this mission a large set of high quality mated sensor data was obtained at what is essentially the docking distance. This data set is perhaps the most accurate truth data in existence for docking proximity sensors in orbit. In this paper, the flight data from Orbital Express is used to test POSE algorithms at 1.22 meters range. Two different POSE algorithms are tested for two different Fields-of-View (FOVs) and two different pixel noise levels. The results of the analysis are used to predict future performance of the POSE algorithms with VNS data.

  1. Benchmarking Sets for Molecular Docking

    PubMed Central

    Huang, Niu; Shoichet, Brian K.; Irwin, John J.

    2012-01-01

    Ligand enrichment among top-ranking hits is a key metric of molecular docking. To avoid bias, decoys should resemble ligands physically, so that enrichment is not simply a separation of gross features, yet be chemically distinct from them, so that they are unlikely to be binders. We have assembled a directory of useful decoys (DUD), with 2950 ligands for 40 different targets. Every ligand has 36 decoy molecules that are physically similar but topologically distinct, leading to a database of 98,266 compounds. For most targets, enrichment was at least half a log better with uncorrected databases such as the MDDR than with DUD, evidence of bias in the former. These calculations also allowed forty-by-forty cross docking, where the enrichments of each ligand set could be compared for all 40 targets, enabling a specificity metric for the docking screens. DUD is freely available online as a benchmarking set for docking at http://blaster.docking.org/dud/. PMID:17154509

  2. Differential Maturation of the Two Regulated Secretory Pathways in Human iPSC-Derived Neurons.

    PubMed

    Emperador Melero, Javier; Nadadhur, Aishwarya G; Schut, Desiree; Weering, Jan V; Heine, Vivi M; Toonen, Ruud F; Verhage, Matthijs

    2017-03-14

    Neurons communicate by regulated secretion of chemical signals from synaptic vesicles (SVs) and dense-core vesicles (DCVs). Here, we investigated the maturation of these two secretory pathways in micro-networks of human iPSC-derived neurons. These micro-networks abundantly expressed endogenous SV and DCV markers, including neuropeptides. DCV transport was microtubule dependent, preferentially anterograde in axons, and 2-fold faster in axons than in dendrites. SV and DCV secretion were strictly Ca(2+) and SNARE dependent. DCV secretion capacity matured until day in vitro (DIV) 36, with intense stimulation releasing 6% of the total DCV pool, and then plateaued. This efficiency is comparable with mature mouse neurons. In contrast, SV secretion capacity continued to increase until DIV50, with substantial further increase in secretion efficiency and decrease in silent synapses. These data show that the two secretory pathways can be studied in human neurons and that they mature differentially, with DCV secretion reaching maximum efficiency when that of SVs is still low.

  3. Vesicles with a double bilayer.

    PubMed

    Zawada, Zygmunt H

    2004-01-01

    A modified reverse phase evaporation method was used to prepare intermediate unilamellar vesicles coated with an additional membrane, or large vesicles in which several vesicles were coated with a common membrane. In both kinds of vesicle, the outer and inner membranes are usually of different phospholipid composition. The preparation involves the formation of a double emulsion: vesicles in a buffer are emerged in a low-boiling point organic solution of phospholipids. Then the organic solvent is evaporated during the heating and mixing process. As result large unilamellar vesicles (LUVs), about 100 nm in diameter, were coated with an additional membrane from egg lecithin or dipalmitoylphosphatidylcholine and cholesterol. The highest yield of the coating was about 50%. When DPPC was used for coating above the phase transition temperature Tm, the data suggested the formation of vesicles that were slightly larger than the starting LUVs. It might be concluded that many of these had a double bilayer. If the coating was done below Tm, the micrographs suggested the formation of structures resembling multi-vesicular vesicles. They looked like LUV clusters coated with a common membrane.

  4. Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

    PubMed Central

    Li, Feng; Tiwari, Neeraj; Rothman, James E.; Pincet, Frederic

    2016-01-01

    Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state. PMID:27601655

  5. Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion.

    PubMed

    Li, Feng; Tiwari, Neeraj; Rothman, James E; Pincet, Frederic

    2016-09-20

    Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

  6. Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion.

    PubMed

    Wollman, R; Meyer, T

    2012-12-01

    The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

  7. Hydrodynamic interactions between two vesicles

    NASA Astrophysics Data System (ADS)

    Gires, Pierre-Yves; Dyfcom Team

    2011-11-01

    A giant vesicle is a closed elastic membrane containing a liquid, inside another liquid. Its size is around 10 microns. If a suspension of such objects is sheared, they sometimes come close and interact hydrodynamically. We studied how these interactions affect the trajectories of the vesicles. For this, we model the properties of the membrane, assuming that the area of a surface element is constant in the course of time, and that it resists bending. We also assume that the inside and outside fluids are Newtonian, and are in the creeping regime. To solve the partial differential equations arising from this model, we used two methods : an asymptotic expansion around spherical shapes for vesicles far away from each other (3d case), and a boudary integral method (2d case). We find that vesicles repel, and that this repulsion decreases with initial transverse distance. We compare our results with experimental results performed with vesicles flowing in microfluidic devices.

  8. Molecular recognition of bilayer vesicles.

    PubMed

    Voskuhl, Jens; Ravoo, Bart Jan

    2009-02-01

    Vesicles have been a versatile topic of research in chemistry ever since the discovery that, besides phospholipids, synthetic amphiphiles can also form molecular bilayers enclosing a small aqueous compartment. Non-covalent interactions of receptors and ligands or hosts and guests at vesicle surfaces resemble recognition processes at biological membranes, including cell recognition, adhesion and fusion. Molecular recognition at membranes is often mediated by a multivalent instead of a monovalent interaction. This tutorial review describes the basics as well as the latest developments in biomimetic supramolecular chemistry of bilayer vesicles. We describe how molecular recognition can mediate the interaction between vesicles, and how the biomimetic supramolecular chemistry of vesicles furthers our understanding of biological membranes.

  9. An anchor-dependent molecular docking process for docking small flexible molecules into rigid protein receptors.

    PubMed

    Lin, Thy-Hou; Lin, Guan-Liang

    2008-08-01

    A molecular docking method designated as ADDock, anchor-dependent molecular docking process for docking small flexible molecules into rigid protein receptors, is presented in this article. ADDock makes the bond connection lists for atoms based on anchors chosen for building molecular structures for docking small flexible molecules or ligands into rigid active sites of protein receptors. ADDock employs an extended version of piecewise linear potential for scoring the docked structures. Since no translational motion for small molecules is implemented during the docking process, ADDock searches the best docking result by systematically changing the anchors chosen, which are usually the single-edge connected nodes or terminal hydrogen atoms of ligands. ADDock takes intact ligand structures generated during the docking process for computing the docked scores; therefore, no energy minimization is required in the evaluation phase of docking. The docking accuracy by ADDock for 92 receptor-ligand complexes docked is 91.3%. All these complexes have been docked by other groups using other docking methods. The receptor-ligand steric interaction energies computed by ADDock for some sets of active and inactive compounds selected and docked into the same receptor active sites are apparently separated. These results show that based on the steric interaction energies computed between the docked structures and receptor active sites, ADDock is able to separate active from inactive compounds for both being docked into the same receptor.

  10. A python-based docking program utilizing a receptor bound ligand shape: PythDock.

    PubMed

    Chung, Jae Yoon; Cho, Seung Joo; Hah, Jung-Mi

    2011-09-01

    PythDock is a heuristic docking program that uses Python programming language with a simple scoring function and a population based search engine. The scoring function considers electrostatic and dispersion/repulsion terms. The search engine utilizes a particle swarm optimization algorithm. A grid potential map is generated using the shape information of a bound ligand within the active site. Therefore, the searching area is more relevant to the ligand binding. To evaluate the docking performance of PythDock, two well-known docking programs (AutoDock and DOCK) were also used with the same data. The accuracy of docked results were measured by the difference of the ligand structure between x-ray structure, and docked pose, i.e., average root mean squared deviation values of the bound ligand were compared for fourteen protein-ligand complexes. Since the number of ligands' rotational flexibility is an important factor affecting the accuracy of a docking, the data set was chosen to have various degrees of flexibility. Although PythDock has a scoring function simpler than those of other programs (AutoDock and DOCK), our results showed that PythDock predicted more accurate poses than both AutoDock4.2 and DOCK6.2. This indicates that PythDock could be a useful tool to study ligand-receptor interactions and could also be beneficial in structure based drug design.

  11. Saturated fatty acids alter the late secretory pathway by modulating membrane properties.

    PubMed

    Payet, Laurie-Anne; Pineau, Ludovic; Snyder, Ellen C R; Colas, Jenny; Moussa, Ahmed; Vannier, Brigitte; Bigay, Joelle; Clarhaut, Jonathan; Becq, Frédéric; Berjeaud, Jean-Marc; Vandebrouck, Clarisse; Ferreira, Thierry

    2013-12-01

    Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

  12. Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth.

    PubMed

    Wang, Hao; Tse, Yu C; Law, Angus H Y; Sun, Samuel S M; Sun, Yong-Bin; Xu, Zeng-Fu; Hillmer, Stefan; Robinson, David G; Jiang, Liwen

    2010-03-01

    Vacuolar sorting receptors (VSRs) are type-I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type-IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans-Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP-VSR/GFP-LIVSR is located throughout the pollen tubes, excepting the apical clear-zone region, whereas GFP-LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.

  13. Avl9p, a Member of a Novel Protein Superfamily, Functions in the Late Secretory Pathway

    PubMed Central

    Schekman, Randy

    2007-01-01

    The branching of exocytic transport routes in both yeast and mammalian cells has complicated studies of the late secretory pathway, and the mechanisms involved in exocytic cargo sorting and exit from the Golgi and endosomes are not well understood. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit a strong secretory phenotype and are therefore difficult to isolate. In a genetic screen designed to isolate such mutants, we identified a novel conserved protein, Avl9p, the absence of which conferred lethality in a vps1Δ apl2Δ strain background (lacking a dynamin and an adaptor-protein complex 1 subunit). Depletion of Avl9p in this strain resulted in secretory defects as well as accumulation of Golgi-like membranes. The triple mutant also had a depolarized actin cytoskeleton and defects in polarized secretion. Overexpression of Avl9p in wild-type cells resulted in vesicle accumulation and a post-Golgi defect in secretion. Phylogenetic analysis indicated evolutionary relationships between Avl9p and regulators of membrane traffic and actin function. PMID:17229886

  14. The Actomyosin Ring Recruits Early Secretory Compartments to the Division Site in Fission Yeast

    PubMed Central

    Vjestica, Aleksandar; Tang, Xin-Zi

    2008-01-01

    The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring. PMID:18184749

  15. Text Mining for Protein Docking.

    PubMed

    Badal, Varsha D; Kundrotas, Petras J; Vakser, Ilya A

    2015-12-01

    The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking). Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu). The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features) approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound benchmark set

  16. Text Mining for Protein Docking

    PubMed Central

    Badal, Varsha D.; Kundrotas, Petras J.; Vakser, Ilya A.

