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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. Secretory vesicles transiently dock and fuse at the porosome to discharge contents during cell secretion.

    PubMed

    Jena, Bhanu P

    2009-12-16

    In contrast with the observation in electron micrographs of partially empty vesicles in cells following secretion, it has been believed since the 1950s that during cell secretion, secretory vesicles completely merge at the cell plasma membrane, resulting in the diffusion of intravesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. In the interim, a large body of work has been published arguing both for and against the complete merger of secretory vesicle membrane at the cell plasma membrane during secretion. The only definitive determination of the mechanism of cell secretion remained in its direct observation at nanometre resolution in live cells. In the past decade, this finally became a reality through the power and scope of the atomic force microscope, which has made it possible to resolve a major conundrum in cell biology. This paradigm shift in our understanding of cell secretion is briefly outlined here.

  4. 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

  5. Secretory vesicle swelling by atomic force microscopy.

    PubMed

    Cho, Sang-Joon; Jena, Bhanu P

    2006-01-01

    The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remained unknown. Earlier studies from our laboratory demonstrated the association of the alpha-subunit of heterotrimeric GTP-binding protein G(alphai3) with zymogen granule membrane and implicated its involvement in vesicle swelling. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and found to be KCl sensitive. However, Ca2+ had no effect on zymogen granule size. Taken together, these results indicated that zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G(alphai3) protein. Subsequently, our studies demonstrated that the water channel aquaporin-1 (AQP1) is also present at the zymogen granule membrane and participates in rapid GTP-induced and G(alphai3)-mediated vesicular water gating and swelling. Isolated zymogen granules exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of zymogen granules with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxy-terminal domain of AQP1 blocked GTP-stimulable swelling of vesicles. Our results demonstrate that AQPI associated at the zymogen granule membrane is involved in basal GTP-induced and G(alphai3)-mediated rapid gating of water into zymogen granules of the exocrine pancreas.

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

    PubMed

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

    2008-01-01

    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.

  7. 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.

  8. Neurexin-1α contributes to insulin-containing secretory granule docking.

    PubMed

    Mosedale, Merrie; Egodage, Sonya; Calma, Rei C; Chi, Nai-Wen; Chessler, Steven D

    2012-02-24

    Neurexins are a family of transmembrane, synaptic adhesion molecules. In neurons, neurexins bind to both sub-plasma membrane and synaptic vesicle-associated constituents of the secretory machinery, play a key role in the organization and stabilization of the presynaptic active zone, and help mediate docking of synaptic vesicles. We have previously shown that neurexins, like many other protein constituents of the neurotransmitter exocytotic machinery, are expressed in pancreatic β cells. We hypothesized that the role of neurexins in β cells parallels their role in neurons, with β-cell neurexins helping to mediate insulin granule docking and secretion. Here we demonstrate that β cells express a more restricted pattern of neurexin transcripts than neurons, with a clear predominance of neurexin-1α expressed in isolated islets. Using INS-1E β cells, we found that neurexin-1α interacts with membrane-bound components of the secretory granule-docking machinery and with the granule-associated protein granuphilin. Decreased expression of neurexin-1α, like decreased expression of granuphilin, reduces granule docking at the β-cell membrane and improves insulin secretion. Perifusion of neurexin-1α KO mouse islets revealed a significant increase in second-phase insulin secretion with a trend toward increased first-phase secretion. Upon glucose stimulation, neurexin-1α protein levels decrease. This glucose-induced down-regulation may enhance glucose-stimulated insulin secretion. We conclude that neurexin-1α is a component of the β-cell secretory machinery and contributes to secretory granule docking, most likely through interactions with granuphilin. Neurexin-1α is the only transmembrane component of the docking machinery identified thus far. Our findings provide new insights into the mechanisms of insulin granule docking and exocytosis.

  9. 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

  10. Potential of yeast secretory vesicles in biodelivery systems.

    PubMed

    Kutralam-Muniasamy, Gurusamy; Flores-Cotera, Luis B; Perez-Guevara, Fermin

    2015-06-01

    Membranous vesicular organelles (MVOs), such as secretory vesicles and exosomes, perform a variety of biological functions ranging from secretion to cellular communication in eukaryotic cells. Exosomes, particularly those of mammalian cells, have been widely studied as potential carriers in human therapeutic applications. However, no study has yet demonstrated the use of yeast secretory vesicles for such applications. Therefore, we explore here the current state of knowledge on yeast secretory vesicles and their potential use in therapeutic delivery systems. We focus on the characteristics shared by exosomes and yeast secretory vesicles to provide insights into the use of the latter as delivery vehicles. From this perspective, we speculate on the potential application of post-Golgi vesicles (PGVs) in the biomedical field. PMID:25843637

  11. 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

  12. 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

  13. Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 β-CellsV⃞

    PubMed Central

    Varadi, Aniko; Tsuboi, Takashi; Rutter, Guy A.

    2005-01-01

    The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic β-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by ∼50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in β-cells. PMID:15788565

  14. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    PubMed

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

    2016-08-17

    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

  15. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    PubMed

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

    2016-08-17

    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.

  16. 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

  17. Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

    PubMed Central

    Mitchell, Kathryn J.; Pinton, Paolo; Varadi, Aniko; Tacchetti, Carlo; Ainscow, Edward K.; Pozzan, Tullio; Rizzuto, Rosario; Rutter, Guy A.

    2001-01-01

    The role of dense core secretory vesicles in the control of cytosolic-free Ca2+ concentrations ([Ca2+]c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet β-cells: (a) increases in [Ca2+]c cause a prompt increase in intravesicular-free Ca2+ concentration ([Ca2+]SV), which is mediated by a P-type Ca2+-ATPase distinct from the sarco(endo) plasmic reticulum Ca2+-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2+ pumps; (b) steady state Ca2+ concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca2+; (c) inositol (1,4,5) trisphosphate has no impact on [Ca2+]SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca2+]SV. Thus, secretory vesicles represent a dynamic Ca2+ store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca2+-induced Ca2+ release from vesicles docked at the plasma membrane could participate in triggering exocytosis. PMID:11571310

  18. Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

    PubMed

    Mitchell, K J; Pinton, P; Varadi, A; Tacchetti, C; Ainscow, E K; Pozzan, T; Rizzuto, R; Rutter, G A

    2001-10-01

    The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

  19. 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

  20. Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking

    PubMed Central

    VanRheenen, Susan M.; Cao, Xiaochun; Lupashin, Vladimir V.; Barlowe, Charles; Gerard Waters, M.

    1998-01-01

    SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex. PMID:9606204

  1. 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. PMID:24489879

  2. 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

  3. 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

  4. 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

  5. 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

  6. Intracellular calcium signalling in rat parotid acinar cells that lack secretory vesicles.

    PubMed Central

    Liu, P; Scott, J; Smith, P M

    1998-01-01

    Secretory vesicles from pancreatic acinar cells have recently been shown to release Ca2+ after stimulation with Ins(1,4,5)P3 [Gerasimenko, Gerasimenko, Belan and Petersen, (1996) Cell 84, 473-480]. These observations have been used in support of the hypothesis that Ca2+ release from secretory vesicles could be an important component of stimulus secretion coupling in exocrine acinar cells. In the rat, ligation of the parotid duct causes a reversible atrophy of the parotid gland. Most notably, after atrophy the acinar cells are reduced in size and no longer contain secretory vesicles [Liu, Smith, and Scott (1996) J. Dent. Res. 74, 900]. We have measured cytosolic free-Ca2+ concentration ([Ca2+]i) in single, acutely isolated, rat parotid acinar cells, and compared Ca2+ mobilization in response to acetylcholine (ACh) stimulation in cells obtained from control animals to that in cells lacking secretory vesicles obtained after atrophy of the parotid gland. Application of 50-5000 nM ACh to control cells gave rise to a typical, dose-dependent, biphasic increase in [Ca2+]i, of which the later, plateau, phase was acutely dependent on the extracellular Ca2+ concentration. An identical pattern of response was observed with cells obtained from atrophic glands. Low concentrations of ACh (10-100 nM) occasionally produced [Ca2+]i oscillations of a similar pattern in cells from both control and atrophic glands. We were able to show that Ca2+ rises first in the apical pole of the cell and the increase then spreads to the rest of the cell in cells from control glands but not in cells from atrophic glands. However, at present we are unable to determine whether this is due to the lack of secretory vesicles or whether the separation is too small to measure in the smaller acinar cells obtained from atrophic glands. We conclude therefore, that secretory vesicles make no significant contribution to overall Ca2+ mobilization in rat parotid acinar cells, nor are they required for oscillatory

  7. Unique biological function of cathepsin L in secretory vesicles for biosynthesis of neuropeptides.

    PubMed

    Funkelstein, Lydiane; Beinfeld, Margery; Minokadeh, Ardalan; Zadina, James; Hook, Vivian

    2010-12-01

    Neuropeptides are essential for cell-cell communication in the nervous and neuroendocrine systems. Production of active neuropeptides requires proteolytic processing of proneuropeptide precursors in secretory vesicles that produce, store, and release neuropeptides that regulate physiological functions. This review describes recent findings indicating the prominent role of cathepsin L in secretory vesicles for production of neuropeptides from their protein precursors. The role of cathepsin L in neuropeptide production was discovered using the strategy of activity-based probes for proenkephalin-cleaving activity for identification of the enzyme protein by mass spectrometry. The novel role of cathepsin L in secretory vesicles for neuropeptide production has been demonstrated in vivo by cathepsin L gene knockout studies, cathepsin L gene expression in neuroendocrine cells, and notably, cathepsin L localization in neuropeptide-containing secretory vesicles. Cathepsin L is involved in producing opioid neuropeptides consisting of enkephalin, β-endorphin, and dynorphin, as well as in generating the POMC-derived peptide hormones ACTH and α-MSH. In addition, NPY, CCK, and catestatin neuropeptides utilize cathepsin L for their biosynthesis. The neuropeptide-synthesizing functions of cathepsin L represent its unique activity in secretory vesicles, which contrasts with its role in lysosomes. Interesting evaluations of protease gene knockout studies in mice that lack cathepsin L compared to those lacking PC1/3 and PC2 (PC, prohormone convertase) indicate the key role of cathepsin L in neuropeptide production. Therefore, dual cathepsin L and prohormone convertase protease pathways participate in neuropeptide production. Significantly, the recent new findings indicate cathepsin L as a novel 'proprotein convertase' for production of neuropeptides that mediate cell-cell communication in health and disease.

  8. Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process

    PubMed Central

    Donovan, Kirk W.

    2015-01-01

    Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion. PMID:26169352

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

    PubMed

    Cho, Sang-Joon; Sattar, A K M Abdus; Jeong, Eun-Hwan; Satchi, Mylan; Cho, Jin Ah; Dash, Sudhansu; Mayes, Mary Sue; Stromer, Marvin H; Jena, Bhanu P

    2002-04-01

    The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G(alpha)i3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid GTP-induced vesicular water gating and swelling. Isolated ZGs exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of ZGs with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulatable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxyl-terminal domain of AQP1 blocks GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the ZG membrane is involved in basal as well as GTP-induced rapid gating of water in ZGs of the exocrine pancreas.

  10. 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.

  11. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length.

    PubMed

    Schott, Daniel H; Collins, Ruth N; Bretscher, Anthony

    2002-01-01

    Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.

  12. 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

  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. Molecular mechanisms involved in secretory vesicle recruitment to the plasma membrane in beta-cells.

    PubMed

    Varadi, Aniko; Ainscow, E K; Allan, V J; Rutter, G A

    2002-04-01

    Glucose stimulates the release of insulin in part by activating the recruitment of secretory vesicles to the cell surface. While this movement is known to be microtubule-dependent, the molecular motors involved are undefined. Active kinesin was found to be essential for vesicle translocation in live beta-cells, since microinjection of cDNA encoding dominant-negative KHC(mut) (motor domain of kinesin heavy chain containing a Thr(93)-->Asn point mutation) blocked vesicular movements. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Thus, vesicles released during the first phase of insulin secretion exist largely within a translocation-independent pool. Kinesin-driven anterograde movement of vesicles is then necessary for the sustained (second phase) of insulin release. Kinesin may, therefore, represent a novel target for increases in intracellular ATP concentrations in response to elevated extracellular glucose and may be involved in the ATP-sensitive K+channel-independent stimulation of secretion by the sugar.

  15. Synaptic Vesicle Docking: Sphingosine Regulates Syntaxin1 Interaction with Munc18

    PubMed Central

    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. PMID:19390577

  16. Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression.

    PubMed

    Funkelstein, Lydiane; Toneff, Thomas; Hwang, Shin-Rong; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

    2008-07-01

    Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore, in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.

  17. UNC-31/CAPS docks and primes dense core vesicles in C. elegans neurons.

    PubMed

    Lin, Xian-Guang; Ming, Min; Chen, Mao-Rong; Niu, Wei-Pin; Zhang, Yong-Deng; Liu, Bei; Jiu, Ya-Ming; Yu, Jun-Wei; Xu, Tao; Wu, Zheng-Xing

    2010-07-01

    UNC-31 or its mammalian homologue, Ca(2+)-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca(2+)-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca(2+) level (pre-flash Ca(2+) was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca(2+) evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex. PMID:20515653

  18. Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

    PubMed Central

    Yasothornsrikul, Sukkid; Greenbaum, Doron; Medzihradszky, Katalin F.; Toneff, Thomas; Bundey, Richard; Miller, Ruthellen; Schilling, Birgit; Petermann, Ivonne; Dehnert, Jessica; Logvinova, Anna; Goldsmith, Paul; Neveu, John M.; Lane, William S.; Gibson, Bradford; Reinheckel, Thomas; Peters, Christoph; Bogyo, Matthew; Hook, Vivian

    2003-01-01

    Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of [Met]enkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormone-processing sites. Cellular studies showed the colocalization of cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L gene knockout mice, [Met]enkephalin levels in brain were reduced significantly; this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of [Met]enkephalin, an endogenous peptide neurotransmitter. PMID:12869695

  19. 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

  20. 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

  1. 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

  2. p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway.

    PubMed

    Nickel, W; Sohn, K; Bünning, C; Wieland, F T

    1997-10-14

    A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8-p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8-p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8-p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8-p23 fusion protein within the ER-Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi. PMID:9326620

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

    PubMed

    Hornick, C A; Hamilton, R L; Spaziani, E; Enders, G H; Havel, R J

    1985-05-01

    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.