    2015-01-01

    The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking). Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu). The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features) approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound benchmark set

  17. Wine glasses and hourglasses: Non-adaptive complexity of vesicle traffic in microbial eukaryotes.

    PubMed

    Mani, Somya; Thattai, Mukund

    Microbial eukaryotes present a stunning diversity of endomembrane organization. From specialized secretory organelles such as the rhoptries and micronemes of apicomplexans, to peroxisome-derived metabolic compartments such as the glycosomes of kinetoplastids, different microbial taxa have explored different solutions to the compartmentalization and processing of cargo. The basic secretory and endocytic system, comprising the ER, Golgi, endosomes, and plasma membrane, as well as diverse taxon-specific specialized endomembrane organelles, are coupled by a complex network of cargo transport via vesicle traffic. It is tempting to connect form to function, ascribing biochemical roles to each compartment and vesicle of such a system. Here we argue that traffic systems of high complexity could arise through non-adaptive mechanisms via purely physical constraints, and subsequently be exapted for various taxon-specific functions. Our argument is based on a Boolean mathematical model of vesicle traffic: we specify rules of how compartments exchange vesicles; these rules then generate hypothetical cells with different types of endomembrane organization. Though one could imagine a large number of hypothetical vesicle traffic systems, very few of these are consistent with molecular interactions. Such molecular constraints are the bottleneck of a metaphorical hourglass, and the rules that make it through the bottleneck are expected to generate cells with many special properties. Sampling at random from among such rules represents an evolutionary null hypothesis: any properties of the resulting cells must be non-adaptive. We show by example that vesicle traffic systems generated in this random manner are reminiscent of the complex trafficking apparatus of real cells. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Lead-dependent deposits in diverse synaptic vesicles: suggestive evidence for the presence of anionic binding sites

    SciTech Connect

    Sulzer, D.; Piscopo, I.; Ungar, F.; Holtzman, E.

    1987-09-01

    We have observed electron dense deposits dependent on incubation of aldehyde-fixed tissues with lead ions within synaptic vesicles of several types of neurons that differ in the neurotransmitters utilized and in the secretory granules of the adrenal medulla. Evidently, vesicle components that can interact with lead ions are widespread. A plausible explanation for the occurrence of the deposits is the presence of anionic binding sites within the vesicles. This would agree well with other biochemical, cytochemical, and immunocytochemical evidence, such as that indicating the presence of sulfated macromolecules in certain synaptic vesicles. Anionic binding sites could play significant roles by participating in processes such as Ca/sup 2 +/ storage, stabilization of pH gradients, or the control of osmotic phenomena.

  19. Docking Mechanism on Progress 52

    NASA Image and Video Library

    2014-02-03

    ISS038-E-041175 (3 Feb. 2014) --- This close-up view shows the docking mechanism of the unpiloted Russian ISS Progress 52 resupply ship as it undocks from the International Space Station's Pirs Docking Compartment at 11:21 a.m. (EST) on Feb. 3, 2014. The Progress backed away to a safe distance from the orbital complex to begin several days of tests to study thermal effects of space on its attitude control system. Filled with trash and other unneeded items, the Russian resupply ship will be commanded to re-enter Earth's atmosphere Feb. 11 and disintegrate harmlessly over the Pacific Ocean.

  20. Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

    PubMed

    Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

    2016-12-01

    Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases.

  1. [Secretory immunoglobulin A in amniotic fluid].

    PubMed

    Briese, V; Straube, W; Brock, J; Lorenz, U

    1983-01-01

    Secretory immunoglobulin A (S-IgA) was estimated in amniotic fluid samples by means of the single radial immunodiffusion according to Mancini. A monospecific antiserum against human secretory component was used. 163 amniotic fluid samples from normal pregnancies and risk pregnancies respectively were investigated. Within the 3rd trimenon the S-IgA content in amniotic fluid increased significantly. With respect to literature and examinations performed previously a connection between S-IgA content in amniotic fluid and fetal lung maturity seems to be possible.

  2. Quality control in the secretory assembly line.

    PubMed Central

    Helenius, A

    2001-01-01

    As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway. PMID:11260794

  3. NASA Docking System (NDS) Technical Integration Meeting

    NASA Technical Reports Server (NTRS)

    Lewis, James L.

    2010-01-01

    This slide presentation reviews the NASA Docking System (NDS) as NASA's implementation of the International Docking System Standard (IDSS). The goals of the NDS, is to build on proven technologies previously demonstrated in flight and to advance the state of the art of docking systems by incorporating Low Impact Docking System (LIDS) technology into the NDS. A Hardware Demonstration was included in the meeting, and there was discussion about software, NDS major system interfaces, integration information, schedule, and future upgrades.

  4. Dysregulations of Synaptic Vesicle Trafficking in Schizophrenia.

    PubMed

    Egbujo, Chijioke N; Sinclair, Duncan; Hahn, Chang-Gyu

    2016-08-01

    Schizophrenia is a serious psychiatric illness which is experienced by about 1 % of individuals worldwide and has a debilitating impact on perception, cognition, and social function. Over the years, several models/hypotheses have been developed which link schizophrenia to dysregulations of the dopamine, glutamate, and serotonin receptor pathways. An important segment of these pathways that have been extensively studied for the pathophysiology of schizophrenia is the presynaptic neurotransmitter release mechanism. This set of molecular events is an evolutionarily well-conserved process that involves vesicle recruitment, docking, membrane fusion, and recycling, leading to efficient neurotransmitter delivery at the synapse. Accumulated evidence indicate dysregulation of this mechanism impacting postsynaptic signal transduction via different neurotransmitters in key brain regions implicated in schizophrenia. In recent years, after ground-breaking work that elucidated the operations of this mechanism, research efforts have focused on the alterations in the messenger RNA (mRNA) and protein expression of presynaptic neurotransmitter release molecules in schizophrenia and other neuropsychiatric conditions. In this review article, we present recent evidence from schizophrenia human postmortem studies that key proteins involved in the presynaptic release mechanism are dysregulated in the disorder. We also discuss the potential impact of dysfunctional presynaptic neurotransmitter release on the various neurotransmitter systems implicated in schizophrenia.

  5. Mass spectrometry of extracellular vesicles.

    PubMed

    Pocsfalvi, Gabriella; Stanly, Christopher; Vilasi, Annalisa; Fiume, Immacolata; Capasso, Giovambattista; Turiák, Lilla; Buzas, Edit I; Vékey, Károly

    2016-01-01

    The review briefly summaries main features of extracellular vesicles, a joint terminology for exosomes, microvesicles, and apoptotic vesicles. These vesicles are in the center of interest in biology and medical sciences, and form a very active field of research. Mass spectrometry (MS), with its specificity and sensitivity, has the potential to identify and characterize molecular composition of these vesicles; but as yet there are only a limited, but fast-growing, number of publications that use MS workflows in this field. MS is the major tool to assess protein composition of extracellular vesicles: qualitative and quantitative proteomics approaches are both reviewed. Beside proteins, lipid and metabolite composition of vesicles might also be best assessed by MS techniques; however there are few applications as yet in this respect. The role of alternative analytical approaches, like gel-based proteomics and antibody-based immunoassays, are also mentioned. The objective of the review is to give an overview of this fast-growing field to help orient MS-based research on extracellular vesicles. © 2015 Wiley Periodicals, Inc.

  6. The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening.

    PubMed

    Lai, Ying; Lou, Xiaochu; Jho, Yongseok; Yoon, Tae-Young; Shin, Yeon-Kyun

    2013-11-15

    Syt1 (synaptotagmin 1), a major Ca2+ sensor for fast neurotransmitter release, contains tandem Ca2+-binding C2 domains (C2AB), a single transmembrane α-helix and a highly charged 60-residue-long linker in between. Using single-vesicle-docking and content-mixing assays we found that the linker region of Syt1 is essential for its two signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. The linker contains the basic-amino-acid-rich N-terminal region and the acidic-amino-acid-rich C-terminal region. When the charge segregation was disrupted, fusion pore opening was slowed, whereas docking was unchanged. Intramolecular disulfide cross-linking between N- and C-terminal regions of the linker or deletion of 40 residues from the linker reduced docking while enhancing pore opening, although the changes were subtle. EPR analysis showed Ca2+-induced line broadening reflecting a conformational change in the linker region. Thus the results of the present study suggest that the electrostatically bipartite linker region may extend for docking and fold to facilitate pore opening.

  7. Voss with docking probe in Service module

    NASA Image and Video Library

    2001-05-30

    ISS002-E-6478 (30 May 2001) --- James S. Voss, Expedition Two flight engineer, handles a spacecraft docking probe in the Service Module. The docking probe assists with the docking of the Soyuz and Progress vehicles to the International Space Station. The image was taken with a digital still camera.

  8. The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

    PubMed Central

    Riquelme, Meritxell; Bredeweg, Erin L.; Callejas-Negrete, Olga; Roberson, Robert W.; Ludwig, Sarah; Beltrán-Aguilar, Alejandro; Seiler, Stephan; Novick, Peter; Freitag, Michael

    2014-01-01

    Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis. PMID:24523289

  9. Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes, is essential for the biogenesis of secretory organelles.

    PubMed

    Morlon-Guyot, Juliette; Pastore, Sandra; Berry, Laurence; Lebrun, Maryse; Daher, Wassim

    2015-08-01

    Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome-like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin-like receptor and syntaxin-6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab-GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps-C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal-related compartments, the vacuole and immature apical secretory organelles. Conditional knock-down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock-down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite-specific functions in the biogenesis of their unique secretory organelles.

  10. Expedition 24 Docks to ISS

    NASA Image and Video Library

    2010-06-17

    Alexei Krasnov, Director of Manned Space Programs Department, ROSCOSMOS, answers a reporter’s question during a Soyuz post-docking press conference at the Russian Mission Control Center in Korolev, Russia on Friday, June 18, 2010. Photo Credit: (NASA/Carla Cioffi)

  11. Boom Rendezvous Alternative Docking Approach

    NASA Technical Reports Server (NTRS)

    Bonometti, Joseph A.

    2006-01-01

    Space rendezvous and docking has always been attempted with primarily one philosophic methodology. The slow matching of one vehicle's orbit by a second vehicle and then a final closing sequence that ends in matching the orbits with perfect precision and with near zero relative velocities. The task is time consuming, propellant intensive, risk inherent (plume impingement, collisions, fuel depletion, etc.) and requires substantial hardware mass. The historical background and rationale as to why this approach is used is discussed in terms of the path-not-taken and in light of an alternate methodology. Rendezvous and docking by boom extension is suggested to have inherent advantages that today s technology can readily exploit. Extension from the primary spacecraft, beyond its inherent large inertia, allows low inertia connections to be made rapidly and safely. Plume contamination issues are eliminated as well as the extra propellant mass and risk required for the final thruster (docking) operations. Space vehicle connection hardware can be significantly lightened. Also, docking sensors and controls require less fidelity; allowing them to be more robust and less sensitive. It is the potential safety advantage and mission risk reduction that makes this approach attractive, besides the prospect of nominal time and mass savings.