  4. Pob1 ensures cylindrical cell shape by coupling two distinct rho signaling events during secretory vesicle targeting.

    PubMed

    Nakano, Kentaro; Toya, Mika; Yoneda, Aki; Asami, Yukiko; Yamashita, Akira; Kamasawa, Naomi; Osumi, Masako; Yamamoto, Masayuki

    2011-06-01

    Proper cell morphogenesis requires the co-ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1-664 has a curious lemon-like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin-polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon-like cell morphology similar to pob1-664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin-mediated vesicle transport and exocyst-mediated vesicle tethering during secretory vesicle targeting.

  5. Glucose metabolism and glutamate analog acutely alkalinize pH of insulin secretory vesicles of pancreatic beta-cells.

    PubMed

    Eto, Kazuhiro; Yamashita, Tokuyuki; Hirose, Kenzo; Tsubamoto, Yoshiharu; Ainscow, Edward K; Rutter, Guy A; Kimura, Satoshi; Noda, Mitsuhiko; Iino, Masamitsu; Kadowaki, Takashi

    2003-08-01

    We studied acute changes of secretory vesicle pH in pancreatic beta-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 +/- 0.10% at 149 +/- 16 s with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by approximately 0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+ influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethyl ester (GME), a plasma membrane-permeable analog of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM without changing cellular ATP content or cytosolic Ca2+ concentration ([Ca2+]). Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 +/- 0.19% at 38 +/- 2 s. These results indicated that the acutely alkalinizing effect of glucose on beta-cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may have potential relevance to the alteration of secretory function by glutamate.

  6. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  7. Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesicle-mediated import toward the plastids

    PubMed Central

    Baslam, Marouane; Oikawa, Kazusato; Kitajima-Koga, Aya; Kaneko, Kentaro; Mitsui, Toshiaki

    2016-01-01

    ABSTRACT The diversity of protein targeting pathways to plastids and their regulation in response to developmental and metabolic status is a key issue in the regulation of cellular function in plants. The general import pathways that target proteins into and across the plastid envelope with changes in gene expression are critical for plant development by regulating the response to physiological and metabolic changes within the cell. Glycoprotein targeting to complex plastids involves routing through the secretory pathway, among others. However, the mechanisms of trafficking via this system remain poorly understood. The present article discusses our results in site-specific N-glycosylation of nucleotide pyrophosphatase/phosphodiesterases (NPPs) glycoproteins and highlights protein delivery in Golgi/plastid pathway via the secretory pathway. Furthermore, we outline the hypotheses that explain the mechanism for importing vesicles trafficking with nucleus-encoded proteins into plastids. PMID:27700755

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

    PubMed

    Kasula, Ravikiran; Chai, Ye Jin; Bademosi, Adekunle T; Harper, Callista B; Gormal, Rachel S; Morrow, Isabel C; Hosy, Eric; Collins, Brett M; Choquet, Daniel; Papadopulos, Andreas; Meunier, Frédéric A

    2016-09-26

    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 (Munc18-1(Δ317-333)) 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 Munc18-1(WT) 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 Munc18-1(Δ317-333), 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

  9. 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

    2013-09-01

    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.

  10. The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

    PubMed Central

    Omiatek, Donna M.; Bressler, Amanda J.; Cans, Ann-Sofie; Andrews, Anne M.; Heien, Michael L.; Ewing, Andrew G.

    2013-01-01

    Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the “weak base hypothesis” that amphetamine reduces synaptic vesicle transmitter and quantal size. PMID:23486177

  11. 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.

  12. 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. PMID:25160003

  13. Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

    PubMed Central

    1993-01-01

    Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087- 2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N- ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP

  14. Use of SNAREs for the immobilization of poly-3-hydroxyalkanoate polymerase type II of Pseudomonas putida CA-3 in secretory vesicles of Saccharomyces cerevisiae ATCC 9763.

    PubMed

    Muniasamy, Gurusamy; Pérez-Guevara, Fermín

    2014-02-20

    Polyhydroxyalkanoate (PHA) synthase, the key enzyme in polyester biosynthesis of bacteria, has been targeted to various organelles in yeasts and plants using respective signal peptides. Here, we report that the sequences derived from SNARE domains efficiently target and integrate the PHA synthase from Pseudomonas putida CA-3 to the membrane of secretory vesicles in Saccharomyces cerevisiae. The studies with the enhanced green fluorescent protein confirm the localization of synthase enzyme in the vesicles of S. cerevisiae. PMID:24368219

  15. 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

  16. Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles.

    PubMed Central

    Calhoun, B C; Goldenring, J R

    1997-01-01

    The tubulovesicles of gastric parietal cells sequester H+/K+-ATPase molecules within resting parietal cells. Stimulation of parietal cell secretion elicits delivery of intracellular H+/K+-ATPase to the apically oriented secretory canaliculus. Previous investigations have suggested that this process requires the regulated fusion of intracellular tubulovesicles with the canalicular target membrane. We have sought to investigate the presence of critical putative regulators of vesicle fusion on immunoisolated gastric parietal cell tubulovesicles. Highly purified tubulovesicles were prepared by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the alpha subunit of H+/K+-ATPase. Western blot analysis revealed the presence of Rab11, Rab25, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs) on immunoisolated vesicles. The same cohort of proteins was recovered on vesicles immunoisolated with monoclonal antibodies against SCAMPs and VAMP-2. In contrast, whereas immunoreactivities for syntaxin 1A/1B and synaptosome-associated protein (SNAP-25) were present in gradient-isolated vesicles, none of the immunoreactivity was associated with immunoisolated vesicles. The observation of VAMP-2 and two Rab proteins on immunoisolated H+/K+-ATPase-containing tubulovesicles supports the role for tubulovesicles in a regulated vesicle fusion process. In addition, the presence of SCAMPs along with Rab11 and Rab25 implicates the tubulovesicles as a critical apical recycling vesicle population. PMID:9230141

  17. 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.

  18. 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

  19. Analysis of Arf1 GTPase-Dependent Membrane Binding and Remodeling Using the Exomer Secretory Vesicle Cargo Adaptor.

    PubMed

    Paczkowski, Jon E; Fromme, J Christopher

    2016-01-01

    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

  20. 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

  1. C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals.

    PubMed

    Ye, Xuan; Zeng, Huiqing; Ning, Gang; Reiter, Jeremy F; Liu, Aimin

    2014-02-11

    The primary cilium plays critical roles in vertebrate development and physiology, but the mechanisms underlying its biogenesis remain poorly understood. We investigated the molecular function of C2 calcium-dependent domain containing 3 (C2cd3), an essential regulator of primary cilium biogenesis. We show that C2cd3 is localized to the centriolar satellites in a microtubule- and Pcm1-dependent manner; however, C2cd3 is dispensable for centriolar satellite integrity. C2cd3 is also localized to the distal ends of both mother and daughter centrioles and is required for the recruitment of five centriolar distal appendage proteins: Sclt1, Ccdc41, Cep89, Fbf1, and Cep164. Furthermore, loss of C2cd3 results in failure in the recruitment of Ttbk2 to the ciliary basal body as well as the removal of Cp110 from the ciliary basal body, two critical steps in initiating ciliogenesis. C2cd3 is also required for recruiting the intraflagellar transport proteins Ift88 and Ift52 to the mother centriole. Consistent with a role in distal appendage assembly, C2cd3 is essential for ciliary vesicle docking to the mother centriole. Our results suggest that C2cd3 regulates cilium biogenesis by promoting the assembly of centriolar distal appendages critical for docking ciliary vesicles and recruiting other essential ciliogenic proteins. PMID:24469809

  2. 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

  3. Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study.

    PubMed

    Zhao, C M; Chen, D; Lintunen, M; Panula, P; Håkanson, R

    1999-12-01

    ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm

  4. 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.

  5. 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

  6. WDR8 is a centriolar satellite and centriole-associated protein that promotes ciliary vesicle docking during ciliogenesis.

    PubMed

    Kurtulmus, Bahtiyar; Wang, Wenbo; Ruppert, Thomas; Neuner, Annett; Cerikan, Berati; Viol, Linda; Dueñas-Sánchez, Rafael; Gruss, Oliver J; Pereira, Gislene

    2016-02-01

    Ciliogenesis initiates at the mother centriole through a series of events that include membrane docking, displacement of cilia-inhibitory proteins and axoneme elongation. Centriolar proteins, in particular at distal and subdistal appendages, carry out these functions. Recently, cytoplasmic complexes named centriolar satellites have also been shown to promote ciliogenesis. Little is known about the functional and molecular relationship between appendage proteins, satellites and cilia biogenesis. Here, we identified the WD-repeat protein 8 (WDR8, also known as WRAP73) as a satellite and centriolar component. We show that WDR8 interacts with the satellite proteins SSX2IP and PCM1 as well as the centriolar proximal end component Cep135. Cep135 is required for the recruitment of WDR8 to centrioles. Depletion experiments revealed that WDR8 and Cep135 have strongly overlapping functions in ciliogenesis. Both are indispensable for ciliary vesicle docking to the mother centriole and for unlocking the distal end of the mother centriole from the ciliary inhibitory complex CP110-Cep97. Our data thus point to an important function of centriolar proximal end proteins in ciliary membrane biogenesis, and establish WDR8 and Cep135 as two factors that are essential for the initial steps of ciliation. PMID:26675238

  7. 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

  8. 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

  9. 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

  10. Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

    PubMed

    Harrison, V J; Barnes, K; Turner, A J; Wood, E; Corder, R; Vane, J R

    1995-07-01

    Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.

  11. 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. PMID:26637356

  12. 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.

  13. Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

    PubMed

    Tanguy, Emeline; Carmon, Ophélie; Wang, Qili; Jeandel, Lydie; Chasserot-Golaz, Sylvette; Montero-Hadjadje, Maité; Vitale, Nicolas

    2016-06-01

    The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

  14. Vesicle associated membrane protein 8 (VAMP8)-mediated zymogen granule exocytosis is dependent on endosomal trafficking via the constitutive-like secretory pathway.

    PubMed

    Messenger, Scott W; Falkowski, Michelle A; Thomas, Diana D H; Jones, Elaina K; Hong, Wanjin; Gaisano, Herbert Y; Giasano, Herbert Y; Boulis, Nicholas M; Groblewski, Guy E

    2014-10-01

    Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in VAMP8 knock-out (-/-) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ∼50% of the response. Analysis of VAMP8(-/-) acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2-3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8(-/-) acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8(-/-) acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14-16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis.

  15. The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation.

    PubMed

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

    2008-03-01

    Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-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 P(4)-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 beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

  16. 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.

  17. Pharmacological evaluation and docking studies of α,β-unsaturated carbonyl based synthetic compounds as inhibitors of secretory phospholipase A₂, cyclooxygenases, lipoxygenase and proinflammatory cytokines.

    PubMed

    Bukhari, Syed Nasir Abbas; Lauro, Gianluigi; Jantan, Ibrahim; Bifulco, Giuseppe; Amjad, Muhammad Wahab

    2014-08-01

    Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α,β-unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A₂ (sPLA₂), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six α,β-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA₂ activity, with IC₅₀ values in the range of 2.19-8.76 μM. Nine compounds 1-4 and 10-14 displayed inhibition of COX-1 with IC₅₀ values ranging from 0.37 to 1.77 μM (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these α,β-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents.

  18. Mouse Norovirus Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway▿ †

    PubMed Central

    Hyde, Jennifer L.; Sosnovtsev, Stanislav V.; Green, Kim Y.; Wobus, Christiane; Virgin, Herbert W.; Mackenzie, Jason M.

    2009-01-01

    Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development. PMID:19587041

  19. Granuphilin exclusively mediates functional granule docking to the plasma membrane

    PubMed Central

    Mizuno, Kouichi; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

    2016-01-01

    In regulated exocytosis, it is generally assumed that vesicles must stably “dock” at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca2+ -triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic β cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules. PMID:27032672

  20. 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.

  1. SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis

    PubMed Central

    Lang, Thorsten; Bruns, Dieter; Wenzel, Dirk; Riedel, Dietmar; Holroyd, Phillip; Thiele, Christoph; Jahn, Reinhard

    2001-01-01

    During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion. PMID:11331586

  2. 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. PMID:25988755

  3. 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

  4. 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).

  5. Interactions of pathogen-containing compartments with the secretory pathway.

    PubMed

    Canton, Johnathan; Kima, Peter E

    2012-11-01

    A subgroup of intracellular pathogens reside and replicate within membrane-bound compartments often termed pathogen-containing compartments (PCC). PCCs navigate around a wide range of host cell vesicles and organelles. In light of the perils of engaging with vesicles of the endocytic pathway, most PCCs modulate their interactions with endocytic vesicles while a few avoid those interactions. The secretory pathway constitutes another important grouping of vesicles and organelles in host cells. Although the negative consequences of engaging with the secretory pathway are not known, there is evidence that PCCs interact differentially with vesicles and organelles in this pathway as well. In this review, we consider three prokaryote pathogens and two protozoan parasites for which there is information on the interactions of their PCCs with the secretory pathway. Current understandings of the molecular interactions as well as the metabolic benefits that accompany those interactions are discussed. Not unexpectedly, our understanding of the extent of these interactions is variable. An underlying theme that is brought to the fore is that PCCs establish preferential interactions with distinct compartments of the secretory pathway.

  6. Auxilin facilitates membrane traffic in the early secretory pathway.

    PubMed

    Ding, Jingzhen; Segarra, Verónica A; Chen, Shuliang; Cai, Huaqing; Lemmon, Sandra K; Ferro-Novick, Susan

    2016-01-01

    Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

  7. 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

  8. Synaptic vesicle pools: an update.

    PubMed

    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

  9. Temporal separation of vesicle release from vesicle fusion during exocytosis.

    PubMed

    Troyer, Kevin P; Wightman, R Mark

    2002-08-01

    During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation. PMID:12034731

  10. 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.

  11. Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells.

    PubMed

    Hao, Zhenhua; Wei, Lisi; Feng, Yaqin; Chen, Xiaowei; Du, Wen; Ma, Jing; Zhou, Zhuan; Chen, Liangyi; Li, Wei

    2015-04-01

    The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs.

  12. Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells.

    PubMed

    Hao, Zhenhua; Wei, Lisi; Feng, Yaqin; Chen, Xiaowei; Du, Wen; Ma, Jing; Zhou, Zhuan; Chen, Liangyi; Li, Wei

    2015-04-01

    The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs. PMID:25673877

  13. 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

  14. Dynamin-2 Function and Dysfunction Along the Secretory Pathway

    PubMed Central

    González-Jamett, Arlek M.; Momboisse, Fanny; Haro-Acuña, Valentina; Bevilacqua, Jorge A.; Caviedes, Pablo; Cárdenas, Ana María

    2013-01-01

    Dynamin-2 is a ubiquitously expressed mechano-GTPase involved in different stages of the secretory pathway. Its most well-known function relates to the scission of nascent vesicles from the plasma membrane during endocytosis; however, it also participates in the formation of new vesicles from the Golgi network, vesicle trafficking, fusion processes and in the regulation of microtubule, and actin cytoskeleton dynamics. Over the last 8 years, more than 20 mutations in the dynamin-2 gene have been associated to two hereditary neuromuscular disorders: Charcot–Marie–Tooth neuropathy and centronuclear myopathy. Most of these mutations are grouped in the pleckstrin homology domain; however, there are no common mutations associated with both disorders, suggesting that they differently impact on dynamin-2 function in diverse tissues. In this review, we discuss the impact of these disease-related mutations on dynamin-2 function during vesicle trafficking and endocytotic processes. PMID:24065954

  15. The Secretory Pathway Kinases

    PubMed Central

    Sreelatha, Anju; Kinch, Lisa N.; Tagliabracci, Vincent S.

    2015-01-01

    Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. PMID:25862977

  16. 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.

  17. Biogenesis of extracellular vesicles in yeast

    PubMed Central

    Oliveira, Débora L; Nakayasu, Ernesto S; Joffe, Luna S; Guimarães, Allan J; Sobreira, Tiago JP; Nosanchuk, Joshua D; Cordero, Radames JB; Frases, Susana; Casadevall, Arturo; Almeida, Igor C; Nimrichter, Leonardo

    2010-01-01

    The cellular events required for unconventional protein secretion in eukaryotic pathogens are beginning to be revealed. In fungi, extracellular release of proteins involves passage through the cell wall by mechanisms that are poorly understood. In recent years, several studies demonstrated that yeast cells produce vesicles that traverse the cell wall to release a wide range of cellular components into the extracellular space. These studies suggested that extracellular vesicle release involves components of both conventional and unconventional secretory pathways, although the precise mechanisms required for this process are still unknown. We discuss here cellular events that are candidates for regulating this interesting but elusive event in the biology of yeast cells. PMID:21331232

  18. Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

    PubMed Central

    Joffe, Luna S.; Guimarães, Allan J.; Sobreira, Tiago J. P.; Nosanchuk, Joshua D.; Cordero, Radames J. B.; Frases, Susana; Casadevall, Arturo; Almeida, Igor C.; Nimrichter, Leonardo; Rodrigues, Marcio L.

    2010-01-01

    Background Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. Conclusions/Significance Our results suggest that both conventional and unconventional pathways of secretion are required for

  19. 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.

  20. Vesicles Are Persistent Features of Different Plastids.

    PubMed

    Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

    2016-10-01

    Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage.

  1. Vesicles Are Persistent Features of Different Plastids.

    PubMed

    Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

    2016-10-01

    Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. PMID:27405297

  2. 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. PMID:27575269

  3. 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

  4. 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

  5. Ultrastructure and innervation of water buffalo (Bubalus bubalis) seminal vesicle.

    PubMed

    Abou-Elmagd, A; Kujat, R; Wrobel, K H

    1992-01-01

    The lining epithelium of secretory end pieces and central glandular duct in the seminal vesicle of the water buffalo (Bubalus bubalis) consists of columnar principal and small polymorphous basal cells. A system of intercellular and even intracellular canaliculi enlarges the secretory surface. The most prominent organelle of the columnar principal cells is the granular endoplasmic reticulum, forming large aggregates of parallel lamellae. Using antibodies against the neural cell adhesion molecule L1 and the neural marker protein gene product 9.5 (PGP 9.5), the innervation pattern of the seminal vesicle becomes evident. The muscular layer surrounding the propria contains a dense network of unmyelinated fibers. Thicker bundles traverse the muscular layer to reach the propria. Around glandular secretory tubules and below the epithelial lining of the glandular duct a tightly woven subepithelial plexus is observed which sends short penetrating branches into the basal zone of the epithelium. These intraepithelial nerves are devoid of Schwann cells and basal lamina (naked axons) and are situated within the intercellular spaces between principal and basal cells. Acetylcholinesterase histochemistry with short (1-2 h) incubation times, dopamine-beta-hydroxylase immunohistochemistry and ultrastructural study of transmitter-containing vesicles was performed. The results suggest that muscular contraction in the seminal vesicle is predominantly under the influence of the sympathetic nervous system, whereas secretory epithelial function is regulated by both sympathetic and parasympathetic fibers.

  6. 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

  7. 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

  8. 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.

  9. Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins.

    PubMed

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

    2016-03-01

    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.

  10. 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

  11. Abnormal apocrine secretory cell mitochondria in a Huntington disease patient.

    PubMed

    Sidiropoulos, Christos; LeWitt, Peter; Hashimoto, Ken

    2012-12-15

    Over two decades, a 42-year old woman experienced the gradual onset of choreic involuntary movements, dystonia, and tics. Decreased caudate nucleus metabolism on 2-deoxyglucose PET scan and a heterozygous 49-CAG repeat expansion within the HTT gene established the diagnosis of HD, although no other family history was known. An axillary skin biopsy revealed a distinctive abnormality of mitochondria limited to the apocrine secretory cells on electron microscopy. All mitochondria were transformed into rounded structures with disrupted cristae and prominent myelin figures; many were enlarged up to 4 times the normal. Cytoplasm of apocrine secretory cells showed an abundance of lipid vacuoles, empty vesicles, and dense bodies. Biopsied skeletal muscle histology (light microscopy) was normal, as was a mitochondrial metabolism study. Biopsies from other HD patients have shown similar mitochondrial changes in cerebral neurons, muscle, fibroblasts, and lymphoblasts, adding to evidence for a systemic disturbance of mitochondria in HD.

  12. Dry Dock No. 3. View of head of Dry Dock ...

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

    Dry Dock No. 3. View of head of Dry Dock with stair to right of shot. View facing west - U.S. Naval Base, Pearl Harbor, Dry Dock No. 3, On northern shoreline of shipyard, west of Dry Dock Nos. 1 & 2, near the intersection of Avenue G and Sixth Street, Pearl City, Honolulu County, HI

  13. 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.

  14. Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

    PubMed Central

    1994-01-01

    Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed. PMID:7798314

  15. Transcriptional regulation of secretory capacity by bZip transcription factors

    PubMed Central

    FOX, Rebecca M.

    2015-01-01

    Cells of specialized secretory organs expand their secretory pathways to accommodate the increased protein load necessary for their function. The endoplasmic reticulum (ER), the Golgi apparatus and the secretory vesicles, expand not only the membrane components but also the protein machinery required for increased protein production and transport. Increased protein load causes an ER stress response akin to the Unfolded Protein Response (UPR). Recent work has implicated several bZip transcription factors in the regulation of protein components of the early secretory pathway necessary to alleviate this stress. Here, we highlight eight bZip transcription factors in regulating secretory pathway component genes. These include components of the three canonical branches of the UPR–ATF4, XBP1, and ATF6, as well as the five members of the Creb3 family of transcription factors. We review findings from both invertebrate and vertebrate model systems suggesting that all of these proteins increase secretory capacity in response to increased protein load. Finally, we propose that the Creb3 family of factors may have a dual role in secretory cell differentiation by also regulating the pathways necessary for cell cycle exit during terminal differentiation. PMID:25821458

  16. Expedition 30 Docking

    NASA Video Gallery

    The Soyuz TMA-03M spacecraft carrying NASA astronaut Don Pettit, Russian cosmonaut Oleg Kononenko and European Space Agency astronaut Andre Kuipers docks to the International Space Station’s Rass...

  17. Engineered Asymmetric Synthetic Vesicles

    NASA Astrophysics Data System (ADS)

    Lu, Li; Chiarot, Paul

    2013-11-01

    Synthetic vesicles are small, fluid-filled spheres that are enclosed by a bilayer of lipid molecules. They can be used as models for investigating membrane biology and as delivery vehicles for pharmaceuticals. In practice, it is difficult to simultaneously control membrane asymmetry, unilamellarity, vesicle size, vesicle-to-vesicle uniformity, and luminal content. Membrane asymmetry, where each leaflet of the bilayer is composed of different lipids, is of particular importance as it is a feature of most natural membranes. In this study, we leverage microfluidic technology to build asymmetric vesicles at high-throughput. We use the precise flow control offered by microfluidic devices to make highly uniform emulsions, with controlled internal content, that serve as templates to build the synthetic vesicles. Flow focusing, dielectrophoretic steering, and interfacial lipid self-assembly are critical procedures performed on-chip to produce the vesicles. Fluorescent and confocal microscopy are used to evaluate the vesicle characteristics.

  18. Calcium-containing phosphopeptides pave the secretory pathway for efficient protein traffic and secretion in fungi.

    PubMed

    Martín, Juan F

    2014-01-01

    Casein phosphopeptides (CPPs) containing chelated calcium drastically increase the secretion of extracellular homologous and heterologous proteins in filamentous fungi. Casein phosphopeptides released by digestion of alpha - and beta-casein are rich in phosphoserine residues (SerP). They stimulate enzyme secretion in the gastrointestinal tract and enhance the immune response in mammals, and are used as food supplements. It is well known that casein phosphopeptides transport Ca2+ across the membranes and play an important role in Ca2+ homeostasis in the cells. Addition of CPPs drastically increases the production of heterologous proteins in Aspergillus as host for industrial enzyme production. Recent proteomics studies showed that CPPs alter drastically the vesicle-mediated secretory pathway in filamentous fungi, apparently because they change the calcium concentration in organelles that act as calcium reservoirs. In the organelles calcium homeostasis a major role is played by the pmr1 gene, that encodes a Ca2+/Mn2+ transport ATPase, localized in the Golgi complex; this transporter controls the balance between intra-Golgi and cytoplasmic Ca2+ concentrations. A Golgi-located casein kinase (CkiA) governs the ER to Golgi directionality of the movement of secretory proteins by interacting with the COPII coat of secretory vesicles when they reach the Golgi. Mutants defective in the casein-2 kinase CkiA show abnormal targeting of some secretory proteins, including cytoplasmic membrane amino acid transporters that in ckiA mutants are miss-targeted to vacuolar membranes. Interestingly, addition of CPPs increases a glyceraldehyde-3-phpshate dehydrogenase protein that is known to associate with microtubules and act as a vesicle/membrane fusogenic agent. In summary, CPPs alter the protein secretory pathway in fungi adapting it to a deregulated protein traffic through the organelles and vesicles what results in a drastic increase in secretion of heterologous and also of

  19. Cell secretion mediated by granule-associated vesicle transport: a glimpse at evolution.

    PubMed

    Crivellato, Enrico; Nico, Beatrice; Gallo, Valentina Patrizia; Ribatti, Domenico

    2010-07-01

    Regulated secretion allows extrusion of cell products stored in specialized membrane-bound organelles called secretory granules or secretory vesicles. Regulated secretion provides basic functions in living organisms, and in a phylogenetic perspective, it is recognizable in the most primitive eukaryotic forms. This article is an attempt to trace the evolutionary history of a special type of secretory pattern, which has been referred to as vesicle-mediated degranulation or piecemeal degranulation (PMD). First described in the early 70s of the last century in inflammatory cells, such as the basophils, mast cells, and eosinophils, this regulated secretory route has subsequently been recognized in endocrine cells, in particular in the chromaffin cells of the adrenal medulla. This vesicle-mediated degranulation is held to mobilize small and specific aliquots of granule-associated material for selective paracrine or endocrine transport to the cell exterior. PMD has been identified in many vertebrate classes. By contrast, no data are available for invertebrates. We speculate that this pattern of cell secretion emerged early in phylogenesis, when the first metazoans appeared. In this review article, we will first revise the concept of vesicle-mediated degranulation in the light of the most recent experimental discoveries and theoretical implications. Then, the distribution of this secretory mode among vertebrates and its molecular basis will be highlighted. Finally, the potential occurrence of PMD in invertebrates, its biological significance from an evolutionary perspective and the future direction of investigations will be briefly sketched.

  20. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    PubMed

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses. PMID:26539890

  1. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    PubMed

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses.