  12. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    The crews of Expedition 18 and 19 are seen on a large TV screen in the Russian Mission Control Center in Korolev, Russia, Saturday, March 28, 2009 shortly after the Soyuz TMA-14 spacecraft docked to the International Space Station and delivered Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  13. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    View from the balcony of the Russian Mission Control Center in Korolev, Russia moments before the Soyuz TMA-14 docks to the International Space Station on Saturday, March 28, 2009. A view of the International Space Station from Soyuz onboard cameras is visible in the upper right display. Photo Credit: (NASA/Bill Ingalls)

  14. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Michelle Barratt, right, prepares to talk on the phone to her husband onboard the International Space Station from the Russian Mission Control Center, Korolev, Russia, Saturday, March 28, 2009. The Soyuz TMA-14 spacecraft docked to the International Space Station and delivered Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  15. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    View from the balcony of the Russian Mission Control Center in Korolev, Russia moments before the Soyuz TMA-14 docks to the International Space Station on Saturday, March 28, 2009. A view of the International Space Station from Soyuz onboard cameras is visible in the upper display. Photo Credit: (NASA/Bill Ingalls)

  16. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    A large TV screen in Russian Mission Control Center in Korolev, Russia shows Cosmonaut Yury Lonchakov, right, welcoming Expedition 19 Flight Engineer Michael R. Barratt onboard the International Space Station after he fellow crew members Expedition 19 Commander Gennady I. Padalka and Spaceflight Participant Charles Simonyi docked their Soyuz TMA-14 spacecraft on Saturday, March 28, 2009. Photo Credit: (NASA/Bill Ingalls)

  17. Expedition 40 Post Docking Statement

    NASA Image and Video Library

    2014-05-29

    General Director of the Russian Federal Space Agency, Roscosmos, left, and William Gerstenmaier, NASA Associate Administrator for Human Exploration and Operations, right, are seen during a joint statement after the docking of the Soyuz TMA-13M spacecraft with the International Space Station on Thursday, May 29, 2014 at the Baikonur Hotel in Baikonur, Kazakhstan. Photo Credit: (NASA/Joel Kowsky)

  18. Expedition 40 Post Docking Statement

    NASA Image and Video Library

    2014-05-29

    Oleg Ostapenko, General Director of the Russian Federal Space Agency, Roscosmos, left, and William Gerstenmaier, NASA Associate Administrator for Human Exploration and Operations, right, shake hands following a joint statement after the docking of the Soyuz TMA-13M spacecraft with the International Space Station on Thursday, May 29, 2014 at the Baikonur Hotel in Baikonur, Kazakhstan. Photo Credit: (NASA/Joel Kowsky)

  19. Preparations for TMA-14 docking

    NASA Image and Video Library

    2009-07-01

    ISS020-E-016092 (1 July 2009) --- Cosmonaut Gennady Padalka (partially visible in the reflection of a mirror), Expedition 20 commander, uses an onboard laptop-based simulator in the Zvezda Service Module of the International Space Station to prepare for the relocation of the Soyuz TMA-14 spacecraft from Zvezda?s aft port to the Pirs Docking Compartment on July 2, 2009.

  20. Structural disorder provides increased adaptability for vesicle trafficking pathways.

    PubMed

    Pietrosemoli, Natalia; Pancsa, Rita; Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (~23%) than the other two, COPI (~9%) and COPII (~8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest

  1. Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

    PubMed Central

    Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and

  2. Hydra Rendezvous and Docking Sensor

    NASA Technical Reports Server (NTRS)

    Roe, Fred; Carrington, Connie

    2007-01-01

    The U.S. technology to support a CEV AR&D activity is mature and was developed by NASA and supporting industry during an extensive research and development program conducted during the 1990's and early 2000 time frame at the Marshall Space Flight Center. Development and demonstration of a rendezvous/docking sensor was identified early in the AR&D Program as the critical enabling technology that allows automated proxinity operations and docking. A first generation rendezvous/docking sensor, the Video Guidance Sensor (VGS) was developed and successfully flown on STS 87 and again on STS 95, proving the concept of a video-based sensor. Advances in both video and signal processing technologies and the lessons learned from the two successful flight experiments provided a baseline for the development of a new generation of video based rendezvous/docking sensor. The Advanced Video Guidance Sensor (AVGS) has greatly increased performance and additional capability for longer-range operation. A Demonstration Automatic Rendezvous Technology (DART) flight experiment was flown in April 2005 using AVGS as the primary proximity operations sensor. Because of the absence of a docking mechanism on the target satellite, this mission did not demonstrate the ability of the sensor to coltrold ocking. Mission results indicate that the rendezvous sensor operated successfully in "spot mode" (2 km acquisition of the target, bearing data only) but was never commanded to "acquire and track" the docking target. Parts obsolescence issues prevent the construction of current design AVGS units to support the NASA Exploration initiative. This flight proven AR&D technology is being modularized and upgraded with additional capabilities through the Hydra project at the Marshall Space Flight Center. Hydra brings a unique engineering approach and sensor architecture to the table, to solve the continuing issues of parts obsolescence and multiple sensor integration. This paper presents an approach to

  3. Early Exposure to General Anesthesia Disrupts Spatial Organization of Presynaptic Vesicles in Nerve Terminals of the Developing Rat Subiculum.

    PubMed

    Lunardi, N; Oklopcic, A; Prillaman, M; Erisir, A; Jevtovic-Todorovic, V

    2015-10-01

    Exposure to general anesthesia (GA) during critical stages of brain development induces widespread neuronal apoptosis and causes long-lasting behavioral deficits in numerous animal species. Although several studies have focused on the morphological fate of neurons dying acutely by GA-induced developmental neuroapoptosis, the effects of an early exposure to GA on the surviving synapses remain unclear. The aim of this study is to study whether exposure to GA disrupts the fine regulation of the dynamic spatial organization and trafficking of synaptic vesicles in presynaptic terminals. We exposed postnatal day 7 (PND7) rat pups to a clinically relevant anesthetic combination of midazolam, nitrous oxide, and isoflurane and performed a detailed ultrastructural analysis of the synaptic vesicle architecture at presynaptic terminals in the subiculum of rats at PND 12. In addition to a significant decrease in the density of presynaptic vesicles, we observed a reduction of docked vesicles, as well as a reduction of vesicles located within 100 nm from the active zone, in animals 5 days after an initial exposure to GA. We also found that the synaptic vesicles of animals exposed to GA are located more distally with respect to the plasma membrane than those of sham control animals and that the distance between presynaptic vesicles is increased in GA-exposed animals compared to sham controls. We report that exposure of immature rats to GA during critical stages of brain development causes significant disruption of the strategic topography of presynaptic vesicles within the nerve terminals of the subiculum.

  4. Reversible Recruitment of a Homeostatic Reserve Pool of Synaptic Vesicles Underlies Rapid Homeostatic Plasticity of Quantal Content

    PubMed Central

    Pinter, Martin J.; Rich, Mark M.

    2016-01-01

    priming/docking of a small homeostatic reserve pool of vesicles that normally have slow-release kinetics. Following priming, the reserve pool of vesicles is released synchronously with the normal readily releasable pool of synaptic vesicles. This is the first description of this unique pool of synaptic vesicles. PMID:26791213

  5. An endosomal tether undergoes an entropic collapse to bring vesicles together.

    PubMed

    Murray, David H; Jahnel, Marcus; Lauer, Janelle; Avellaneda, Mario J; Brouilly, Nicolas; Cezanne, Alice; Morales-Navarrete, Hernán; Perini, Enrico D; Ferguson, Charles; Lupas, Andrei N; Kalaidzidis, Yannis; Parton, Robert G; Grill, Stephan W; Zerial, Marino

    2016-09-01

    An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.

  6. An endosomal tether undergoes an entropic collapse to bring vesicles together

    PubMed Central

    Lauer, Janelle; Avellaneda, Mario J.; Brouilly, Nicolas; Cezanne, Alice; Morales-Navarrete, Hernán; Perini, Enrico D.; Ferguson, Charles; Lupas, Andrei N.; Kalaidzidis, Yannis; Parton, Robert G.; Grill, Stephan W.; Zerial, Marino

    2016-01-01

    An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors1–4. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs alone5,6,7. Here, we addressed the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA16,7 recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harboring Rab5. Surprisingly, structural analysis revealed that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers we confirmed that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measured its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induced prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle toward the target membrane to initiate docking and fusion. PMID:27556945

  7. Temperature-Dependent Differences between Readily Releasable and Reserve Pool Vesicles in Chromaffin Cells

    PubMed Central

    Haynes, Christy L.; Siff, Lauren N.; Wightman, R. Mark

    2007-01-01

    Summary Statistical differences between amperometric traces recorded from chromaffin cells using K+ and Ba2+ secretagogues support the assertion that readily releasable pool (RRP) and reserve pool (RP) vesicles can be probed with pool-specific secretagogues. Release from the RRP was evoked by K+ while release from the RP was evoked by Ba2+. Similar temperature-dependent changes in spike area and half-width for both pools suggest that the content of RRP and RP vesicles is similar and packaged in the same way. Differences between the vesicle pools were revealed in the temperature dependence of spike frequency. While the burst spike frequency of the RRP, which is comprised of pre-docked and primed vesicles, increased 2.8% per °C, the RP spike frequency increased 12% per °C. This difference is attributed to a temperature dependent mobilization of the RP. Furthermore, the RP exhibited more foot events at room temperature than the RRP but this difference was not apparent at 37°C. This trend suggests that RP vesicle membranes have a compromised surface tension compared to RRP vesicles. Collectively, the changes of release characteristics with temperature reveal distinctions between the RRP and the RP. PMID:17467077

  8. Signaling from the Secretory Granule to the Nucleus: Uhmk1 and PAM

    PubMed Central

    Francone, Victor P.; Ifrim, Marius F.; Rajagopal, Chitra; Leddy, Christopher J.; Wang, Yanping; Carson, John H.; Mains, Richard E.; Eipper, Betty A.

    2010-01-01

    Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine α-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent. PMID:20573687

  9. Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex.

    PubMed

    Xu, Guo Xin; Tan, Chao; Wei, Xiao Jing; Gao, Xiao Yan; Zheng, Hui Qiong

    2011-04-01

    The distribution and development of secretory cells and crystal cells in young shoot apexes of water hyacinth were investigated through morphological and cytological analysis. The density of secretory cells and crystal cells were high in parenchyma tissues around the vascular bundles of shoot apexes. Three developmental stages of the secretory cells can be distinguished under transmission electron microscopy. Firstly, a large number of electron-dense vesicles formed in the cytoplasm, then fused with the tonoplast and released into the vacuole in the form of electron-dense droplets. As these droplets fused together, a large mass of dark material completely filled the vacuole. To this end, a secretion storage vacuole (SSV) formed. Secondly, an active secretion stage accompanied with degradation of the large electron-dense masses through an ill-defined autophagic process at periphery and in the limited internal regions of the SSV. Finally, after most storage substances were withdrawn, the materials remaining in the spent SSV consisted of an electron-dense network structure. The distribution and development of crystal cells in shoot apical tissue of water hyacinth were also studied by light and electron microscopy. Crystals initially formed at one site in the vacuole, where tube-like membrane structures formed crystal chambers. The chamber enlarged as the crystal grew in bidirectional manner and formed needle-shaped raphides. Most of these crystals finally occurred as raphide bundles, and the others appeared as block-like rhombohedral crystals in the vacuole. These results suggest that the formation of both secretory cells and crystal cells are involved in the metamorphosis of vacuoles and a role for vacuoles in water hyacinth rapid growth and tolerance.

  10. Adhesion energy can regulate vesicle fusion and stabilize partially fused states.

    PubMed

    Long, Rong; Hui, Chung-Yuen; Jagota, Anand; Bykhovskaia, Maria

    2012-07-07

    Release of neurotransmitters from nerve terminals occurs by fusion of synaptic vesicles with the plasma membrane, and this process is highly regulated. Although major molecular components that control docking and fusion of vesicles to the synaptic membrane have been identified, the detailed mechanics of this process is not yet understood. We have developed a mathematical model that predicts how adhesion forces imposed by docking and fusion molecular machinery would affect the fusion process. We have computed the membrane stress that is produced by adhesion-driven vesicle bending and find that it is compressive. Further, our computations of the membrane curvature predict that strong adhesion can create a metastable state with a partially opened pore that would correspond to the 'kiss and run' release mode. Our model predicts that the larger the vesicle size, the more likely the metastable state with a transiently opened pore. These results contribute to understanding the mechanics of the fusion process, including possible clamping of the fusion by increasing molecular adhesion, and a balance between 'kiss and run' and full collapse fusion modes.