  2. Secretory end-feet, extracerebral cells, and cerebral sense organs in certain limicole oligochaete annelids.

    PubMed

    Golding, D W; Whittle, A C

    1975-01-01

    Secretory end-feet (or SEF) systems are present in Limnodrilus and Stylodrilus but are less highly organized than those of polychaetes. SEF contain secretory vesicles and abundant mitochondria. Typical neurosecretory terminals are not found within the brain although "neurosecretory" perikarya are present in all four species studied. In Limnodrilus, Stylodrilus and Enchytraeus extracerebral cells, of probable neurosecretory function, are invested by the pericapsular epithelium. Characteristically such cells bear several cilia. In these species and in Stylaria a pair of sensory cell groups is located anteriorly within the brain. These cells are ciliated but lack associated supporting cells. PMID:170709

  3. 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

  4. Extracellular Vesicles as New Players in Cellular Senescence.

    PubMed

    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

  5. Secretory protein trafficking in Giardia intestinalis.

    PubMed

    Hehl, Adrian B; Marti, Matthias

    2004-07-01

    Early diverged extant organisms, which may serve as convenient laboratory models to look for and study evolutionary ancient features of eukaryotic cell biology, are rare. The diplomonad Giardia intestinalis, a protozoan parasite known to cause diarrhoeal disease, has become an increasingly popular object of basic research in cell biology, not least because of a genome sequencing project nearing completion. Commensurate with its phylogenetic status, the Giardia trophozoite has a very basic secretory system and even lacks hallmark structures such as a morphologically identifiable Golgi apparatus. The cell's capacity for protein sorting is nevertheless unimpeded, exemplified by its ability to cope with massive amounts of newly synthesized cyst wall proteins and glycans, which are sorted to dedicated Golgi-like compartments termed encystation-specific vesicles (ESVs) generated from endoplasmic reticulum (ER)-derived transport intermediates. This soluble bulk cargo is kept strictly separate from constitutively transported variant surface proteins during export, a function that is dependent on the stage-specific recognition of trafficking signals. Encysting Giardia therefore provide a unique system for the study of unconventional, Golgi-independent protein trafficking mechanisms in the broader context of eukaryotic endomembrane organization and evolution. PMID:15225300

  6. Alterations in hepatic pericanalicular cytoplasm during enhanced bile secretory activity.

    PubMed

    Jones, A L; Schmucker, D L; Mooney, J S; Ockner, R K; Adler, R D

    1979-04-01

    In an attempt to demonstrate the morphology of the bile secretory apparatus, male rats were restrained and maintained on an isocaloric diet with (experimental) and without (control) taurocholate, which was continuously infused via a duodenal cannula. This method of taurocholate administration promotes a 2-fold increase in the bile acid pool size and bile secretory rate and increases the transport maximum of taurocholate by approximately 50%. After 48 hours, the livers from both the control and experimental animals were perfusion-fixed and whole hepatocytes as well as pericanalicular cytoplasm (defined as a 1-micron. wide zone of cytoplasm adjacent to the bile canaliculus) in both centrolobular and periportal cells were subjected to a stereologic analysis. Although taurocholate infusion produced relatively few changes in the amounts of organelles or inclusionswithin hepatocytes, it caused highly significant increases in the amount ofGolgi-rich area, Golgi membranes, and the number of vesicles with diameters greater than 1000 A in the pericanalicular area of cytoplasm. In addition to these changes, which occurred in both central and periportal zones, decreases in the volume of lysosomes and the surface area of smooth surfaced endoplasmic reticulum were observed. These data provide new evidence that the "bile secretory apparatus" may encompass several hepatocellular components which include the Golgi complex and a vesicular transport system.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. DOCK8 Deficiency

    MedlinePlus

    ... on ClinicalTrials.gov . Related Links Primary Immune Deficiency Diseases (PIDDs) Immune System ​​​​​​​ Javascript Error Your browser JavaScript is turned ... Scientists Identify Genetic Cause of Previously Undefined Primary Immune Deficiency Disease Signs and Symptoms DOCK8 deficiency causes persistent skin ...

  16. 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

  17. Nanotubes from gelly vesicles

    NASA Astrophysics Data System (ADS)

    Kremer, S.; Campillo, C.; Pepin-Donat, B.; Viallat, A.; Brochard-Wyart, F.

    2008-05-01

    Hydrodynamic extrusions of tethers from giant unilamellar vesicles (GUV) enclosing a poly-N-isopropylacrylamide (polyNIPAM) gel are studied. The collapse of the gel upon heating induces a deswelling of the GUV, showing that the membrane is linked to the polymer network. The gelly vesicle is attached to a micro-rod and submitted to a flow (velocity U). Above a threshold velocity (U>Uc) a tether is extruded and reaches a stationary length L∞simeτ0U in a characteristic time τ0. The vesicle behaves like an entropic spring with a tether length L∞ proportional to the Stokes friction force. Compared to viscous "sol" vesicles, gelly vesicle are much stiffer: L∞ and τ0 being hundred times smaller. We conclude that the mobility of lipids is reduced, only a small portion of the vesicle area being free to flow into the tube.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Coexistence of intracytoplasmic lumens and membrane-bound vesicles in an invasive carcinoma arising in a cystosarcoma phyllodes.

    PubMed

    Gilks, B; Tavassoli, F A

    1988-01-01

    An unusual invasive breast carcinoma, arising in a cystosarcoma phyllodes and characterized by a variable cytoplasmic appearance and mucin content, was evaluated to determine the nature of the secretory material within the cells as well as the type of secretory organelle at the ultrastructural level. Histochemical studies revealed both acidic (sialic acid) and neutral mucin within the tumor cells. Ultrastructural analysis revealed secretory material within membrane-bound vesicles in some cells and within intracytoplasmic lumens in others; some cells contained both membrane-bound vesicles and intracytoplasmic lumens simultaneously. The Golgi derivation of the intracytoplasmic lumens was supported by their presence within or near hyperplastic Golgi complexes. The histochemical characteristics of the secretory material is correlated with their ultrastructural site of accumulation.

  6. Synaptic vesicle endocytosis.

    PubMed

    Saheki, Yasunori; De Camilli, Pietro

    2012-09-01

    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.

  7. 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

  8. Intermembrane Docking Reactions Are Regulated by Membrane Curvature

    PubMed Central

    Kunding, Andreas H.; Mortensen, Michael W.; Christensen, Sune M.; Bhatia, Vikram K.; Makarov, Ivan; Metzler, Ralf; Stamou, Dimitrios

    2011-01-01

    The polymorphism of eukaryotic cellular membranes is a tightly regulated and well-conserved phenotype. Recent data have revealed important regulatory roles of membrane curvature on the spatio-temporal localization of proteins and in membrane fusion. Here we quantified the influence of membrane curvature on the efficiency of intermembrane docking reactions. Using fluorescence microscopy, we monitored the docking of single vesicle–vesicle pairs of different diameter (30–200 nm) and therefore curvature, as mediated by neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and streptavidin-biotin. Surprisingly, the intermembrane docking efficiency exhibited an ∼30–60 fold enhancement as a function of curvature. In comparison, synaptotagmin and calcium accelerate SNARE-mediated fusion in vitro by a factor of 2–10. To explain this finding, we formulated a biophysical model. On the basis of our findings, we propose that membrane curvature can regulate intermembrane tethering reactions and consequently any downstream process, including the fusion of vesicles and possibly viruses with their target membranes. PMID:22261058

  9. 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

  10. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    PubMed

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion. PMID:27498562

  11. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    PubMed

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion.

  12. 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.

  13. 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

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

    PubMed

    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. Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia.

    PubMed

    Reiner, D S; McCaffery, M; Gillin, F D

    1990-10-01

    Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.

  16. Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

    PubMed

    Stenbeck, G; Schreiner, R; Herrmann, D; Auerbach, S; Lottspeich, F; Rothman, J E; Wieland, F T

    1992-12-14

    Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae. PMID:1360908

  17. Immunocytochemical Evidence for Golgi Vesicle Involvement in Milk Fat Globule Secretion.

    PubMed

    Wooding, F B Peter; Sargeant, Timothy J

    2015-12-01

    The exact mechanism of secretion of the milk fat globule (MFG) from the mammary secretory cell is still controversial. We have previously suggested close involvement of Golgi vesicles in this process. This paper provides direct immunocytochemical evidence that butyrophilin is present in the Golgi stack and vesicles in ovine and caprine mammary glands. We suggest that it is the butyrophilin in the Golgi vesicle membrane that forms the specific association with the adipophilin on the lipid surface in the cytoplasm. Exocytosis of the associated Golgi vesicle will then initiate the process of MFG secretion. Further exocytosis of associated Golgi vesicles will continue and complete the process. Areas of the plasmalemma that have butyrophilin delivered by previous non-lipid associated Golgi exocytoses may also contribute to the process of forming the milk fat globule membrane (MFGM). PMID:26374828

  18. Orchestrated content release from Drosophila glue-protein vesicles by a contractile actomyosin network.

    PubMed

    Rousso, Tal; Schejter, Eyal D; Shilo, Ben-Zion

    2016-02-01

    Releasing content from large vesicles measuring several micrometres in diameter poses exceptional challenges to the secretory system. An actomyosin network commonly coats these vesicles, and is thought to provide the necessary force mediating efficient cargo release. Here we describe the spatial and temporal dynamics of the formation of this actomyosin coat around large vesicles and the resulting vesicle collapse, in live Drosophila melanogaster salivary glands. We identify the Formin family protein Diaphanous (Dia) as the main actin nucleator involved in generating this structure, and uncover Rho as an integrator of actin assembly and contractile machinery activation comprising this actomyosin network. High-resolution imaging reveals a unique cage-like organization of myosin II on the actin coat. This myosin arrangement requires branched-actin polymerization, and is critical for exerting a non-isotropic force, mediating efficient vesicle contraction.

  19. 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.

  20. 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.

  1. 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.

  2. Structure and mechanism of COPI vesicle biogenesis.

    PubMed

    Jackson, Lauren P

    2014-08-01

    Distinct trafficking pathways within the secretory and endocytic systems ensure prompt and precise delivery of specific cargo molecules to different cellular compartments via small vesicular (50-150nm) and tubular carriers. The COPI vesicular coat is required for retrograde trafficking from the cis-Golgi back to the ER and within the Golgi stack. Recent structural data have been obtained from X-ray crystallographic studies on COPI coat components alone and on COPI subunits in complex with either cargo motifs or Arf1, and from reconstructions of COPI coated vesicles by electron tomography. These studies provide important molecular information and indicate key differences in COPI coat assembly as compared with clathrin-based and COPII-based coats. PMID:24840894

  3. Docking challenge: protein sampling and molecular docking performance.

    PubMed

    Elokely, Khaled M; Doerksen, Robert J

    2013-08-26

    Computational tools are essential in the drug design process, especially in order to take advantage of the increasing numbers of solved X-ray and NMR protein-ligand structures. Nowadays, molecular docking methods are routinely used for prediction of protein-ligand interactions and to aid in selecting potent molecules as a part of virtual screening of large databases. The improvements and advances in computational capacity in the past decade have allowed for further developments in molecular docking algorithms to address more complicated aspects such as protein flexibility. The effects of incorporation of active site water molecules and implicit or explicit solvation of the binding site are other relevant issues to be addressed in the docking procedures. Using the right docking algorithm at the right stage of virtual screening is most important. We report a staged study to address the effects of various aspects of protein flexibility and inclusion of active site water molecules on docking effectiveness to retrieve (and to be able to predict) correct ligand poses and to rank docked ligands in relation to their biological activity for CHK1, ERK2, LpxC, and UPA. We generated multiple conformers for the ligand and compared different docking algorithms that use a variety of approaches to protein flexibility, including rigid receptor, soft receptor, flexible side chains, induced fit, and multiple structure algorithms. Docking accuracy varied from 1% to 84%, demonstrating that the choice of method is important.

  4. Secretory breast cancer. Case report

    PubMed Central

    LOMBARDI, A.; MAGGI, S.; BERSIGOTTI, L.; LAZZARIN, G.; NUCCETELLI, E.; AMANTI, C.

    2013-01-01

    Summary: Secretory carcinoma of the breast is a rare tumor initially described in children but occurring equally in adult population. This unusual breast cancer subtype has a generally favorable prognosis, although several cases have been described in adults with increased aggressiveness and a risk of metastases. However, surgery is still considered the most appropriate treatment for this pathology. We describe the case of a 50 – year-old woman who has undergone a breast conservative surgery for a little tumor, preoperatively diagnosticated by a fine needle aspiration biopsy (FNAB) as a well differentiated infiltrating carcinoma. PMID:23660165

  5. Secretory breast cancer. Case report.

    PubMed

    Lombardi, A; Maggi, S; Bersigotti, L; Lazzarin, G; Nuccetelli, E; Amanti, C

    2013-04-01

    Secretory carcinoma of the breast is a rare tumor initially described in children but occurring equally in adult population. This unusual breast cancer subtype has a generally favorable prognosis, although several cases have been described in adults with increased aggressiveness and a risk of metastases. However, surgery is still considered the most appropriate treatment for this pathology. We describe the case of a 50 -year-old woman who has undergone a breast conservative surgery for a little tumor, preoperatively diagnosticated by a fine needle aspiration biopsy (FNAB) as a well differentiated infiltrating carcinoma.

  6. 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

  7. 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.

  8. Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins

    PubMed Central

    Rivera, Johanna; Cordero, Radames J. B.; Nakouzi, Antonio S.; Frases, Susana; Nicola, André; Casadevall, Arturo

    2010-01-01

    Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria. PMID:20956325

  9. Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins.

    PubMed

    Rivera, Johanna; Cordero, Radames J B; Nakouzi, Antonio S; Frases, Susana; Nicola, André; Casadevall, Arturo

    2010-11-01

    Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27084478

  11. Rescoring ligand docking poses.

    PubMed

    Zhong, Shijun; Zhang, Youping; Xiu, Zhilong

    2010-05-01

    The ranking of ligand docking poses according to certain scoring systems to identify the best fit is the most important step in virtual database screening for drug discovery. By focusing on method development strategy, this review provides possibilities for constructing rescoring approaches based on an overview of recent developments in the field. These developments can be classified into three categories. The first category involves a scaling approach that employs a factor to scale the primary scoring function. These scaling factors are defined with respect to the geometrical match between the location of a ligand and the target binding site, or defined according to a molecular weight distribution consistent with the empirical range of molecular weights of drug-like compounds. The second category involves consensus scoring approaches that use multiple scoring functions to rank the ligand poses retained in a docking procedure, based on the preliminary ranking according to a primary scoring function. The final category involves the addition of selected accuracy-oriented energy terms, such as the solvent effect and quantum mechanics/molecular mechanics treatments. PMID:20443166

  12. PC12 Cells that Lack Synaptotagmin I Exhibit Loss of a Subpool of Small Dense Core Vesicles

    PubMed Central

    Adams, Robert D.; Harkins, Amy B.

    2014-01-01

    Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca2+ sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells. PMID:25517150

  13. Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

    PubMed Central

    1994-01-01

    Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs. PMID:7962100

  14. RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking.

    PubMed

    Mukherjee, Debarati; Sen, Arpita; Aguilar, R Claudio

    2014-01-01

    Cell polarity, the asymmetric distribution of proteins and lipids, is essential for a variety of cellular functions. One mechanism orchestrating cell polarity is polarized vesicle trafficking; whereby cargo loaded secretory vesicles are specifically transported to predetermined areas of the cell. The evolutionarily conserved exocyst complex and its small GTPase regulators play crucial roles in spatiotemporal control of polarized vesicle trafficking. In studies on neuronal membrane remodeling and synaptic plasticity, conserved mechanisms of exocyst regulation and cargo recycling during polarized vesicle trafficking are beginning to emerge as well. Recently, our lab demonstrated that RhoGTPase-binding proteins in both yeast (Bem3) and mammals (Ocrl1) are also required for the efficient traffic of secretory vesicles to sites of polarized growth and signaling. Together with our studies, we highlight the evolutionary conservation of the basic elements essential for polarized vesicle traffic across different cellular functions and model systems. In conclusion, we emphasize that studies on RhoGTPase-binding proteins in these processes should be included in the next level of investigation, for a more complete understanding of their hitherto unknown roles in polarized membrane traffic and exocyst regulation.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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).

  20. 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.

  1. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

    PubMed Central

    1981-01-01

    Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles. PMID:7298720

  2. Electrohydrodynamics Of Multicomponent Vesicles

    NASA Astrophysics Data System (ADS)

    Gera, Prerna; Salac, David

    2015-11-01

    The addition of cholesterol into a lipid membrane induces the formation of distinct domains. These domains try to minimize the overall energy of the system by coalescence and migration. The application of electric fields will induce flow of these membrane domains and influence the rate at which they coarsen. In this work the electrohydrodynamics of multicomponent vesicles is numerically modelled. The method uses a Cahn-Hilliard-Cook model of the lipid domains restricted to a deforming three-dimensional vesicle and will be briefly discussed. Sample results will be presented and compared to experimental observations. This work supported by NSF Grant #1253739.

  3. RCAN1 regulates vesicle recycling and quantal release kinetics via effects on calcineurin activity.

    PubMed

    Zanin, Mark P; Mackenzie, Kimberly D; Peiris, Heshan; Pritchard, Melanie A; Keating, Damien J

    2013-02-01

    We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over-expressing RCAN1 (RCAN1(ox)), but not in wild-type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK-506, replicates this effect in WT cells but has no additional effect in RCAN1(ox) cells. When we chronically expose WT cells to cyclosporine A and FK-506 we find that catecholamine release per vesicle and pre-spike foot (PSF) signal parameters are decreased, similar to that in RCAN1(ox) cells. Inhibiting calcineurin activity in RCAN1(ox) cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1(ox) cells, the smaller vesicle and dense core size we observe in RCAN1(ox) cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.

  4. Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cells.

    PubMed

    Varadi, Aniko; Ainscow, Edward K; Allan, Victoria J; Rutter, Guy A

    2002-11-01

    Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal beta-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHC(mut)) blocked all vesicular movements with velocity >0.4 micro m second(-1), which demonstrates that kinesin activity was essential for vesicle motility in live beta-cells. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.

  5. Electron microscopic observations on the epithelium of ram seminal vesicles.

    PubMed Central

    Plöen, L

    1980-01-01

    The ultrastructure of the secretory cells of the ram seminal vesicle was studied on material fixed by immersion or by vascular perfusion. The signs of apocrine secretion seen after immersion fixation did not appear after perfusion fixation and are therefore interpreted as artefacts. Instead, vacuoles with a granule in them were seen. Such vacuoles were observed in the Golgi apparatus and in the apical cytoplasm. Further indications of merocrine secretion were also found. It therefore appears that protein secretion in the ram seminal vesicle follows the typical pattern of serous glands. The possibility that fructose is extruded with the protein as the vacuoles open at the luminal cell surface is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7410195

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties.

    PubMed

    Hübner, Kirsten; Windoffer, Reinhard; Hutter, Harald; Leube, Rudolf E

    2002-01-01

    Tetraspan vesicle membrane proteins (TVPs) are characterized by four transmembrane regions and cytoplasmically located end domains. They are ubiquitous and abundant components of vesicles in most, if not all, cells of multicellular organisms. TVP-containing vesicles shuttle between various membranous compartments and are localized in biosynthetic and endocytotic pathways. Based on gene organization and amino acid sequence similarities TVPs can be grouped into three distinct families that are referred to as physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In mammals synaptophysin, synaptoporin, pantophysin, and mitsugumin29 constitute the physins, synaptogyrin 1-4 the gyrins, and SCAMP1-5 the SCAMPs. Members of each family are cell-type-specifically synthesized resulting in unique patterns of TVP coexpression and subcellular colocalization. TVP orthologs have been identified in most multicellular organisms, including diverse animal and plant species, but have not been detected in unicellular organisms. They are subject to protein modification, most notably to phosphorylation, and are part of multimeric complexes. Experimental evidence is reviewed showing that TVPs contribute to vesicle trafficking and membrane morphogenesis. PMID:11893164

  11. 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

  12. 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.

  13. 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

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

    PubMed

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

    2016-01-01

    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

  15. Reduced release probability prevents vesicle depletion and transmission failure at dynamin mutant synapses.

    PubMed

    Lou, Xuelin; Fan, Fan; Messa, Mirko; Raimondi, Andrea; Wu, Yumei; Looger, Loren L; Ferguson, Shawn M; De Camilli, Pietro

    2012-02-21

    Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.

  16. Biogenesis of extracellular vesicles in yeast: Many questions with few answers.

    PubMed

    Oliveira, Débora L; Nakayasu, Ernesto S; Joffe, Luna S; Guimarães, Allan J; Sobreira, Tiago Jp; Nosanchuk, Joshua D; Cordero, Radames Jb; Frases, Susana; Casadevall, Arturo; Almeida, Igor C; Nimrichter, Leonardo; Rodrigues, Marcio L

    2010-11-01

    The cellular events required for unconventional protein secretion in eukaryotic pathogens are beginning to be revealed. In fungi, extracellular release of proteins involves passage through the cell wall by mechanisms that are poorly understood. In recent years, several studies demonstrated that yeast cells produce vesicles that traverse the cell wall to release a wide range of cellular components into the extracellular space. These studies suggested that extracellular vesicle release involves components of both conventional and unconventional secretory pathways, although the precise mechanisms required for this process are still unknown. We discuss here cellular events that are candidates for regulating this interesting but elusive event in the biology of yeast cells.

  17. 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

  18. 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.

  19. Bacterial vesicles in marine ecosystems.

    PubMed

    Biller, Steven J; Schubotz, Florence; Roggensack, Sara E; Thompson, Anne W; Summons, Roger E; Chisholm, Sallie W

    2014-01-10

    Many heterotrophic bacteria are known to release extracellular vesicles, facilitating interactions between cells and their environment from a distance. Vesicle production has not been described in photoautotrophs, however, and the prevalence and characteristics of vesicles in natural ecosystems is unknown. Here, we report that cultures of Prochlorococcus, a numerically dominant marine cyanobacterium, continuously release lipid vesicles containing proteins, DNA, and RNA. We also show that vesicles carrying DNA from diverse bacteria are abundant in coastal and open-ocean seawater samples. Prochlorococcus vesicles can support the growth of heterotrophic bacterial cultures, which implicates these structures in marine carbon flux. The ability of vesicles to deliver diverse compounds in discrete packages adds another layer of complexity to the flow of information, energy, and biomolecules in marine microbial communities.

  20. A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

    PubMed

    Grefen, Christopher; Karnik, Rucha; Larson, Emily; Lefoulon, Cécile; Wang, Yizhou; Waghmare, Sakharam; Zhang, Ben; Hills, Adrian; Blatt, Michael R

    2015-01-01

    Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

  1. 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.

  2. Transgelin: an androgen-dependent protein identified in the seminal vesicles of three Saharan rodents.

    PubMed

    Kaci-Ouchfoun, Naïma; Izemrane, Djamila; Boudrissa, Abdelkrim; Gernigon, Thérèse; Khammar, Farida; Exbrayat, Jean Marie

    2013-10-15

    During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.

  3. 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.

  4. 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

  5. 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

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

    PubMed

    Topalidou, Irini; Cattin-Ortolá, Jérôme; Pappas, Andrea L; Cooper, Kirsten; Merrihew, Gennifer E; MacCoss, Michael J; Ailion, Michael

    2016-05-01

    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.

  7. 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.

  8. 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.

  9. 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

  10. Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs

    PubMed Central

    Kim, Jae-Yeol; Choi, Bong-Kyu; Choi, Mal-Gi; Kim, Sun-Ae; Lai, Ying; Shin, Yeon-Kyun; Lee, Nam Ki

    2012-01-01

    Synaptotagmin-1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway. PMID:22407297

  11. 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

  12. Ultrastructural patterns of secretory activity in poison cutaneous glands of larval and juvenile Dendrobates auratus (Amphibia, Anura).

    PubMed

    Angel, R; Delfino, G; Parra, G J

    2003-01-01

    A transmission electron-microscope study has been performed on larval and juvenile skin of the Central American arrow-frog Dendrobates auratus to investigate early secretory processes and maturational changes in the serous (poison) glands. Poison biosynthesis involves the endoplasmic reticulum (both smooth and rough types), as well as Golgi stacks which release early serous product as secretory vesicles (or pre-granules). These vesicles contain fine-grained material, along with single electron-opaque bodies, spheroidal in shape, that accompany the grained product throughout its post-Gogian, maturational change. The first steps of this process involve condensation and lead to the formation of secretory granules with a glomerular-like substructure, resulting from a thick, random aggregation of rods (secretory granule subunits). Advanced maturational activity causes the loss of peculiar granule substructure: the dense bodies split into fragments, whereas the thick glomerular arrangement becomes looser, until the secretory product changes into a dispersed material. This ultrastructural study revealed biosynthesis and maturation processes in close sequence, suggesting the poison of D. auratus contains proteins and/or peptides as well as lipophilic compounds. Molecules of both these classes are known to perform several roles relevant to survival strategies in extant anurans. Furthermore, the ephemeral granules with a glomerular-like substructure detected in tadpoles and froglets exhibit the complex patterns of mature poisons in adult specimens of other anurans: Hylidae and related families. This agrees with current trends in the taxonomy of these advanced frogs and underlines the pertinence of an ontogenetic approach in investigating anuran phylogenesis. PMID:12467659

  13. 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.

  14. Modeling F-actin cortex influence on the secretory properties of neuroendocrine cells

    PubMed Central

    Gil, Amparo

    2011-01-01

    Chromaffin cells are considered as one of the most valuable models to study regulated exocytosis. In these cells, like in other neuroendocrine systems, an intricate cortical cytoskeleton acts as a retentive network impeding vesicle access to plasma membrane. Therefore, during stimulation this structure suffers a transient reorganization allowing active transport of vesicles toward secretory sites. Interestingly, a combination of confocal microscopy studies and mathematical modeling is showing us new aspects of the influence of cortical cytoskeleton in shaping the secretory properties of excitable cells. In this new vision the F-actin-myosin II cortical cytoskeleton is organized forming polygonal cages with the molecular machinery of exocytosis composed by SNARE proteins and voltage-dependent calcium channels associating with its border. In this way the cytoskeleton not only holds together the essential elements acting during secretion, but we proposed that could also act as a structural factor opposing to the free diffusion of the calcium signal and therefore sustains high levels of the intracellular signal triggering exocytosis. PMID:21966558

  15. Evolution of chloroplast vesicle transport.

    PubMed

    Westphal, Sabine; Soll, Jürgen; Vothknecht, Ute C

    2003-02-01

    Vesicle traffic plays a central role in eukaryotic transport. The presence of a vesicle transport system inside chloroplasts of spermatophytes raises the question of its phylogenetic origin. To elucidate the evolution of this transport system we analyzed organisms belonging to different lineages that arose from the first photosynthetic eukaryote, i.e. glaucocystophytes, chlorophytes, rhodophytes, and charophytes/embryophytes. Intriguingly, vesicle transport is not apparent in any group other than embryophytes. The transfer of this eukaryotic-type vesicle transport system from the cytosol into the chloroplast thus seems a late evolutionary development that was acquired by land plants in order to adapt to new environmental challenges.

  16. 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

  17. 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.

  18. Disruption of the fusion of Leishmania parasitophorous vacuoles with ER vesicles results in the control of the infection.

    PubMed

    Canton, Johnathan; Ndjamen, Blaise; Hatsuzawa, Kiyotaka; Kima, Peter E

    2012-06-01

    Parasitophorous vacuoles (PV) that harbour Leishmania parasites acquire some characteristics from fusion with host cell vesicles. Recent studies have shown that PVs acquire and display resident endoplasmic reticulum (ER) molecules. We investigated the importance of ER molecules to PV biology by assessing the consequence of blocking the fusion of PVs with vesicles that originate from the early secretory pathway. This was achieved by targeting the N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that mediate the fusion of early secretory vesicles. In the presence of dominant negative variants of sec22b or some of its known cognate partners, D12 and syntaxin 18, PVs failed to distend and harboured fewer parasites. These observations were confirmed in studies in which each of the SNAREs listed above including the intermediate compartment ER/Golgi SNARE, syntaxin 5, was knocked down. The knock-down of these SNARES had little or no measurable effect on the morphology of the ER or on activated secretion even though they resulted in a more significant reduction of PV size. Moreover, the knock-down of the ER/Golgi SNAREs resulted in significant reduction in parasite replication. Taken together, these studies provide further evidence that PVs acquire ER components by fusing with vesicles derived from the early secretory pathway; disruption of this interaction results in inhibition of the development of PVs as well as the limitation of parasite replication within infected cells.

  19. 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

  20. 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

  1. Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

    PubMed

    Yamagami, Kanako; Yamamoto, Takaharu; Sakai, Shota; Mioka, Tetsuo; Sano, Takamitsu; Igarashi, Yasuyuki; Tanaka, Kazuma

    2015-01-01

    In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

  2. Preeclampsia and Extracellular Vesicles.

    PubMed

    Gilani, Sarwat I; Weissgerber, Tracey L; Garovic, Vesna D; Jayachandran, Muthuvel

    2016-09-01

    Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers. PMID:27590522

  3. A vesicle carrier that mediates peroxisome protein traffic from the endoplasmic reticulum.

    PubMed

    Lam, Sheung Kwan; Yoda, Naofumi; Schekman, Randy

    2010-12-14

    Pex19p, a soluble cytoplasmic transport protein, is required for the traffic of the peroxisomal membrane proteins Pex3p and Pex15p from the endoplasmic reticulum (ER) to the peroxisome. We documented Pex15p traffic from the ER using a chimeric protein containing a C-terminal glycosylation acceptor peptide. Pex15Gp expressed in wild-type yeast cells is N-glycosylated and functions properly in the peroxisome. In contrast, pex19Δ-mutant cells accumulate the glycoprotein Pex15Gp in the ER. We developed a cell-free preperoxisomal vesicle-budding reaction in which Pex15Gp and Pex3p are packaged into small vesicles in the presence of cytosol, Pex19p, and ATP. Secretory vesicle budding (COPII) detected by the packaging of a SNARE protein (soluble N-ethylmaleimide-sensitive attachment protein receptor) occurs in the same incubation but does not depend on Pex19p. Conversely a dominant GTPase mutant Sar1p which inhibits COPII has no effect on Pex3p packaging. Pex15Gp and Pex3p budded vesicles sediment as low-buoyant-density membranes on a Nycodenz gradient and copurify by affinity isolation using native but not Triton X-100-treated budded vesicles. ER-peroxisome transport vesicles appear to rely on a novel budding mechanism requiring Pex19p and additional unknown factors.