  11. Adhesion energy can regulate vesicle fusion and stabilize partially fused states

    PubMed Central

    Long, Rong; Hui, Chung-Yuen; Jagota, Anand; Bykhovskaia, Maria

    2012-01-01

    Release of neurotransmitters from nerve terminals occurs by fusion of synaptic vesicles with the plasma membrane, and this process is highly regulated. Although major molecular components that control docking and fusion of vesicles to the synaptic membrane have been identified, the detailed mechanics of this process is not yet understood. We have developed a mathematical model that predicts how adhesion forces imposed by docking and fusion molecular machinery would affect the fusion process. We have computed the membrane stress that is produced by adhesion-driven vesicle bending and find that it is compressive. Further, our computations of the membrane curvature predict that strong adhesion can create a metastable state with a partially opened pore that would correspond to the ‘kiss and run’ release mode. Our model predicts that the larger the vesicle size, the more likely the metastable state with a transiently opened pore. These results contribute to understanding the mechanics of the fusion process, including possible clamping of the fusion by increasing molecular adhesion, and a balance between ‘kiss and run’ and full collapse fusion modes. PMID:22258550

  12. Differential roles for snapin and synaptotagmin in the synaptic vesicle cycle.

    PubMed

    Yu, Szi-Chieh; Klosterman, Susan M; Martin, Ashley A; Gracheva, Elena O; Richmond, Janet E

    2013-01-01

    Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin null mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

  13. Structural Evidence for Common Ancestry of the Nuclear Pore Complex and Vesicle Coats

    SciTech Connect

    Brohawn, S.; Leksa, N; Spear, E; Rajashankar, K; Schwartz, T

    2008-01-01

    Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)bulletSeh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.

  14. A fast, single-vesicle fusion assay mimics physiological SNARE requirements

    PubMed Central

    Karatekin, Erdem; Di Giovanni, Jérôme; Iborra, Cécile; Coleman, Jeff; O'Shaughnessy, Ben; Seagar, Michael; Rothman, James E.

    2010-01-01

    Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5–10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5–11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement. PMID:20133592

  15. Ursodeoxycholic acid attenuates colonic epithelial secretory function

    PubMed Central

    Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

    2013-01-01

    Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl− secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl− secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na+/K+-ATPase activity and basolateral K+ channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg−1) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50–200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

  16. Airway Secretory microRNAome Changes during Rhinovirus Infection in Early Childhood

    PubMed Central

    Gutierrez, Maria J.; Gomez, Jose L.; Perez, Geovanny F.; Pancham, Krishna; Val, Stephanie; Pillai, Dinesh K.; Giri, Mamta; Ferrante, Sarah; Freishtat, Robert; Rose, Mary C.; Preciado, Diego; Nino, Gustavo

    2016-01-01

    Background Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV) to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome) and examine the changes during rhinovirus (RV) infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood. Methods Nasal airway secretions were obtained from children (≤3 yrs. old) during PCR-confirmed RV infections (n = 10) and age-matched controls (n = 10). Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC) differentiated at air-liquid interface (ALI). Bioinformatics tools were used to determine the unified (nasal and bronchial) signature airway secretory miRNAome and changes during RV infection in children. Results Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV. Conclusions Comparative analysis of the airway secretory microRNAome in children indicates that RV infection

  17. Synaptic Vesicle Exocytosis

    PubMed Central

    Südhof, Thomas C.; Rizo, Josep

    2011-01-01

    Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components. PMID:22026965

  18. Benzaldehyde-functionalized Polymer Vesicles

    PubMed Central

    Sun, Guorong; Fang, Huafeng; Cheng, Chong; Lu, Peng; Zhang, Ke; Walker, Amy V.; Taylor, John-Stephen A.; Wooley, Karen L.

    2009-01-01

    Polymer vesicles with diameters of ca. 100-600 nm and bearing benzaldehyde functionalities within the vesicular walls were constructed through self assembly of an amphiphilic block copolymer PEO45-b-PVBA26 in water. The reactivity of the benzaldehyde functionalities was verified by crosslinking the polymersomes, and also by a one-pot crosslinking and functionalization approach to further render the vesicles fluorescent, each via reductive amination. In vitro studies found these labelled nanostructures to undergo cell association. PMID:19309173

  19. Mammalian vesicle trafficking proteins of the endoplasmic reticulum and Golgi apparatus.

    PubMed

    Hay, J C; Hirling, H; Scheller, R H

    1996-03-08

    Vesicle traffic propagates and maintains distinct subcellular compartments and routes secretory products from their site of synthesis to their final destinations. As a basis for the specificity of vesicular transport reactions, each step in the secretory pathway appears to be handled by a distinct set of evolutionarily conserved proteins. Mammalian proteins responsible for vesicle trafficking at early steps in the secretory pathway are not well understood. In this report, we describe rat sec22 (rsec22) and rat bet1 (rbet1), mammalian sequence homologs of yeast proteins identified as mediators of endoplasmic reticulum-to-Golgi protein transport. rsec22 and rbet1 were expressed widely in mammalian tissues, as anticipated for proteins involved in fundamental membrane trafficking reactions. Recombinant rsec22 and rbet1 proteins behaved as integral membrane components of 28 and 18 kDa, respectively, consistent with their primary structures, which contain a predicted transmembrane domain at or near the carboxyl terminus. Recombinant rsec22 and rbet1 had distinct subcellular localizations, with rsec22 residing on endoplasmic reticulum membranes and rbet1 found on Golgi membranes. Studies with brefeldin A and nocodazole indicated that rbet1 function might involve interaction with or retention in the intermediate compartment. The distinct localizations of rsec22 and rbet1 may reflect their participation in opposite directions of membrane flow between the endoplasmic reticulum and Golgi apparatus.

  20. Prediction and scoring of docking poses with pyDock.

    PubMed

    Grosdidier, Solène; Pons, Carles; Solernou, Albert; Fernández-Recio, Juan

    2007-12-01

    The two previous CAPRI experiments showed the success of our rigid-body and refinement approach. For this third edition of CAPRI, we have used a new faster protocol called pyDock, which uses electrostatics and desolvation energy to score docking poses generated with FFT-based algorithms. In target T24 (unbound/model), our best prediction had the highest value of fraction of native contacts (40%) among all participants, although it was not considered as acceptable by the CAPRI criteria. In target T25 (unbound/bound), we submitted a model with medium quality. In target T26 (unbound/unbound), we did not submit any acceptable model (but we would have submitted acceptable predictions if we had included available mutational information about the binding site). For targets T27 (unbound/unbound) and T28 (homo-dimer using model), nobody (including us) submitted any acceptable model. Intriguingly, the crystal structure of target T27 shows an alternative interface that correlates with available biological data (we would have submitted acceptable predictions if we had included this). We also participated in all targets of the SCORERS experiment, with at least acceptable accuracy in all valid cases. We submitted two medium and four acceptable scoring models of T25. Using additional distance restraints (from mutational data), we had two medium and two acceptable scoring models of T26. For target T27, we submitted two acceptable scoring models of the alternative interface in the crystal structure. In summary, CAPRI showed the excellent capabilities of pyDock in identifying near-native docking poses. (c) 2007 Wiley-Liss, Inc.

  1. Extracellular Vesicles in Renal Pathophysiology

    PubMed Central

    Pomatto, Margherita A. C.; Gai, Chiara; Bussolati, Benedetta; Camussi, Giovanni

    2017-01-01

    Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy. PMID:28638822

  2. Extracellular vesicles in renal disease.

    PubMed

    Karpman, Diana; Ståhl, Anne-Lie; Arvidsson, Ida

    2017-09-01

    Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.

  3. Extracellular Vesicles in Renal Pathophysiology.

    PubMed

    Pomatto, Margherita A C; Gai, Chiara; Bussolati, Benedetta; Camussi, Giovanni

    2017-01-01

    Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

  4. New Putative Chloroplast Vesicle Transport Components and Cargo Proteins Revealed Using a Bioinformatics Approach: An Arabidopsis Model

    PubMed Central

    Khan, Nadir Zaman; Lindquist, Emelie; Aronsson, Henrik

    2013-01-01

    Proteins and lipids are known to be transported to targeted cytosolic compartments in vesicles. A similar system in chloroplasts is suggested to transfer lipids from the inner envelope to the thylakoids. However, little is known about both possible cargo proteins and the proteins required to build a functional vesicle transport system in chloroplasts. A few components have been suggested, but only one (CPSAR1) has a verified location in chloroplast vesicles. This protein is localized in the donor membrane (envelope) and vesicles, but not in the target membrane (thylakoids) suggesting it plays a similar role to a cytosolic homologue, Sar1, in the secretory pathway. Thus, we hypothesized that there may be more similarities, in addition to lipid transport, between the vesicle transport systems in the cytosol and chloroplast, i.e. similar vesicle transport components, possible cargo proteins and receptors. Therefore, using a bioinformatics approach we searched for putative chloroplast components in the model plant Arabidopsis thaliana, corresponding mainly to components of the cytosolic vesicle transport system that may act in coordination with previously proposed COPII chloroplast homologues. We found several additional possible components, supporting the notion of a fully functional vesicle transport system in chloroplasts. Moreover, we found motifs in thylakoid-located proteins similar to those of COPII vesicle cargo proteins, supporting the hypothesis that chloroplast vesicles may transport thylakoid proteins from the envelope to the thylakoid membrane. Several putative cargo proteins are involved in photosynthesis, thus we propose the existence of a novel thylakoid protein pathway that is important for construction and maintenance of the photosynthetic machinery. PMID:23573218

  5. Adaptive BP-Dock: An Induced Fit Docking Approach for Full Receptor Flexibility.

    PubMed

    Bolia, Ashini; Ozkan, S Banu

    2016-04-25

    We present an induced fit docking approach called Adaptive BP-Dock that integrates perturbation response scanning (PRS) with the flexible docking protocol of RosettaLigand in an adaptive manner. We first perturb the binding pocket residues of a receptor and obtain a new conformation based on the residue response fluctuation profile using PRS. Next, we dock a ligand to this new conformation by RosettaLigand, where we repeat these steps for several iterations. We test this approach on several protein test sets including difficult unbound docking cases such as HIV-1 reverse transcriptase and HIV-1 protease. Adaptive BP-Dock results show better correlation with experimental binding affinities compared to other docking protocols. Overall, the results imply that Adaptive BP-Dock can easily capture binding induced conformational changes by simultaneous sampling of protein and ligand conformations. This can provide faster and efficient docking of novel targets for rational drug design.

  6. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release

    PubMed Central

    1979-01-01

    We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick- freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better. PMID:38256

  7. Unbinding of adhesive vesicles

    NASA Astrophysics Data System (ADS)

    Brochard-Wyart, FrançOise de Gennes, Pierre-Gilles

    We consider a vesicle, bound on one side to a pipette and sticking on the other side to a flat plate. When a pulling force f is applied to the pipette, the radius Rc of the contact patch decreases, and jumps to zero at a critical value of the force. We present here an extension of the Evans theory for these processes. Then we discuss the dynamics of separation for two distinct cases: (a) nonspecific adhesion; and (b) specific adhesion induced by mobile proteins. To cite this article: F. Brochard-Wyart, P.-G. de Gennes, C. R. Physique 4 (2003). On considère une vésicule qui, aspirée par une pipette d'un coteé adhèe de l'autre sur une surface plane. Lorsqu'on tire sur la pipette avec une force f le rayon du contact adhesif decroit, et s'annule brusquement a une valeur critique de la force. On présente ici une extension de la théorie d'Evans pour interpréter ces processus de détachement. Puis l'on discute la dynamique de la séparation pour deux cas distincts : (a) adhésion non spécifique ; et (b) adhésion spécifique par des protéines mobiles. Pour citer cet article : F. Brochard-Wyart, P.-G. de Gennes, C. R. Physique 4 (2003).

  8. Laser docking system flight experiment

    NASA Technical Reports Server (NTRS)

    Erwin, Harry O.