  4. Role of vesicle-mediated transport pathways in hepatocellular bile secretion.

    PubMed

    Crawford, J M

    1996-05-01

    Bile formation by hepatocytes involves the secretion of organic and inorganic solutes derived from a number of intracellular sources. Plasma-to-bile trafficking of bile salts and proteins, in particular, is a major route for solute movement through the hepatocyte. Intracellular vesicle trafficking is the primary pathway for delivery of plasma proteins to bile, via either fluid-phase or receptor-mediated endocytosis. In contrast, bile salts do not appear to traffic via vesicles. Rather, bile salts appear to promote the insertion of vesicles containing the apical transport proteins into the hepatocyte canalicular membrane. Lysosomal protein also is released into bile by fusion of vesicles or possibly of tubular lysosomes with the canalicular membrane. Structural phospholipid is presumably delivered to the canalicular membrane as part of vesicular traffic, but biliary phosphatidylcholine molecules are more likely delivered via binding to cytosolic transfer proteins. Cholesterol may be delivered either via cystolic proteins or via vesicular trafficking, the latter in conjunction with sphingomyelin recycling to and from the canalicular membrane. Lastly, the primary mechanism for phospholipid secretion into bile appears to be the budding of phospholipid vesicles from the exoplasmic hemileaflet of the hepatocyte canalicular membrane. Thus, vesicle-mediated pathways play a major role in a number of bile secretory mechanisms.

  5. Principles of Flexible Protein-Protein Docking

    PubMed Central

    Andrusier, Nelly; Mashiach, Efrat; Nussinov, Ruth; Wolfson, Haim J.

    2008-01-01

    Treating flexibility in molecular docking is a major challenge in cell biology research. Here we describe the background and the principles of existing flexible protein-protein docking methods, focusing on the algorithms and their rational. We describe how protein flexibility is treated in different stages of the docking process: in the preprocessing stage, rigid and flexible parts are identified and their possible conformations are modeled. This preprocessing provides information for the subsequent docking and refinement stages. In the docking stage, an ensemble of pre-generated conformations or the identified rigid domains may be docked separately. In the refinement stage, small-scale movements of the backbone and side-chains are modeled and the binding orientation is improved by rigid-body adjustments. For clarity of presentation, we divide the different methods into categories. This should allow the reader to focus on the most suitable method for a particular docking problem. PMID:18655061

  6. Giant vesicles: preparations and applications.

    PubMed

    Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale

    2010-05-01

    There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed. PMID:20336703

  7. The juxtamembrane region of synaptotagmin 1 interacts with dynamin 1 and regulates vesicle fission during compensatory endocytosis in endocrine cells.

    PubMed

    McAdam, Robyn L; Varga, Kelly T; Jiang, Zhongjiao; Young, Fiona B; Blandford, Vanessa; McPherson, Peter S; Gong, Liang-Wei; Sossin, Wayne S

    2015-06-15

    Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that is important for the kinetics of both exocytosis and endocytosis, and is thus a candidate molecule to link these two processes. Although the tandem Ca(2+)-binding C2 domains of Syt1 have important roles in exocytosis and endocytosis, the function of the conserved juxtamembrane (jxm) linker region has yet to be determined. We now demonstrate that the jxm region of Syt1 interacts directly with the pleckstrin homology (PH) domain of the endocytic protein dynamin 1. By using cell-attached capacitance recordings with millisecond time resolution to monitor clathrin-mediated endocytosis of single vesicles in neuroendocrine chromaffin cells, we find that loss of this interaction prolongs the lifetime of the fission pore leading to defects in the dynamics of vesicle fission. These results indicate a previously undescribed interaction between two major regulatory proteins in the secretory vesicle cycle and that this interaction regulates endocytosis.

  8. 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.

  9. 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.

  10. 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

  11. Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6).

    PubMed

    Mitchell, Kathryn J; Lai, F Anthony; Rutter, Guy A

    2003-03-28

    We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.

  12. 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

  13. 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. PMID:26048670

  14. 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

  15. Reversible Recruitment of a Homeostatic Reserve Pool of Synaptic Vesicles Underlies Rapid Homeostatic Plasticity of Quantal Content.

    PubMed

    Wang, Xueyong; Pinter, Martin J; Rich, Mark M

    2016-01-20

    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.

  16. 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

  17. 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.

  18. Dimerization of DOCK2 is essential for DOCK2-mediated Rac activation and lymphocyte migration.

    PubMed

    Terasawa, Masao; Uruno, Takehito; Mori, Sayako; Kukimoto-Niino, Mutsuko; Nishikimi, Akihiko; Sanematsu, Fumiyuki; Tanaka, Yoshihiko; Yokoyama, Shigeyuki; Fukui, Yoshinori

    2012-01-01

    The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane. PMID:23050005

  19. Nanotube-Enabled Vesicle-Vesicle Communication: A Computational Model.

    PubMed

    Zhang, Liuyang; Wang, Xianqiao

    2015-07-01

    Cell-to-cell communications via the tunneling nanotubes or gap junction channels are vital for the development and maintenance of multicellular organisms. Instead of these intrinsic communication pathways, how to design artificial communication channels between cells remains a challenging but interesting problem. Here, we perform dissipative particle dynamics (DPD) simulations to analyze the interaction between rotational nanotubes (RNTs) and vesicles so as to provide a novel design mechanism for cell-to-cell communication. Simulation results have demonstrated that the RNTs are capable of generating local disturbance and promote vesicle translocation toward the RNTs. Through ligand pattern designing on the RNTs, we can find a suitable nanotube candidate with a specific ligand coating pattern for forming the RNT-vesicle network. The results also show that a RNT can act as a bridged channel between vesicles, which facilitates substance transfer. Our findings provide useful guidelines for the molecular design of patterned RNTs for creating a synthetic channel between cells. PMID:26266730

  20. Nanotube-Enabled Vesicle-Vesicle Communication: A Computational Model.

    PubMed

    Zhang, Liuyang; Wang, Xianqiao

    2015-07-01

    Cell-to-cell communications via the tunneling nanotubes or gap junction channels are vital for the development and maintenance of multicellular organisms. Instead of these intrinsic communication pathways, how to design artificial communication channels between cells remains a challenging but interesting problem. Here, we perform dissipative particle dynamics (DPD) simulations to analyze the interaction between rotational nanotubes (RNTs) and vesicles so as to provide a novel design mechanism for cell-to-cell communication. Simulation results have demonstrated that the RNTs are capable of generating local disturbance and promote vesicle translocation toward the RNTs. Through ligand pattern designing on the RNTs, we can find a suitable nanotube candidate with a specific ligand coating pattern for forming the RNT-vesicle network. The results also show that a RNT can act as a bridged channel between vesicles, which facilitates substance transfer. Our findings provide useful guidelines for the molecular design of patterned RNTs for creating a synthetic channel between cells.

  1. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    PubMed

    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

  2. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    PubMed

    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

  3. Electron tomographic characterization of a vacuolar reticulum and of six vesicle types that occupy different cytoplasmic domains in the apex of tip-growing Chara rhizoids.

    PubMed

    Limbach, Christoph; Staehelin, L Andrew; Sievers, Andreas; Braun, Markus

    2008-04-01

    We provide a 3D ultrastructural analysis of the membrane systems involved in tip growth of rhizoids of the green alga Chara. Electron tomography of cells preserved by high-pressure freeze fixation has enabled us to distinguish six different types of vesicles in the apical cytoplasm where the tip growth machinery is accommodated. The vesicle types are: dark and light secretory vesicles, plasma membrane-associated clathrin-coated vesicles (PM-CCVs), Spitzenkoerper-associated clathrin-coated vesicles (Sp-CCVs) and coated vesicles (Sp-CVs), and microvesicles. Each of these vesicle types exhibits a distinct distribution pattern, which provides insights into their possible function for tip growth. The PM-CCVs are confined to the cytoplasm adjacent to the apical plasma membrane. Within this space they are arranged in clusters often surrounding tubular plasma membrane invaginations from which CCVs bud. This suggests that endocytosis and membrane recycling are locally confined to specialized apical endocytosis sites. In contrast, exocytosis of secretory vesicles occurs over the entire membrane area of the apical dome. The Sp-CCVs and the Sp-CVs are associated with the aggregate of endoplasmic reticulum membranes in the center of the growth-organizing Spitzenkoerper complex. Here, Sp-CCVs are seen to bud from undefined tubular membranes. The subapical region of rhizoids contains a vacuolar reticulum that extends along the longitudinal cell axis and consists of large, vesicle-like segments interconnected by thin tubular domains. The tubular domains are encompassed by thin filamentous structures resembling dynamin spirals which could drive peristaltic movements of the vacuolar reticulum similar to those observed in fungal hyphae. The vacuolar reticulum appears to serve as a lytic compartment into which multivesicular bodies deliver their internal vesicles for molecular recycling and degradation.

  4. Ca2+ dialogue between acidic vesicles and ER.

    PubMed

    Morgan, Anthony J

    2016-04-15

    Extracellular stimuli evoke the synthesis of intracellular second messengers, several of which couple to the release of Ca(2+)from Ca(2+)-storing organelles via activation of cognate organellar Ca(2+)-channel complexes. The archetype is the inositol 1,4,5-trisphosphate (IP3) and IP3receptor (IP3R) on the endoplasmic reticulum (ER). A less understood, parallel Ca(2+)signalling cascade is that involving the messenger nicotinic acid adenine dinucleotide phosphate (NAADP) that couples to Ca(2+)release from acidic Ca(2+)stores [e.g. endo-lysosomes, secretory vesicles, lysosome-related organelles (LROs)]. NAADP-induced Ca(2+)release absolutely requires organellar TPCs (two-pore channels). This review discusses how ER and acidic Ca(2+)stores physically and functionally interact to generate and shape global and local Ca(2+)signals, with particular emphasis on the two-way dialogue between these two organelles.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Secretory IgA synthesis in Kwashiorkor.

    PubMed

    Beatty, D W; Napier, B; Sinclair-Smith, C C; McCabe, K; Hughes, E J

    1983-09-01

    The synthesis of intestinal secretory IgA was studied in in vitro cultures of duodenal mucosal biopsies from children with Kwashiorkor. Production of secretory IgA was measured by the incorporation of radioactive label and visualized following PAGE and autoradiography. Results obtained before and after nutritional rehabilitation demonstrate an enhanced synthesis of sIgA in children with acute Kwashiorkor. Histological examination of plasma cells in the biopsy tissue confirms a twofold increase in IgA staining plasma cells in acute Kwashiorkor. Peripheral blood B lymphocytes in acute Kwashiorkor however, showed a reduction in IgA synthesis in the acute stage. These results suggest an effective mucosal sIgA response to the increased intestinal antigen load in Kwashiorkor.

  10. Yeast secretory expression of insulin precursors.

    PubMed

    Kjeldsen, T

    2000-09-01

    Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield. PMID:11030562

  11. 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

  12. Islet secretory granules contain cytochrome b561.

    PubMed

    Mackin, R B; Jones, D P; Noe, B D

    1986-08-01

    A cytochrome has been detected in secretory granules prepared from anglerfish islets of Langerhans. The heme moiety was determined to be of the b type, and the dithionite-reduced cytochrome exhibited an alpha-band maximum at 561 nm with an extinction coefficient of 13.8 mM-1 X cm-1. The protein was present at a concentration of 40 +/- 4 pmol/mg of secretory granule protein. The cytochrome was found to be an integral membrane protein and to be reduced by ascorbic acid but not by NADH, NADPH, reduced glutathione (GSH), or succinate. Because of the similarity to previously characterized secretory granule cytochrome b561's from neuroendocrine tissues, this cytochrome is also referred to as cytochrome b561. Although its function has not yet been elucidated, the apparent specificity for ascorbate suggests that it may be a component of the ascorbate-dependent peptidyl-glycine alpha-amidating monooxygenase system that functions in the amidation of islet hormones. PMID:3525285

  13. 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.

  14. The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

    PubMed

    Binotti, Beyenech; Pavlos, Nathan J; Riedel, Dietmar; Wenzel, Dirk; Vorbrüggen, Gerd; Schalk, Amanda M; Kühnel, Karin; Boyken, Janina; Erck, Christian; Martens, Henrik; Chua, John J E; Jahn, Reinhard

    2015-01-01

    Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

  15. 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

  16. Secretory activity is rapidly induced in stigmatic papillae by compatible pollen, but inhibited for self-incompatible pollen in the Brassicaceae.