    1986-01-01

    Experiments necessary in the development of the Laser Docking System (LDS) are described. The LDS would be mounted in the Orbiter payload bay, along with a grid connected by fiber optic link to a computer in the cabin. The tests would be performed to aid in the design of an operational sensor which could track a passive target accurately enough to permit soft docking. Additional data would be gained regarding the LDS performance in space, the effects of Orbiter RCS plume impingement on the target, and refinements needed for the flight hardware. A working model which includes an IR laser steered by galvanometer-driven motors for bouncing beams off retroreflectors mounted on targets is described, together with a 300 ft long indoor test facility. Tests on Orbiter flights would first be in a wholly automatic mode and then in a man-in-the-loop mode.

  9. Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum.

    PubMed Central

    Salama, N R; Chuang, J S; Schekman, R W

    1997-01-01

    The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae. This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150. Here, we report the cloning and characterization of the p150 gene. p150 is encoded by an essential gene. Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene. Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1. p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor. The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus. Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay. Images PMID:9190202

  10. Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways.

    PubMed

    Heidtman, Matthew; Chen, Emy J; Moy, Man-Yu; Isberg, Ralph R

    2009-02-01

    The bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.

  11. Large scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways

    PubMed Central

    Heidtman, Matthew; Chen, Emy J.; Moy, Man-Yu; Isberg, Ralph R.

    2009-01-01

    The bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analyzed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection. PMID:19016775

  12. Mammalian TRAPPIII Complex positively modulates the recruitment of Sec13/31 onto COPII vesicles

    PubMed Central

    Zhao, Shan; Li, Chun Man; Luo, Xiao Min; Siu, Gavin Ka Yu; Gan, Wen Jia; Zhang, Lin; Wu, William K. K.; Chan, Hsiao Chang; Yu, Sidney

    2017-01-01

    The Transport protein particle (TRAPP) complex is a tethering factor for COPII vesicle. Of three forms of TRAPP (TRAPPI, II and III) complexes identified so far, TRAPPIII has been largely considered to play a role in autophagy. While depletion of TRAPPIII specific subunits caused defects in the early secretory pathway and TRAPPIII might interact with components of the COPII vesicle coat, its exact role remains to be determined. In this study, we studied the function of TRAPPIII in early secretory pathway using a TRAPPIII-specific subunit, TRAPPC12, as starting point. We found that TRAPPC12 was localized to the ER exit sites and ERGIC. In cells deleted with TRAPPC12, ERGIC and to a lesser extent, the Golgi became dispersed. ER-to-Golgi transport was also delayed. TRAPPC12, but not TRAPPC8, bound to Sec13/Sec31A tetramer but each Sec protein alone could not interact with TRAPPC12. TRAPPIII positively modulated the assembly of COPII outer layer during COPII vesicle formation. These results identified a novel function of TRAPPIII as a positive modulator of the outer layer of the COPII coat. PMID:28240221

  13. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Michelle Barratt wishes her husband, NASA Astronaut Michael Barratt, a happy wedding anniversary via phone to the International Space Station from the Russian Mission Control Center, Korolev, Russia, Saturday, March 28, 2009. The Soyuz TMA-14 spacecraft docked to the International Space Station and delivered Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  14. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Michelle Barratt, 3rd from left, claps as she watches her husband, NASA Astronaut Mike Barratt, enter the International Space Station live on TV from the Russia Mission Control Center in Korolev, Russia, Saturday, March 28, 2009. The Soyuz TMA-14 docked to the International Space Station carrying Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  15. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    12-year-old Anna Chibiskova of Moscow speaks during the Soyuz post-docking press conference at the Russian mission Control Center in Korolev, Russia on Saturday March 28, 2009. Chibiskova was the winner of an International logo design contest for the Expedition 19 mission. Stas Pyatkin, (not pictured) from the Uglegorsk Amur region, won third place and 12-year-old Keytlin Riley (not pictured) from New York won second place. Photo Credit: (NASA/Bill Ingalls)

  16. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Valery Grin, Deputy Head of State Commission, talks on the phone to the six crew members onboard the International Space Station from the Russian Mission Control Center, Korolev, Russia, Saturday, March 28, 2009. The Soyuz TMA-14 spacecraft docked to the International Space Station and delivered Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  17. Expedition 19 Docks to ISS

    NASA Image and Video Library

    2009-03-27

    Mike Hawes, NASA's Acting Associate Administrator, talks on the phone to the six crew members onboard the International Space Station from the Russian Mission Control Center, Korolev, Russia, Saturday, March 28, 2009. The Soyuz TMA-14 spacecraft docked to the International Space Station and delivered Expedition 19 Commander Gennady I. Padalka, Flight Engineer Michael R. Barratt and Spaceflight Participant Charles Simonyi. Photo Credit: (NASA/Bill Ingalls)

  18. Expedition 40 Post Docking Statement

    NASA Image and Video Library

    2014-05-29

    Jean-Jacques Dordain, Director General of the European Space Agency, left, Oleg Ostapenko, General Director of the Russian Federal Space Agency, Roscosmos, center, and William Gerstenmaier, NASA Associate Administrator for Human Exploration and Operations, right, are seen during a joint statement after the docking of the Soyuz TMA-13M spacecraft with the International Space Station on Thursday, May 29, 2014 at the Baikonur Hotel in Baikonur, Kazakhstan. Photo Credit: (NASA/Joel Kowsky)

  19. Preparations for TMA-14 docking

    NASA Image and Video Library

    2009-07-01

    ISS020-E-016084 (1 July 2009) --- NASA astronaut Michael Barratt, Expedition 20 flight engineer, uses an onboard laptop-based simulator in the Zvezda Service Module of the International Space Station to prepare for the relocation of the Soyuz TMA-14 spacecraft from Zvezda?s aft port to the Pirs Docking Compartment on July 2, 2009. Cosmonaut Roman Romanenko, flight engineer, is visible in the background.

  20. Expedition 32 Docking with ISS

    NASA Image and Video Library

    2012-07-17

    Dina Pandya, Expedition 32 Flight Engineer Sunita Williams’ sister, says hello after her arrival to the International Space Station on Tuesday, July 17, 2012 at the Russian Mission Control Center in Korolev, Russia. The Soyuz docked to the International Space Station with Williams and fellow crew members Soyuz Commander Yuri Malenchenko and JAXA Flight Engineer Akihiko Hoshide two days after they launched from the Baikonur Cosmodrome in Kazakhstan. Photo Credit: (NASA/Carla Cioffi)

  1. Customizing scoring functions for docking.

    PubMed

    Pham, Tuan A; Jain, Ajay N

    2008-05-01

    Empirical scoring functions used in protein-ligand docking calculations are typically trained on a dataset of complexes with known affinities with the aim of generalizing across different docking applications. We report a novel method of scoring-function optimization that supports the use of additional information to constrain scoring function parameters, which can be used to focus a scoring function's training towards a particular application, such as screening enrichment. The approach combines multiple instance learning, positive data in the form of ligands of protein binding sites of known and unknown affinity and binding geometry, and negative (decoy) data of ligands thought not to bind particular protein binding sites or known not to bind in particular geometries. Performance of the method for the Surflex-Dock scoring function is shown in cross-validation studies and in eight blind test cases. Tuned functions optimized with a sufficient amount of data exhibited either improved or undiminished screening performance relative to the original function across all eight complexes. Analysis of the changes to the scoring function suggest that modifications can be learned that are related to protein-specific features such as active-site mobility.

  2. Customizing scoring functions for docking

    NASA Astrophysics Data System (ADS)

    Pham, Tuan A.; Jain, Ajay N.

    2008-05-01

    Empirical scoring functions used in protein-ligand docking calculations are typically trained on a dataset of complexes with known affinities with the aim of generalizing across different docking applications. We report a novel method of scoring-function optimization that supports the use of additional information to constrain scoring function parameters, which can be used to focus a scoring function's training towards a particular application, such as screening enrichment. The approach combines multiple instance learning, positive data in the form of ligands of protein binding sites of known and unknown affinity and binding geometry, and negative (decoy) data of ligands thought not to bind particular protein binding sites or known not to bind in particular geometries. Performance of the method for the Surflex-Dock scoring function is shown in cross-validation studies and in eight blind test cases. Tuned functions optimized with a sufficient amount of data exhibited either improved or undiminished screening performance relative to the original function across all eight complexes. Analysis of the changes to the scoring function suggest that modifications can be learned that are related to protein-specific features such as active-site mobility.

  3. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    PubMed

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  4. Parotid salivary secretory pattern in bulimia nervosa.

    PubMed

    Riad, M; Barton, J R; Wilson, J A; Freeman, C P; Maran, A G

    1991-01-01

    Parotid gland enlargement occurs in about 25% of patients with the binge eating syndrome of bulimia nervosa. The parotid salivary secretory patterns in 28 bulimics were determined in order to investigate the functional abnormality in the glands. Bulimia patients had a reduced resting flow rate. Bulimics who developed sialadenosis (4 patients) had reduced resting and stimulated flow rates. The salivary amylase activity was increased in both the resting and stimulated states in bulimics and the sialadenosis group. The resting total protein levels were greater in the bulimics. The electrolyte and immunoglobulin levels were within normal limits. The possibility of protein and enzymatic secretory disturbances due to autonomic nerve disorders as an explanation for the development of sialadenosis in bulimia nervosa is discussed.

  5. Engineering vesicle trafficking improves the extracellular activity and surface display efficiency of cellulases in Saccharomyces cerevisiae.

    PubMed

    Tang, Hongting; Song, Meihui; He, Yao; Wang, Jiajing; Wang, Shenghuan; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2017-01-01

    Cellulase expression via extracellular secretion or surface display in Saccharomyces cerevisiae is one of the most frequently used strategies for a consolidated bioprocess (CBP) of cellulosic ethanol production. However, the inefficiency of the yeast secretory pathway often results in low production of heterologous proteins, which largely limits cellulase secretion or display. In this study, the components of the vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi and from the Golgi to the plasma membrane, involved in vesicle budding, tethering and fusion, were over-expressed in Clostridium thermocellum endoglucanase (CelA)- and Sacchromycopsis fibuligera β-glucosidase (BGL1)-secreting or -displaying strains. Engineering the targeted components in the ER to Golgi vesicle trafficking, including Sec12p, Sec13p, Erv25p and Bos1p, enhanced the extracellular activity of CelA. However, only Sec13p over-expression increased BGL1 secretion. By contrast, over-expression of the components in the Golgi to plasma membrane vesicle trafficking, including Sso1p, Snc2p, Sec1p, Exo70p, Ypt32p and Sec4p, showed better performance in increasing BGL1 secretion compared to CelA secretion, and the over-expression of these components all increased BGL1 extracellular activity. These results revealed that various cellulases showed different limitations in protein transport, and engineering vesicle trafficking has protein-specific effects. Importantly, we found that engineering the above vesicle trafficking components, particularly from the ER to the Golgi, also improved the display efficiency of CelA and BGL1 when a-agglutinin was used as surface display system. Further analyses illustrated that the display efficiency of a-agglutinin was increased by engineering vesicle trafficking, and the trend was consistent with displayed CelA and BGL1. These results indicated that fusion with a-agglutinin may affect the proteins' properties and alter the rate-limiting step in the

  6. The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments.

    PubMed

    Cui, Xiaoyan; Wei, Taiyun; Chowda-Reddy, R V; Sun, Guangyu; Wang, Aiming

    2010-02-05

    Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication.