    PubMed

    Safavian, Darya; Goring, Daphne R

    2013-01-01

    [In the Brassicaceae, targeted exocytosis to the stigmatic papillar plasma membrane under the compatible pollen grain is hypothesized to be essential for pollen hydration and pollen tube penetration. In contrast, polarized secretion is proposed to be inhibited in the stigmatic papillae during the rejection of self-incompatible pollen. Using transmission electron microscopy (TEM), we performed a detailed time-course of post-pollination events to view the cytological responses of the stigmatic papillae to compatible and self-incompatible pollinations. For compatible pollinations in Arabidopsis thaliana and Arabidopsis lyrata, vesicle secretion was observed at the stigmatic papillar plasma membrane under the pollen grain while Brassica napus stigmatic papillae appeared to use multivesicular bodies (MVBs) for secretion. Exo70A1, a component of the exocyst complex, has been previously implicated in the compatible pollen responses, and disruption of Exo70A1 in both A. thaliana and B. napus resulted in a loss of secretory vesicles/MVBs at the stigmatic papillar plasma membrane. Similarly, for self-incompatible pollinations, secretory vesicles/MVBs were absent from the stigmatic papillar plasma membrane in A. lyrata and B. napus; and furthermore, autophagy appeared to be induced to direct vesicles/MVBs to the vacuole for degradation. Thus, these findings support a model where the basal pollen recognition pathway in the stigmatic papilla promotes exocytosis to accept compatible pollen, and the basal pollen recognition pathway is overridden by the self-incompatibility pathway to prevent exocytosis and reject self-pollen. PMID:24386363

  17. Oncogenic extracellular vesicles in brain tumor progression.

    PubMed

    D'Asti, Esterina; Garnier, Delphine; Lee, Tae H; Montermini, Laura; Meehan, Brian; Rak, Janusz

    2012-01-01

    The brain is a frequent site of neoplastic growth, including both primary and metastatic tumors. The clinical intractability of many brain tumors and their distinct biology are implicitly linked to the unique microenvironment of the central nervous system (CNS) and cellular interactions within. Among the most intriguing forms of cellular interactions is that mediated by membrane-derived extracellular vesicles (EVs). Their biogenesis (vesiculation) and uptake by recipient cells serves as a unique mechanism of intercellular trafficking of complex biological messages including the exchange of molecules that cannot be released through classical secretory pathways, or that are prone to extracellular degradation. Tumor cells produce EVs containing molecular effectors of several cancer-related processes such as growth, invasion, drug resistance, angiogenesis, and coagulopathy. Notably, tumor-derived EVs (oncosomes) also contain oncogenic proteins, transcripts, DNA, and microRNA (miR). Uptake of this material may change properties of the recipient cells and impact the tumor microenvironment. Examples of transformation-related molecules found in the cargo of tumor-derived EVs include the oncogenic epidermal growth factor receptor (EGFRvIII), tumor suppressors (PTEN), and oncomirs (miR-520g). It is postulated that EVs circulating in blood or cerebrospinal fluid (CSF) of brain tumor patients may be used to decipher molecular features (mutations) of the underlying malignancy, reflect responses to therapy, or molecular subtypes of primary brain tumors [e.g., glioma or medulloblastoma (MB)]. It is possible that metastases to the brain may also emit EVs with clinically relevant oncogenic signatures. Thus, EVs emerge as a novel and functionally important vehicle of intercellular communication that can mediate multiple biological effects. In addition, they provide a unique platform to develop molecular biomarkers in brain malignancies. PMID:22934045

  18. 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.

  19. Storage and degradation of secretory proteins in adenomatous and secondary hyperplastic parathyroid cells. An immunoelectron microscope study.

    PubMed

    Berger, G; Berger, F; Billard, F; Danowski, J; Vauzelle, J L

    1989-01-01

    Parathyroid hormone (PTH) and chromogranin A/secretory protein-I (SP-I) have been localized on immunoelectron microscopy in double-fixed tissues from adenomatous and secondary hyperplastic parathyroid glands. Storage organelles, identified on the basis of their consistent labelling, included tow morphologically distinct varieties of granules/vesicles; the mature granules and the progranules. The former consisted of dense, mostly rounded, medium to large-sized bodies which were strongly labelled and predominant in the proximity of the cell membrane. The other variety of body included a spectrum of small pale vesicles/granules which were mainly located in the Golgi area. Because their morphology and their labelling pattern varied other bodies were assumed to be engaged in degradation or cleavage of the secretory proteins. These bodies comprised crinophagic structures, that is to say multivesicular bodies and large Golgi-related vesicles, as well as a number of atypical solid bodies. Whereas most of the granulated cells stored a mature or a maturing population of vesicles/granules, the process of maturation appeared to be either absent or incomplete in a number of cells from some glands. The major defects were frequently associated with an unusual labelling pattern of the Golgi area and selectively affected groups of cells from all the transitional oxyphil cell adenomas. The minor defects concerned individual cells of different types present in both categories of glands. The present data suggest that in hyperfunctioning glands, the type of hormone processing depends on the capacity of each cell in progranule maturation and that the maturation capacity may decrease dramatically in adenomatous or chronically hyperstimulated cells of the transitional oxyphil type. PMID:2505443

  20. Contextual view of building 110 with dry dock 1 visible ...

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

    Contextual view of building 110 with dry dock 1 visible on left; camera facing southeast. - Mare Island Naval Shipyard, Pump House, California Avenue, east side between Dry Dock 1 & Dry Dock 2, near Ninth Street, Vallejo, Solano County, CA

  1. Contextual view of building 110 with dry dock 2 in ...

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

    Contextual view of building 110 with dry dock 2 in foreground; camera facing northeast. - Mare Island Naval Shipyard, Pump House, California Avenue, east side between Dry Dock 1 & Dry Dock 2, near Ninth Street, Vallejo, Solano County, CA

  2. 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

  3. Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment

    PubMed Central

    1992-01-01

    We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C- peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway. PMID:1639842

  4. Catecholamine Storage Vesicles: Role of Core Protein Genetic Polymorphisms in Hypertension

    PubMed Central

    Zhang, Kuixing; Chen, Yuqing; Wen, Gen; Mahata, Manjula; Rao, Fangwen; Fung, Maple M.; Vaingankar, Sucheta; Biswas, Nilima; Gayen, Jiaur R.; Friese, Ryan S.; Mahata, Sushil K.; Hamilton, Bruce A.

    2010-01-01

    Hypertension is a complex trait with deranged autonomic control of the circulation. The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Catecholamine storage vesicles (or chromaffin granules) of the adrenal medulla contain remarkably high concentrations of chromogranins/secretogranins (or “granins”), catecholamines, neuropeptide Y, adenosine triphosphate (ATP), and Ca2+. Within secretory granules, granins are co-stored with catecholamine neurotransmitters and co-released upon stimulation of the regulated secretory pathway. The principal granin family members, chromogranin A (CHGA), chromogranin B (CHGB), and secretogranin II (SCG2), may have evolved from shared ancestral exons by gene duplication. This article reviews human genetic variation at loci encoding the major granins and probes the effects of such polymorphisms on blood pressure, using twin pairs to probe heritability and individuals with the most extreme blood pressure values in the population to study hypertension. PMID:21104344

  5. 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

  6. Dynamic testing of docking system hardware

    NASA Technical Reports Server (NTRS)

    Dorland, W. D.

    1972-01-01

    Extensive dynamic testing was conducted to verify the flight readiness of the Apollo docking hardware. Testing was performed on a unique six degree-of-freedom motion simulator controlled by a computer that calculated the associated spacecraft motions. The test system and the results obtained by subjecting flight-type docking hardware to actual impact loads and resultant spacecraft dynamics are described.

  7. Spacecraft Docks Under Six Hours After Launch

    NASA Video Gallery

    The unpiloted ISS Progress 48 Russian cargo ship docked at 9:18 p.m. EDT Aug. 1 to the Pirs docking compartment of the International Space Station. The resupply spacecraft launched at 3:35 p.m. and...

  8. Self-docking analysis and velocity-aimed control for spacecraft electromagnetic docking

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan-wen; Yang, Le-ping; Zhu, Yan-wei; Ao, Hou-jun; Qi, Da-wei

    2016-06-01

    As a novel and potential supporting technology for on-orbit operation missions, spacecraft electromagnetic docking has not only distinct visible advantages, but also several intrinsic unconspicuous capabilities, such as the self-docking capability which could be exploited to alleviate the burden of the docking controller. Based on theoretical derivation and comparison with the near-field model and numerical simulation, the feasibility of utilizing the far-field electromagnetic force/torque model to spacecraft electromagnetic docking characteristics analysis is firstly verified. Then, the self-docking capability is studied with self-alignment and self-attraction analysis, and the necessary condition for the former and the sufficient condition for the latter are derived. Finally, a velocity-aimed electromagnetic docking control approach based on the self-docking capability and the conservation laws is put forward and verified by numerical simulations.

  9. Computational protein-ligand docking and virtual drug screening with the AutoDock suite.

    PubMed

    Forli, Stefano; Huey, Ruth; Pique, Michael E; Sanner, Michel F; Goodsell, David S; Olson, Arthur J

    2016-05-01

    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 it is applied to structure-based drug design. The methods are fast enough to allow virtual screening of ligand libraries containing tens of thousands of compounds. This protocol covers the docking and virtual screening methods provided by the AutoDock suite of programs, including a basic docking of a drug molecule with an anticancer target, a virtual screen of this target with a small ligand library, docking with selective receptor flexibility, active site prediction and docking with explicit hydration. The entire protocol will require ∼5 h. PMID:27077332

  10. Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles

    PubMed Central

    1996-01-01

    Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content. PMID:8707851

  11. PTools: an opensource molecular docking library

    PubMed Central

    Saladin, Adrien; Fiorucci, Sébastien; Poulain, Pierre; Prévost, Chantal; Zacharias, Martin

    2009-01-01

    Background Macromolecular docking is a challenging field of bioinformatics. Developing new algorithms is a slow process generally involving routine tasks that should be found in a robust library and not programmed from scratch for every new software application. Results We present an object-oriented Python/C++ library to help the development of new docking methods. This library contains low-level routines like PDB-format manipulation functions as well as high-level tools for docking and analyzing results. We also illustrate the ease of use of this library with the detailed implementation of a 3-body docking procedure. Conclusion The PTools library can handle molecules at coarse-grained or atomic resolution and allows users to rapidly develop new software. The library is already in use for protein-protein and protein-DNA docking with the ATTRACT program and for simulation analysis. This library is freely available under the GNU GPL license, together with detailed documentation. PMID:19409097

  12. 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

  13. 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.

  14. Isotonic water transport in secretory epithelia.

    PubMed

    Swanson, C H

    1977-01-01

    The model proposed by Diamond and Bossert [1] for isotonic water transport has received wide acceptance in recent years. It assumes that the local driving force for water transport is a standing osmotic gradient produced in the lateral intercellular spaces of the epithelial cell layer by active solute transport. While this model is based on work done in absorptive epithelia where the closed to open direction of the lateral space and the direction of net transport are the same, it has been proposed that the lateral spaces could also serve as the site of the local osmotic gradients for water transport in secretory epithelia, where the closed to open direction of the lateral space and net transport are opposed, by actively transporting solute out of the space rather than into it. Operation in the backward direction, however, requires a lower than ambient hydrostatic pressure within the lateral space which would seem more likely to cause the space to collapse with loss of function. On the other hand, most secretory epithelia are characterized by transport into a restricted ductal system which is similar to the lateral intercellular space in the absorptive epithelia in that its closed to open direction is the same as that of net transport. In vitro micropuncture studies on the exocrine pancreas of the rabbit indicate the presence of a small but statistically significant increase in juice osmolality, 6 mOsm/kg H(2)O, at the site of electrolyte and water secretion in the smallest extralobular ducts with secretin stimulation which suggests that the ductal system in the secretory epithelia rather than the lateral intercellular space is the site of the local osmotic gradients responsible for isotonic water transport. PMID:331693

  15. Psychological distress and salivary secretory immunity.

    PubMed

    Engeland, C G; Hugo, F N; Hilgert, J B; Nascimento, G G; Junges, R; Lim, H-J; Marucha, P T; Bosch, J A

    2016-02-01

    Stress-induced impairments of mucosal immunity may increase susceptibility to infectious diseases. The present study investigated the association of perceived stress, depressive symptoms, and loneliness with salivary levels of secretory immunoglobulin A (S-IgA), the subclasses S-IgA1, S-IgA2, and their transporter molecule Secretory Component (SC). S-IgA/SC, IgA1/SC and IgA2/SC ratios were calculated to assess the differential effects of stress on immunoglobulin transport versus availability. This study involved 113 university students, in part selected on high scores on the UCLA Loneliness Scale and/or the Beck Depression Inventory. Stress levels were assessed using the Perceived Stress Scale. Unstimulated saliva was collected and analysed for total S-IgA and its subclasses, as well as SC and total salivary protein. Multiple linear regression analyses, adjusted for gender, age, health behaviours, and concentration effects (total protein) revealed that higher perceived stress was associated with lower levels of IgA1 but not IgA2. Perceived stress, loneliness and depressive symptoms were all associated with lower IgA1/SC ratios. Surprisingly, higher SC levels were associated with loneliness and depressive symptoms, indicative of enhanced transport activity, which explained a lower IgA1/SC ratio (loneliness and depression) and IgA2/SC ratio (depression). This is the first study to investigate the effects of protracted psychological stress across S-IgA subclasses and its transporter SC. Psychological stress was negatively associated with secretory immunity, specifically IgA1. The lower immunoglobulin/transporter ratio that was associated with higher loneliness and depression suggested a relative immunoglobulin depletion, whereby availability was not keeping up with enhanced transport demand.

  16. Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast.

    PubMed

    Neiman, A M

    1998-01-12

    Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm. This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei. The effect of mutations in late-acting genes on sporulation was investigated. Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane. The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation. The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20. These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell. Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells. Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems.

  17. 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

  18. Immunomodulatory action of mycobacterial secretory proteins.

    PubMed

    Trajkovic, Vladimir; Natarajan, Krishnamurthy; Sharma, Pawan

    2004-04-01

    The recently discovered RD1 locus encodes proteins that are actively secreted by pathogenic mycobacteria, including Mycobacterium tuberculosis. Since they are missing in non-tuberculous mycobacteria, these proteins are promising not only as candidates for vaccination and diagnostic tests, but also in understanding mycobacterial evasion of protective immunity in susceptible individuals. Here we analyze the possible role of M. tuberculosis secretory proteins in immunity against tuberculosis, with emphasis on their immunomodulatory action and the potential involvement in mycobacterial subversion of the host immune defense.

  19. "Secretory" Carcinoma of the Skin Mimicking Secretory Carcinoma of the Breast: Case Report and Literature Review.