  7. Machine learning in computational docking.

    PubMed

    Khamis, Mohamed A; Gomaa, Walid; Ahmed, Walaa F

    2015-03-01

    The objective of this paper is to highlight the state-of-the-art machine learning (ML) techniques in computational docking. The use of smart computational methods in the life cycle of drug design is relatively a recent development that has gained much popularity and interest over the last few years. Central to this methodology is the notion of computational docking which is the process of predicting the best pose (orientation + conformation) of a small molecule (drug candidate) when bound to a target larger receptor molecule (protein) in order to form a stable complex molecule. In computational docking, a large number of binding poses are evaluated and ranked using a scoring function. The scoring function is a mathematical predictive model that produces a score that represents the binding free energy, and hence the stability, of the resulting complex molecule. Generally, such a function should produce a set of plausible ligands ranked according to their binding stability along with their binding poses. In more practical terms, an effective scoring function should produce promising drug candidates which can then be synthesized and physically screened using high throughput screening process. Therefore, the key to computer-aided drug design is the design of an efficient highly accurate scoring function (using ML techniques). The methods presented in this paper are specifically based on ML techniques. Despite many traditional techniques have been proposed, the performance was generally poor. Only in the last few years started the application of the ML technology in the design of scoring functions; and the results have been very promising. The ML-based techniques are based on various molecular features extracted from the abundance of protein-ligand information in the public molecular databases, e.g., protein data bank bind (PDBbind). In this paper, we present this paradigm shift elaborating on the main constituent elements of the ML approach to molecular docking along

  8. RFP tags for labeling secretory pathway proteins

    SciTech Connect

    Han, Liyang; Zhao, Yanhua; Xu, Pingyong; Huan, Shuangyan

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  9. Clathrin regenerates synaptic vesicles from endosomes

    PubMed Central

    Watanabe, Shigeki; Trimbuch, Thorsten; Camacho-Pérez, Marcial; Rost, Benjamin R.; Brokowski, Bettina; Söhl-Kielczynski, Berit; Felies, Annegret; Davis, M. Wayne; Rosenmund, Christian; Jorgensen, Erik M.

    2014-01-01

    Summary Ultrafast endocytosis can retrieve a single large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 seconds, which in turn become small-diameter synaptic vesicles 5-6 seconds after stimulation. We disrupted clathrin function using RNAi and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results explain in large part discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis. PMID:25296249

  10. When to biopsy seminal vesicles.

    PubMed

    Panach-Navarrete, J; García-Morata, F; Hernández-Medina, J A; Martínez-Jabaloyas, J M

    2015-05-01

    The involvement of seminal vesicles in prostate cancer can affect the prognosis and determine the treatment. The objective of this study was to determine whether we could predict its infiltration at the time of the prostate biopsy to know when to indicate the biopsy of the seminal vesicles. observational retrospective study of 466 patients who underwent seminal vesicle biopsy. The indication for this biopsy was a prostate-specific antigen (PSA) level greater than 10 ng/ml or an asymmetric or obliterated prostatoseminal angle. The following variables were included in the analysis: PSA level, PSA density, prostate volume, number of cores biopsied, suspicious rectal examination, and preservation of the prostatoseminal angle, studying its relationship with the involvement of the seminal vesicles. Forty-one patients (8.8%) had infiltrated seminal vesicles and 425 (91.2%) had no involvement. In the univariate analysis, the cases with infiltration had a higher mean PSA level (P < .01) and PSA density (P < .01), as well as a lower mean prostate volume (P < .01). A suspicious rectal examination (20.7% of the infiltrated vesicles) and the obliteration or asymmetry of the prostatoseminal angle (33.3% of the infiltrated vesicles) were significantly related to the involvement (P < .01). In the multivariate analysis, we concluded that the probability of having infiltrated seminal vesicles is 5.19 times higher if the prostatoseminal angle is not preserved (P < .01), 4.65 times higher for PSA levels >19.60 ng/dL (P < .01) and 2.95 times higher if there is a suspicious rectal examination (P = .014). Furthermore, this probability increases by 1.04 times for each unit of prostate volume lower (P < .01). The ROC curves showed maximum sensitivity and specificity at 19.6 ng/mL for PSA and 0.39 for PSA density. In this series, greater involvement of seminal vesicles was associated with a PSA level ≥20 ng/ml, a suspicious rectal examination and a lack of prostatoseminal angle

  11. Extracellular Vesicles in Lung Disease.

    PubMed

    Kubo, Hiroshi

    2017-07-03

    Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the pathogenesis of lung diseases. These vesicles include exosomes, ectosomes (ie, microparticles, extracellular vesicles, microvesicles, and shedding vesicles), and apoptotic bodies. Exosomes are generated by inward budding of the membrane (endocytosis), subsequent forming of multivesicular bodies, and release by exocytosis. Ectosomes are formed by outward blebbing from the plasma membrane and are then released by proteolytic cleavage from the cell surface. Apoptotic bodies are generated on apoptotic cell shrinkage and death. Extracellular vesicles are released when the cells are activated or undergo apoptosis under inflammatory conditions. The number and types of released EVs are different according to the pathophysiological status of the disease. Therefore, EVs can be novel biomarkers for various lung diseases. EVs contain several molecules, including proteins, mRNA, microRNA, and DNA; they transfer these molecules to distant recipient cells. Circulating EVs modify the targeted cells and influence the microenvironment of the lungs. For this unique capability, EVs are expected to be a new drug delivery system and a novel therapeutic target. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  12. Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

    SciTech Connect

    Wild, G.; Bennett, G.

    1984-08-01

    Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.

  13. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    PubMed

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets.

  14. Model Docking Using Knowledge-Level Analysis

    NASA Astrophysics Data System (ADS)

    Trewhitt, Ethan; Whitaker, Elizabeth; Briscoe, Erica; Weiss, Lora

    This paper presents an initial approach for exploring the docking of social models at the knowledge level. We have prototyped a simple blackboard environment allowing for model docking experimentation. There are research challenges in identifying which models are appropriate to dock and the concepts that they should exchange to build a richer multi-scale view of the world. Our early approach includes docking of societal system dynamics models with individual and organizational behaviors represented in agent-based models. Case-based models allow exploration of historical knowledge by other models. Our research presents initial efforts to attain opportunistic, asynchronous interactions among multi-scale models through investigation and experimentation of knowledge-level model docking. A docked system can supply a multi-scale modeling capability to support a user's what-if analysis through combinations of case-based modeling, system dynamics approaches and agent-based models working together. An example is provided for the domain of terrorist recruiting.

  15. Ellipsoidal Relaxation of Deformed Vesicles

    NASA Astrophysics Data System (ADS)

    Yu, Miao; Lira, Rafael B.; Riske, Karin A.; Dimova, Rumiana; Lin, Hao

    2015-09-01

    Theoretical analysis and experimental quantification on the ellipsoidal relaxation of vesicles are presented. The current work reveals the simplicity and universal aspects of this process. The Helfrich formula is shown to apply to the dynamic relaxation of moderate-to-high tension membranes, and a closed-form solution is derived which predicts the vesicle aspect ratio as a function of time. Scattered data are unified by a time scale, which leads to a similarity behavior, governed by a distinctive solution for each vesicle type. Two separate regimes in the relaxation are identified, namely, the "entropic" and the "constant-tension" regimes. The bending rigidity and the initial membrane tension can be simultaneously extracted from the data analysis, posing the current approach as an effective means for the mechanical analysis of biomembranes.

  16. Scoring docking conformations using predicted protein interfaces

    PubMed Central

    2014-01-01

    Background Since proteins function by interacting with other molecules, analysis of protein-protein interactions is essential for comprehending biological processes. Whereas understanding of atomic interactions within a complex is especially useful for drug design, limitations of experimental techniques have restricted their practical use. Despite progress in docking predictions, there is still room for improvement. In this study, we contribute to this topic by proposing T-PioDock, a framework for detection of a native-like docked complex 3D structure. T-PioDock supports the identification of near-native conformations from 3D models that docking software produced by scoring those models using binding interfaces predicted by the interface predictor, Template based Protein Interface Prediction (T-PIP). Results First, exhaustive evaluation of interface predictors demonstrates that T-PIP, whose predictions are customised to target complexity, is a state-of-the-art method. Second, comparative study between T-PioDock and other state-of-the-art scoring methods establishes T-PioDock as the best performing approach. Moreover, there is good correlation between T-PioDock performance and quality of docking models, which suggests that progress in docking will lead to even better results at recognising near-native conformations. Conclusion Accurate identification of near-native conformations remains a challenging task. Although availability of 3D complexes will benefit from template-based methods such as T-PioDock, we have identified specific limitations which need to be addressed. First, docking software are still not able to produce native like models for every target. Second, current interface predictors do not explicitly consider pairwise residue interactions between proteins and their interacting partners which leaves ambiguity when assessing quality of complex conformations. PMID:24906633

  17. Synchronized Flashing Lights For Approach And Docking

    NASA Technical Reports Server (NTRS)

    Book, Michael L.; Howard, Richard T.; Bryan, Thomas C.; Bell, Joseph L.

    1994-01-01

    Proposed optoelectronic system for guiding vehicle in approaching and docking with another vehicle includes active optical targets (flashing lights) on approached vehicle synchronized with sensor and image-processing circuitry on approaching vehicle. Conceived for use in automated approach and docking of two spacecraft. Also applicable on Earth to manually controlled and automated approach and docking of land vehicles, aircraft, boats, and submersible vehicles, using GPS or terrestrial broadcast time signals for synchronization. Principal advantage: optical power reduced, with consequent enhancement of safety.

  18. Development of robotics facility docking test hardware

    NASA Technical Reports Server (NTRS)

    Loughead, T. E.; Winkler, R. V.

    1984-01-01

    Design and fabricate test hardware for NASA's George C. Marshall Space Flight Center (MSFC) are reported. A docking device conceptually developed was fabricated, and two docking targets which provide high and low mass docking loads were required and were represented by an aft 61.0 cm section of a Hubble space telescope (ST) mockup and an upgrading of an existing multimission modular spacecraft (MSS) mockup respectively. A test plan is developed for testing the hardware.

  19. Synchronized Flashing Lights For Approach And Docking

    NASA Technical Reports Server (NTRS)

    Book, Michael L.; Howard, Richard T.; Bryan, Thomas C.; Bell, Joseph L.

    1994-01-01

    Proposed optoelectronic system for guiding vehicle in approaching and docking with another vehicle includes active optical targets (flashing lights) on approached vehicle synchronized with sensor and image-processing circuitry on approaching vehicle. Conceived for use in automated approach and docking of two spacecraft. Also applicable on Earth to manually controlled and automated approach and docking of land vehicles, aircraft, boats, and submersible vehicles, using GPS or terrestrial broadcast time signals for synchronization. Principal advantage: optical power reduced, with consequent enhancement of safety.

  20. Laser Docking System Radar flight experiment

    NASA Technical Reports Server (NTRS)

    Erwin, Harry O.

    1986-01-01

    Flight experiments to verify the Laser Docking System Radar are discussed. The docking requirements are summarized, and the breadboarded hardware is described, emphasizing the two major scanning concepts being utilized: a mechanical scanning technique employing galvanometer beamsteerers and an electronic scanning technique using an image dissector. The software simulations used to apply hardware solutions to the docking requirements are briefly discussed, the tracking test bed is described, and the objectives of the flight experiment are reviewed.

  1. Progress 48P approaches for docking

    NASA Image and Video Library

    2012-10-31

    ISS033-E-017605 (31 Oct. 2012) --- Russian cosmonauts Yuri Malenchenko (foreground) and Oleg Novitskiy, both Expedition 33 flight engineers, monitor data at the manual TORU docking system controls in the Zvezda Service Module of the International Space Station during approach and docking operations of the unpiloted ISS Progress 49 resupply vehicle. Progress 49 docked automatically to Zvezda’s aft port at 9:33 a.m. (EDT) on Oct. 31, 2012.

  2. Progress 48P approaches for docking

    NASA Image and Video Library

    2012-10-31

    ISS033-E-017632 (31 Oct. 2012) --- Russian cosmonauts Yuri Malenchenko (foreground) and Oleg Novitskiy, both Expedition 33 flight engineers, monitor data at the manual TORU docking system controls in the Zvezda Service Module of the International Space Station during approach and docking operations of the unpiloted ISS Progress 49 resupply vehicle. Progress 49 docked automatically to Zvezda’s aft port at 9:33 a.m. (EDT) on Oct. 31, 2012.

  3. Progress 48P approaches for docking

    NASA Image and Video Library

    2012-10-31

    ISS033-E-016948 (31 Oct. 2012) --- Russian cosmonauts Yuri Malenchenko and Oleg Novitskiy, both Expedition 33 flight engineers, monitor data at the manual TORU docking system controls in the Zvezda Service Module of the International Space Station during approach and docking operations of the unpiloted ISS Progress 49 resupply vehicle. Progress 49 docked automatically to Zvezda’s aft port at 9:33 a.m. (EDT) on Oct. 31, 2012. NASA astronaut Sunita Williams, commander, is visible at top left.

  4. Neurotransmitter Release: The Last Millisecond in the Life of a Synaptic Vesicle

    PubMed Central

    Südhof, Thomas C.

    2013-01-01

    During an action potential, Ca2+ entering a presynaptic terminal triggers synaptic vesicle exocytosis and neurotransmitter release in less than a millisecond. How does Ca2+ stimulate release so rapidly and precisely? Work over the last decades revealed that Ca2+-binding to synaptotagmin triggers release by stimulating synaptotagmin-binding to a core machinery composed of SNARE and SM proteins that mediates membrane fusion during exocytosis. Complexin adaptor proteins assist synaptotagmin by activating and clamping this core fusion machinery. Synaptic vesicles containing synaptotagmin are positioned at the active zone, the site of vesicle fusion, by a protein complex containing RIM proteins. RIM proteins simultaneously activate docking and priming of synaptic vesicles and recruit Ca2+-channels to active zones, thereby connecting in a single complex primed synaptic vesicles to Ca2+-channels. This architecture allows direct flow of Ca2+-ions from Ca2+-channels to synaptotagmin, which then triggers fusion, thus mediating tight millisecond coupling of an action potential to neurotransmitter release. PMID:24183019

  5. Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells

    PubMed Central

    Hosaka, Masahiro; Watanabe, Tsuyoshi; Sakai, Yuko; Uchiyama, Yasuo; Takeuchi, Toshiyuki

    2002-01-01

    Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca2+-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48–111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214–373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells. PMID:12388744

  6. The Role of Secretory Autophagy in Zika Virus Transfer through the Placental Barrier

    PubMed Central

    Zhang, Zhong-Wei; Li, Zi-Lin; Yuan, Shu

    2017-01-01

    Recent studies indicated that the Zika virus genome could be detected in the amniotic fluid and the fetal brain, which confirms that the virus can cross the placental barrier. Secretory autophagy or exosome pathways may participate in this virus transfer. Autophagy modulators regulate autophagosome formation or membrane fusion with lysosomal vesicles and therefore inhibit viral nucleocapsid releasing or virus transfer to the fetus hypothetically. However, some autophagy modulators may enhance virus replication. Autophagy inhibitors may arrest placental development; while exaggeration of autophagy in human placenta may be associated with the fetal growth restriction. Therefore, autophagy modulators should be used carefully due to their complex clinical effects. Alternatively, exosome-specific inhibitors might be also considered, although their safety of both maternal and fetal conditions must be carefully assessed before any advancement to human clinical trials. PMID:28119857

  7. Machine learning optimization of cross docking accuracy.

    PubMed

    Bjerrum, Esben J

    2016-06-01

    Performance of small molecule automated docking programs has conceptually been divided into docking -, scoring -, ranking - and screening power, which focuses on the crystal pose prediction, affinity prediction, ligand ranking and database screening capabilities of the docking program, respectively. Benchmarks show that different docking programs can excel in individual benchmarks which suggests that the scoring function employed by the programs can be optimized for a particular task. Here the scoring function of Smina is re-optimized towards enhancing the docking power using a supervised machine learning approach and a manually curated database of ligands and cross docking receptor pairs. The optimization method does not need associated binding data for the receptor-ligand examples used in the data set and works with small train sets. The re-optimization of the weights for the scoring function results in a similar docking performance with regard to docking power towards a cross docking test set. A ligand decoy based benchmark indicates a better discrimination between poses with high and low RMSD. The reported parameters for Smina are compatible with Autodock Vina and represent ready-to-use alternative parameters for researchers who aim at pose prediction rather than affinity prediction. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Soyuz and Progress docked on ISS

    NASA Image and Video Library

    2014-05-29

    ISS040-E-005999 (29 May 2014) --- Two Russian spacecraft docked to the International Space Station are featured in this image photographed by an Expedition 40 crew member in the International Space Station’s Cupola. The Soyuz 39 (TMA-13M) spacecraft, docked to the Rassvet Mini-Research Module 1 (MRM1), is visible in the foreground. The Progress 55 resupply vehicle, docked to the Pirs Docking Compartment, is visible in the background. A blue and white part of Earth and the blackness of space provide the backdrop for the scene.

  9. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements

    PubMed Central

    Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E.

    2015-01-01

    Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

  10. Lipid transfer between vesicles: effect of high vesicle concentration.

    PubMed Central

    Almeida, P F

    1999-01-01

    The problem of the desorption of a lipid molecule from a lipid vesicle (donor) and its incorporation into another vesicle (acceptor) at high acceptor concentrations, which has been investigated experimentally (Jones, J. D. and Thompson, T. E., 1990. Biochemistry, 29:1593-1600), is analyzed here from a theoretical point of view, formulated in terms of the diffusion equation with appropriate boundary conditions. The goal is to determine whether or not the observed acceleration of the off-rate from a donor is caused by interaction with an acceptor vesicle at short range, or is simply the result of statistical effects due the proximity of the acceptor and its influence on the probability of the test lipid returning to the donor. We establish a correspondence between the theoretical parameters and the experimental, thermodynamic and dynamic variables entering the problem. The solution shows that, because of the extremely high Gibbs activation energy for desorption of a phospholipid, the process would always be first-order, even at very high vesicle concentrations. This means that acceleration of the off-rate must be due to donor-acceptor interactions at short distances, as proposed in the experimental work. PMID:10096890

  11. LigDockCSA: protein-ligand docking using conformational space annealing.

    PubMed

    Shin, Woong-Hee; Heo, Lim; Lee, Juyong; Ko, Junsu; Seok, Chaok; Lee, Jooyoung

    2011-11-30

    Protein-ligand docking techniques are one of the essential tools for structure-based drug design. Two major components of a successful docking program are an efficient search method and an accurate scoring function. In this work, a new docking method called LigDockCSA is developed by using a powerful global optimization technique, conformational space annealing (CSA), and a scoring function that combines the AutoDock energy and the piecewise linear potential (PLP) torsion energy. It is shown that the CSA search method can find lower energy binding poses than the Lamarckian genetic algorithm of AutoDock. However, lower-energy solutions CSA produced with the AutoDock energy were often less native-like. The loophole in the AutoDock energy was fixed by adding a torsional energy term, and the CSA search on the refined energy function is shown to improve the docking performance. The performance of LigDockCSA was tested on the Astex diverse set which consists of 85 protein-ligand complexes. LigDockCSA finds the best scoring poses within 2 Å root-mean-square deviation (RMSD) from the native structures for 84.7% of the test cases, compared to 81.7% for AutoDock and 80.5% for GOLD. The results improve further to 89.4% by incorporating the conformational entropy.

  12. Numbers of presynaptic Ca(2+) channel clusters match those of functionally defined vesicular docking sites in single central synapses.

    PubMed

    Miki, Takafumi; Kaufmann, Walter A; Malagon, Gerardo; Gomez, Laura; Tabuchi, Katsuhiko; Watanabe, Masahiko; Shigemoto, Ryuichi; Marty, Alain

    2017-06-27

    Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such "simple synapses" indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Cav2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Cav2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1-10) and 2.03 at 4 wk (range: 1-4), whereas the mean numbers of Cav2.1 clusters were 2.84 at 2 wk (range: 1-8) and 2.37 at 4 wk (range: 1-5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 μm(2) to 0.0234 μm(2)), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels.

  13. Expedition 32 Docking with ISS

    NASA Image and Video Library

    2012-07-17

    Expedition 32 Flight Engineer Sunita Williams’ sister and friend brought a photo of William’s dog “Gorby” in support of her arrival to the International Space Station on Tuesday, July 17, 2012 at the Russian Mission Control Center in Korolev, Russia. The Soyuz docked to the International Space Station with Williams and fellow crew members Soyuz Commander Yuri Malenchenko and JAXA Flight Engineer Akihiko Hoshide two days after they launched from the Baikonur Cosmodrome in Kazakhstan. Photo Credit: (NASA/Carla Cioffi)

  14. Expedition 40 Post Docking Statement

    NASA Image and Video Library

    2014-05-29

    William Gerstenmaier, NASA Associate Administrator for Human Exploration and Operations, speaks during a joint statement with Jean-Jacques Dordain, Director General of the European Space Agency, and Oleg Ostapenko, General Director of the Russian Federal Space Agency, Roscosmos, following the docking of the Soyuz TMA-13M spacecraft with the International Space Station on Thursday, May 29, 2014 at the Baikonur Hotel in Baikonur, Kazakhstan. The three spoke on the importance of international cooperation in human spaceflight. Photo Credit: (NASA/Joel Kowsky)

  15. Expedition 40 Post Docking Statement

    NASA Image and Video Library

    2014-05-29

    Jean-Jacques Dordain, Director General of the European Space Agency, left, Oleg Ostapenko, General Director of the Russian Federal Space Agency, Roscosmos, center, and William Gerstenmaier, NASA Associate Administrator for Human Exploration and Operations, right, are seen during a joint statement after the docking of the Soyuz TMA-13M spacecraft with the International Space Station on Thursday, May 29, 2014 at the Baikonur Hotel in Baikonur, Kazakhstan. The three spoke on the importance of international cooperation in human spaceflight. Photo Credit: (NASA/Joel Kowsky)

  16. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence.

    PubMed Central

    Rubartelli, A; Cozzolino, F; Talio, M; Sitia, R

    1990-01-01

    Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2328723

  17. Myosin 2 Maintains an Open Exocytic Fusion Pore in Secretory Epithelial Cells

    PubMed Central

    Bhat, Purnima

    2009-01-01

    Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (−)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (−)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening. PMID:19158378

  18. Spontaneous charged lipid transfer between lipid vesicles.

    PubMed

    Richens, Joanna L; Tyler, Arwen I I; Barriga, Hanna M G; Bramble, Jonathan P; Law, Robert V; Brooks, Nicholas J; Seddon, John M; Ces, Oscar; O'Shea, Paul

    2017-10-03

    An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

  19. Synaptic vesicle distribution by conveyor belt.

    PubMed

    Moughamian, Armen J; Holzbaur, Erika L F

    2012-03-02

    The equal distribution of synaptic vesicles among synapses along the axon is critical for robust neurotransmission. Wong et al. show that the continuous circulation of synaptic vesicles throughout the axon driven by molecular motors ultimately yields this even distribution.

  20. Purified Kinesin Promotes Vesicle Motility and Induces Active Sliding Between Microtubules In vitro

    NASA Astrophysics Data System (ADS)

    Urrutia, Raul; McNiven, Mark A.; Albanesi, Joseph P.; Murphy, Douglas B.; Kachar, Bechara

    1991-08-01

    We examined the ability of kinesin to support the movement of adrenal medullary chromaffin granules on microtubules in a defined in vitro system. We found that kinesin and ATP are all that is required to support efficient (33% vesicle motility) and rapid (0.4-0.6 μ m/s) translocation of secretory granule membranes on microtubules in the presence of a low-salt motility buffer. Kinesin also induced the formation of microtubule asters in this buffer, with the plus ends of microtubules located at the center of each aster. This observation indicates that kinesin is capable of promoting active sliding between microtubules toward their respective plus ends, a movement analogous to that of anaphase b in the mitotic spindle. The fact that vesicle translocation, microtubule sliding, and microtubule-dependent kinesin ATPase activities are all enhanced in low-salt buffer establishes a functional parallel between this translocator and other motility ATPases, myosin, and dynein.

  1. How cancer cells dictate their microenvironment: present roles of extracellular vesicles.

    PubMed

    Naito, Yutaka; Yoshioka, Yusuke; Yamamoto, Yusuke; Ochiya, Takahiro

    2017-02-01

    Intercellular communication plays an important role in cancer initiation and progression through secretory molecules, including growth factors and cytokines. Recent advances have revealed that small membrane vesicles, termed extracellular vesicles (EVs), served as a regulatory agent in the intercellular communication of cancer. EVs enable the transfer of functional molecules, including proteins, mRNA and microRNAs (miRNAs), into recipient cells. Cancer cells utilize EVs to dictate the unique phenotype of surrounding cells, thereby promoting cancer progression. Against such "education" by cancer cells, non-tumoral cells suppress cancer initiation and progression via EVs. Therefore, researchers consider EVs to be important cues to clarify the molecular mechanisms of cancer biology. Understanding the functions of EVs in cancer progression is an important aspect of cancer biology that has not been previously elucidated. In this review, we summarize experimental data that indicate the pivotal roles of EVs in cancer progression.

  2. Influence of experimental hypokinesia on gastric secretory function

    NASA Technical Reports Server (NTRS)

    Markova, O. O.; Vavryshchuk, V. I.; Rozvodovskyy, V. I.; Proshcheruk, V. A.

    1980-01-01

    The gastric secretory function of rats was studied in 4, 8, 16 and 30 day hypokinesia. Inhibition of both the gastric juice secretory and acid producing functions was found. The greatest inhibition was observed on day 8 of limited mobility. By days 16 and 30 of the experiment, a tendency of the gastric secretory activity to return to normal was observed, although it remained reduced.

  3. Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition

    PubMed Central

    Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

    2016-01-01

    Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition. PMID:27882938

  4. Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants.

    PubMed

    Avaro, Sandrine; Belgareh-Touzé, Naïma; Sibella-Argüelles, Carla; Volland, Christiane; Haguenauer-Tsapis, Rosine

    2002-03-15

    We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.

  5. The electrostatic basis for the interfacial binding of secretory phospholipases A2.

    PubMed Central

    Scott, D L; Mandel, A M; Sigler, P B; Honig, B

    1994-01-01

    Biochemical and structural data suggest that electrostatic forces play a critical role in the binding of secretory phospholipases A2 to substrate aggregates (micelles, vesicles, monolayers, and membranes). This initial binding (adsorption) of the enzyme to the interface is kinetically distinct from the subsequent binding of substrate to the buried active site. Thus, in the absence of specific active-site interactions, electrostatic forces operating at the molecular surface may orient and hold the enzyme at the interface. We have calculated the electrostatic potentials for 10 species of secretory phospholipases A2 whose atomic coordinates have been determined by x-ray crystallography. Most of these enzymes show a marked electrostatic sidedness that is accentuated to a variable degree by the presence of the essential cofactor calcium ion. This asymmetry suggests a discrete interfacial binding region on the protein's surface, the location of which is in general agreement with proposals derived from the results of chemical modification, mutational, and crystallographic experiments. Images FIGURE 2 FIGURE 3 FIGURE 3 FIGURE 3 FIGURE 4 FIGURE 4 FIGURE 5 FIGURE 5 PMID:7948668

  6. Mechanisms of COPI vesicle formation

    PubMed Central

    Hsu, Victor W.; Yang, Jia-Shu

    2009-01-01

    Coat Protein I (COPI) is one of the most intensely investigated coat complexes. Numerous studies have contributed to a general understanding of how coat proteins act to initiate intracellular vesicular transport. This review highlights key recent findings that have shaped our current understanding of how COPI vesicles are formed. PMID:19854177

  7. 5. Top surface of dock showing indented section (bay) on ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. Top surface of dock showing indented section (bay) on SW side; looking NW. Ferry in background is at Winslow ferry dock. - Pacific Creosoting Plant, West Dock, 5350 Creosote Place, Northeast, Bremerton, Kitsap County, WA

  8. Calcium mediation of the pig jejunal secretory response.

    PubMed Central

    Forsyth, G W; Wong, P H; Maenz, D D

    1985-01-01

    The involvement of Ca++ ions as secretory mediators in pig jejunal epithelia has been investigated with an in vitro system. Omission of Ca++ from the Ringer-HCO3 bathing media on both sides of the tissue had minor effects on the basal electrical activity of pig jejunal mucosa. There were only slight decreases in transepithelial potential difference and increases in conductance with Ca++ free media. Low EGTA concentrations which reversibly blocked potential difference responses to secretory agents also had minimal effects on basal electrical activity. The in vitro secretory responses to A23187, to theophylline, and to Escherichia coli heat-stable enterotoxin were all eliminated by Ca++ depletion and restored by replacing normal Ca++ concentrations in the bathing media. Dantrolene prevented the secretory response but not the potential difference increases caused by heat-stable enterotoxin and A23187, suggesting that intracellular Ca++ stores may be reservoirs of secretory signal agent. Verapamil only blocked the secretory response to heat-stable enterotoxin. Chlorpromazine had negligible effects on basal conditions, but totally blocked both the secretory response and the Ca++-dependent effects of A23187 and heat-stable enterotoxin on potential difference. The response to theophylline was only partially inhibited by chlorpromazine, implying some involvement of both cAMP and Ca++ as secretory signals for theophylline. Cytoplasmic Ca++ concentrations appear to be at least as important as cyclic nucleotides in regulating the secretory effects of pig jejunum. PMID:2410089

  9. Rab proteins: the key regulators of intracellular vesicle transport.

    PubMed

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  11. Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

    PubMed Central

    Mukherjee, Debarati; Sen, Arpita; Boettner, Douglas R.; Fairn, Gregory D.; Schlam, Daniel; Bonilla Valentin, Fernando J.; Michael McCaffery, J.; Hazbun, Tony; Staiger, Chris J.; Grinstein, Sergio; Lemmon, Sandra K.; Claudio Aguilar, R.

    2013-01-01

    Summary Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking. PMID:23943876

  12. Quantitative subcellular study of apical pole membranes from chicken oxyntic cells in resting and HCl secretory state.

    PubMed

    Koenig, C S; Dabiké, M; Bronfman, M

    1987-12-01

    Vertebrate oxyntic cells, responsible for gastric HCl production, undergo a remarkable morphological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular membranes, the tubular system, occupies the luminal cytoplasm. Actin filaments frame a cortical network between the tubular system and the luminal plasma membrane. With the onset of HCl secretion, the tubular system becomes incorporated into the luminal plasma membrane. Villous processes containing microfilaments fill the secretory surface. This morphological reorganization of membranes and cytoskeletal matrix could regulate HCl secretion by translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface. In this paper, we describe the isolation of membranes that selectively belong to the tubular system or to the cytoplasmic processes of the secretory surface of chicken oxyntic cells. Chicken oxyntic cells are the main cellular component of the proventricular glands. A resting state was obtained after cimetidine treatment, whereas the HCl-secretory state was induced by histamine. We present a comparative analysis of resting and stimulated chicken gastric glands by quantitative subcellular fractionation. The HCl secretory state was related to specific modifications in membrane fractions derived from the secretory pole of oxyntic cells. Morphological and functional reorganization of oxyntic cells was closely correlated with changes in: the sedimentation pattern of the marker enzyme of the apical pole membrane (K-NPPase), the total activity of K-NPPase and nonmitochondrial Mg-ATPase, the valinomycin dependence of K-ATPase, and polypeptides that cosediment in purified membrane fractions. Changes in the distribution pattern of K-NPPase after fractionation of histamine-stimulated glands were consistent with the replacement of the small vesicles typical of resting glands by

  13. Morphology of nematic and smectic vesicles

    PubMed Central

    Xing, Xiangjun; Shin, Homin; Bowick, Mark J.; Yao, Zhenwei; Jia, Lin; Li, Min-Hui

    2012-01-01

    Recent experiments on vesicles formed from block copolymers with liquid-crystalline side chains reveal a rich variety of vesicle morphologies. The additional internal order (“structure”) developed by these self-assembled block copolymer vesicles can lead to significantly deformed vesicles as a result of the delicate interplay between two-dimensional ordering and vesicle shape. The inevitable topological defects in structured vesicles of spherical topology also play an essential role in controlling the final vesicle morphology. Here we develop a minimal theoretical model for the morphology of the membrane structure with internal nematic/smectic order. Using both analytic and numerical approaches, we show that the possible low free energy morphologies include nano-size cylindrical micelles (nano-fibers), faceted tetrahedral vesicles, and ellipsoidal vesicles, as well as cylindrical vesicles. The tetrahedral vesicle is a particularly fascinating example of a faceted liquid-crystalline membrane. Faceted liquid vesicles may lead to the design of supramolecular structures with tetrahedral symmetry and new classes of nano-carriers. PMID:22431595

  14. Secretory protein decondensation as a distinct, Ca2+-mediated event during the final steps of exocytosis in Paramecium cells

    PubMed Central

    1981-01-01

    The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed. PMID:7204486

  15. Intermolecular domain docking in the hairpin ribozyme

    PubMed Central

    Sumita, Minako; White, Neil A.; Julien, Kristine R.; Hoogstraten, Charles G.

    2013-01-01

    The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH3)63+. RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a “double conformational capture” model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding. PMID:23324606

  16. New docking target taped to middeck locker

    NASA Image and Video Library

    1996-09-20

    STS79-E-5104 (20 September 1996) --- The STS-79 crew members removed the docking target from the Docking Module (DM) and attached it to a locker door to photograph it and examine a slight peel on the surface, during Flight Day 5.

  17. GalaxyDock BP2 score: a hybrid scoring function for accurate protein-ligand docking

    NASA Astrophysics Data System (ADS)

    Baek, Minkyung; Shin, Woong-Hee; Chung, Hwan Won; Seok, Chaok

    2017-07-01

    Protein-ligand docking is a useful tool for providing atomic-level understanding of protein functions in nature and design principles for artificial ligands or proteins with desired properties. The ability to identify the true binding pose of a ligand to a target protein among numerous possible candidate poses is an essential requirement for successful protein-ligand docking. Many previously developed docking scoring functions were trained to reproduce experimental binding affinities and were also used for scoring binding poses. However, in this study, we developed a new docking scoring function, called GalaxyDock BP2 Score, by directly training the scoring power of binding poses. This function is a hybrid of physics-based, empirical, and knowledge-based score terms that are balanced to strengthen the advantages of each component. The performance of the new scoring function exhibits significant improvement over existing scoring functions in decoy pose discrimination tests. In addition, when the score is used with the GalaxyDock2 protein-ligand docking program, it outperformed other state-of-the-art docking programs in docking tests on the Astex diverse set, the Cross2009 benchmark set, and the Astex non-native set. GalaxyDock BP2 Score and GalaxyDock2 with this score are freely available at http://galaxy.seoklab.org/softwares/galaxydock.html.

  18. Computational protein-ligand docking and virtual drug screening with the AutoDock suite

    PubMed Central

    Forli, Stefano; Huey, Ruth; Pique, Michael E.; Sanner, Michel; Goodsell, David S.; Olson, Arthur J.

    2016-01-01

    Computational docking can be used to predict bound conformations and free energies of binding for small molecule ligands to macromolecular targets. Docking is widely used for the study of biomolecular interactions and mechanisms, and is applied to structure-based drug design. The methods are fast enough to allow virtual screening of ligand libraries containing tens of thousa