    PubMed

    Huang, Sixia; Liu, Yan; Su, Jing; Liu, Jianying; Guo, Xiaoning; Mei, Fang; Zheng, Jie; Liao, Songlin

    2016-09-01

    Secretory carcinoma is a unique kind of adenocarcinoma. It has distinct histological features and a special genetic change, that is, t (12; 15) (p13; q25) translocation which leads to the expression of the ETV6-NTRK3 fusion gene. Secretory carcinoma has been found to occur both in the breast and salivary gland. Here the authors present a case of 22-year-old woman with a unique cutaneous neoplasm located at the axilla. The tumor was characterized histologically with the formation of round to ovoid microcysts and papillary structure, which was similar to the secretory carcinoma of the breast and salivary gland. Furthermore, the gene sequence analysis of reverse-transcription polymerase chain reaction products demonstrated the expression of the ETV6-NTRK3 fusion gene. To the authors' knowledge, this is the first case of secretory carcinoma from the skin which has the same genetic change as those from the breast and salivary gland. Local excision was performed on this patient. She had been followed up for nearly 1 year. No recurrence or metastasis was found yet. PMID:26981741

  20. 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

  1. 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.

  2. Advanced Docking Berthing System Update

    NASA Technical Reports Server (NTRS)

    Lewis, James

    2006-01-01

    In FY05 the Exploration Systems Technology Maturation Program selected the JSC advanced mating systems development to continue as an in-house project. In FY06, as a result of ESAS Study (60 Day Study) the CEV Project (within the Constellation Program) has chosen to continue the project as a GFE Flight Hardware development effort. New requirement for CEV to travel and dock with the ISS in 2011/12 in support of retiring the Shuttle and reducing the gap of time where US does not have any US based crew launch capability. As before, long-duration compatible seal-on-seal technology (seal-on-seal to support androgynous interface) has been identified as a risk mitigation item.

  3. Computational methods for molecular docking

    SciTech Connect

    Klebe, G.; Lengauer, T.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. Recently, it has been demonstrated that the knowledge of the three-dimensional structure of the protein can be used to derive new protein ligands with improved binding properties. This tutorial focuses on the following questions: What is its binding affinity toward a particular receptor? What are putative conformations of a ligand at the binding site? What are the similarities of different ligands in terms of their recognition capabilities? Where and in which orientation will a ligand bind to the active site? How is a new putative protein ligand selected? An overview is presented of the algorithms which are presently used to handle and predict protein-ligand interactions and to dock small molecule ligands into proteins.

  4. 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

  5. Membrane trafficking. The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins.

    PubMed

    Wong, Mie; Munro, Sean

    2014-10-31

    The Golgi apparatus is a multicompartment central sorting station at the intersection of secretory and endocytic vesicular traffic. The mechanisms that permit cargo-loaded transport vesicles from different origins to selectively access different Golgi compartments are incompletely understood. We developed a rerouting and capture assay to investigate systematically the vesicle-tethering activities of 10 widely conserved golgin coiled-coil proteins. We find that subsets of golgins with distinct localizations on the Golgi surface have capture activities toward vesicles of different origins. These findings demonstrate that golgins act as tethers in vivo, and hence the specificity we find to be encoded in this tethering is likely to make a major contribution to the organization of membrane traffic at the Golgi apparatus.

  6. Ultrastructural localization of glycodelin oligosaccharides Le-x and Le-y in human seminal vesicles by immunogold staining

    PubMed Central

    Piludu, M; Cossu, M; De Lisa, A; Piras, M; Lantini, M S

    2007-01-01

    Histo-blood group antigens Le-x and Le-y are oligosaccharidic terminals that characterize many glycoproteins in the human tissues. In seminal plasma, they are expressed as part of the so-called glycodelin S, which is suggested to regulate sperm capacitation/decapacitation. It has recently been demonstrated that the core protein of glycodelin S is secreted by seminal vesicles. Here we show that epithelial cells of human seminal vesicles also release the Le-x and Le-y antigens. The presence of these substances in secretory material was revealed by means of an immunogold staining method in normal surgical samples. The results suggest that glycodelin S is secreted by seminal vesicles in its finished glycosylated form. Moreover, antigen reactivity was also revealed associated with plasma membranes. PMID:17331183

  7. Laser docking sensor engineering model

    NASA Astrophysics Data System (ADS)

    Dekome, Kent; Barr, Joseph M.

    NASA JSC has been involved in the development of Laser sensors for the past ten years in order to support future rendezvous and docking missions, both manned and unmanned. Although many candidate technologies have been breadboarded and evaluated, no sensor hardware designed specifically for rendezvous and docking applications has been demonstrated on-orbit. It has become apparent that representative sensors need to be flown and demonstrated as soon as possible, with minimal cost, to provide the capability of the technology in meeting NASA's future AR&C applications. Technology and commercial component reliability have progressed to where it is now feasible to fly hardware as a detailed test objective minimizing the overall cost and development time. This presentation will discuss the ongoing effort to convert an existing in-house developed breadboard to an engineering model configuration suitable for flight. The modifications include improving the ranger resolution and stability with an in-house design, replacing the rack mounted galvanometric scanner drivers with STD-bus cards, replacing the system controlling personal computer with a microcontroller, and repackaging the subsystems as appropriate. The sensor will use the performance parameters defined in previous JSC requirements working groups as design goals and be built to withstand the space environment where fiscally feasible. Testing of the in-house ranger design is expected to be completed in October. The results will be included in the presentation. Preliminary testing of the ranging circuitry indicates a range resolution of 4mm is possible. The sensor will be mounted in the payload bay on a shelf bracket and have command, control, and display capabilities using the payload general support computer via an RS422 data line.

  8. Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

    NASA Astrophysics Data System (ADS)

    Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

    2000-04-01

    Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

  9. Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides.

    PubMed

    Yanagimoto, Chikatoshi; Harada, Masaru; Kumemura, Hiroto; Koga, Hironori; Kawaguchi, Takumi; Terada, Kunihiko; Hanada, Shinichiro; Taniguchi, Eitaro; Koizumi, Yukio; Koyota, Souichi; Ninomiya, Haruaki; Ueno, Takato; Sugiyama, Toshihiro; Sata, Michio

    2009-01-15

    Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.

  10. Small GTPases in vesicle trafficking.

    PubMed

    Molendijk, Arthur J; Ruperti, Benedetto; Palme, Klaus

    2004-12-01

    Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.

  11. Effects of wood preservative leachates from docks

    SciTech Connect

    Wendt, P.H.; Van Dolah, R.F.; Bobo, M.Y.; Mathews, T.D.

    1994-12-31

    Recent evidence indicates that the wood preservative commonly used in dock pilings (chromated copper arsenate or CCA) is highly toxic to several estuarine organisms in laboratory experiments. Increasing demand for residential docks prompted a field study intended to complement these earlier laboratory investigations. Objectives of the study were to: (1) examine concentrations of Cu, Cr, and As in sediments and oysters from intertidal locations in several creeks with and without high densities of docks; (2) examine the bioaccumulation of wood preservative leachates by laboratory-reared oysters transferred to field sites near and distant from newly constructed docks; and (3) investigate the acute toxicity of wood preservative leachates for several species of estuarine fishes and invertebrates exposed to these compounds in the field. Preliminary results indicate that sediment concentrations of all three metals were well below ER-L levels reported by Long and Morgan at all but one dock site. In an ancillary study, 24h LC{sub 50} bioassays were performed using rotifers (Brachionus plicatilis) which were exposed to pore water from sediments in creeks with and without docks. Toxicities of bulk sediments from the same sites were examined using Microtox which measures decreases in bioluminescence of marine bacteria (Photobacterium phosphoreum) as a function of sediment concentration. Neither the rotifer nor the Microtox bioassays showed any significant differences in toxicity between creeks with and without docks.

  12. 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.

  13. Zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity

    PubMed Central

    Thorn, Peter; Fogarty, Kevin E.; Parker, Ian

    2004-01-01

    The dynamics of the fusion pore that forms between a secretory vesicle and the plasma membrane are important in the regulation of both exocytosis and endocytosis. Here, we describe characteristics of fusion during zymogen granule exocytosis in exocrine pancreatic acinar cells. By using fluorescence recovery after photobleaching techniques, we show that the fusion pore remains open to allow free aqueous exchange with the vesicle lumen. There is no lipid interchange between the plasma and granule membranes during this time, and at the end of its life, the intact granule shrinks in situ, probably by a gradual pinching off of membrane patches. We propose that the protracted fusion pore lifetime is adapted to permit compound exocytosis, whereby the lingering primary granule acts as a conduit through which the contents of a secondary granule can be released. The lack of lipid intermixing may then facilitate selective recycling of granule membrane and preservation of apical membrane integrity. PMID:15090649

  14. Secretory pattern of canine growth hormone

    SciTech Connect

    French, M.B.; Vaitkus, P.; Cukerman, E.; Sirek, A.; Sirek, O.V.

    1987-02-01

    The aim of this paper was to define the secretory pattern of growth hormone (GH) under basal conditions in fasted, conscious, male dogs accustomed to handling. Blood samples were withdrawn from a cephalic vein at 15-min intervals. In this way, any ultradian rhythms, if present, could be detected within the frequency range of 0.042-2 cycles/h. In addition, samples were drawn at either 1- or 2.5-min intervals for 2.5 or 5 h to determine whether frequency components greater than 2 cycles/h were present. GH was measured by radioimmunoassay and the raw data were submitted to time series analysis employing power spectral estimation by means of fast Fourier transformation techniques. Peak plasma levels were up to 12 times higher than the baseline concentration of approx. 1 ng/ml. Spectral analysis revealed an endogenous frequency of 0.22 cycles/h, i.e., a periodicity of 4.5 h/cycle. The results indicate that under basal conditions the secretory bursts of canine GH are limited to one peak every 4.5 h.

  15. Copper trafficking to the secretory pathway.

    PubMed

    Lutsenko, Svetlana

    2016-09-01

    Copper (Cu) is indispensible for growth and development of human organisms. It is required for such fundamental and ubiquitous processes as respiration and protection against reactive oxygen species. Cu also enables catalytic activity of enzymes that critically contribute to the functional identity of many cells and tissues. Pigmentation, production of norepinephrine by the adrenal gland, the key steps in the formation of connective tissue, neuroendocrine signaling, wound healing - all these processes require Cu and depend on Cu entering the secretory pathway. To reach the Cu-dependent enzymes in a lumen of the trans-Golgi network and various vesicular compartments, Cu undertakes a complex journey crossing the extracellular and intracellular membranes and staying firmly on course while traveling in a cytosol. The proteins that assist Cu in this journey by mediating its entry, distribution, and export, have been identified. The accumulating data also indicate that the current model of cellular Cu homeostasis is still a "skeleton" that has to be fleshed out with many new details. This review summarizes recent data on the mechanisms responsible for Cu transfer to the secretory pathway. The emerging new concepts and gaps in our knowledge are discussed. PMID:27603756

  16. Advanced Docking System With Magnetic Initial Capture

    NASA Technical Reports Server (NTRS)

    Lewis, James L.; Carroll, Monty B.; Morales, Ray; Le, Thang

    2004-01-01

    An advanced docking system is undergoing development to enable softer, safer docking than was possible when using prior docking systems. This system is intended for original use in docking of visiting spacecraft and berthing the Crew Return Vehicle at the International Space Station (ISS). The system could also be adapted to a variety of other uses in outer space and on Earth, including mating submersible vehicles, assembling structures, and robotic berthing/handling of payloads and cargo. Heretofore, two large spacecraft have been docked by causing the spacecraft to approach each other at a speed sufficient to activate capture latches - a procedure that results in large docking loads and is made more difficult because of the speed. The basic design and mode of operation of the present advanced docking system would eliminate the need to rely on speed of approach to activate capture latches, thereby making it possible to reduce approach speed and thus docking loads substantially. The system would comprise an active subsystem on one spacecraft and a passive subsystem on another spacecraft with which the active subsystem will be docked. The passive subsystem would include an extensible ring containing magnetic striker plates and guide petals. The active subsystem would include mating guide petals and electromagnets containing limit switches and would be arranged to mate with the magnetic striker plates and guide petals of the passive assembly. The electromagnets would be carried on (but not rigidly attached to) a structural ring that would be instrumented with load sensors. The outputs of the sensors would be sent, along with position information, as feedback to an electronic control subsystem. The system would also include electromechanical actuators that would extend or retract the ring upon command by the control subsystem.

  17. 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.

  18. Pearling of lipid vesicles induced by nanoparticles.

    PubMed

    Yu, Yan; Granick, Steve

    2009-10-14

    We show that cationic nanoparticles encapsulated within vesicles of phosphocholine lipid can induce pearling. The dynamic process occurs as two stages: formation of tubular protrusions followed by pearling instability. The breakup into individual vesicles can be controlled by nanoparticle concentration.

  19. Rosetta Ligand docking with flexible XML protocols.

    PubMed

    Lemmon, Gordon; Meiler, Jens

    2012-01-01

    RosettaLigand is premiere software for predicting how a protein and a small molecule interact. Benchmark studies demonstrate that 70% of the top scoring RosettaLigand predicted interfaces are within 2Å RMSD from the crystal structure [1]. The latest release of Rosetta ligand software includes many new features, such as (1) docking of multiple ligands simultaneously, (2) representing ligands as fragments for greater flexibility, (3) redesign of the interface during docking, and (4) an XML script based interface that gives the user full control of the ligand docking protocol. PMID:22183535

  20. Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi.

    PubMed

    Moreno, Yovany; Nabhan, Joseph F; Solomon, Jonathan; Mackenzie, Charles D; Geary, Timothy G

    2010-11-16

    Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system.

  1. Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues.

    PubMed

    Rasila, Tiina; Lehtonen, Alexandra; Kanerva, Kristiina; Mäkitie, Laura T; Haglund, Caj; Andersson, Leif C

    2016-01-01

    Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated. PMID:26963840

  2. 21 CFR 866.5380 - Free secretory component immuno-logical test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques free secretory component (normally a portion of the secretory IgA antibody molecule) in body fluids. Measurement of free secretory component (protein molecules) aids in the diagnosis...

  3. 21 CFR 866.5380 - Free secretory component immuno-logical test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... immunochemical techniques free secretory component (normally a portion of the secretory IgA antibody molecule) in body fluids. Measurement of free secretory component (protein molecules) aids in the diagnosis...

  4. Ellipsoidal relaxation of electrodeformed vesicles

    NASA Astrophysics Data System (ADS)

    Yu, Miao; Lin, Hao; Lira, Rafael; Dimova, Rumiana; Riske, Karin

    2015-11-01

    Electrodeformation has been extensively applied to investigate the mechanical behavior of vesicles and cells. While the deformation process often exhibits complex behavior and reveals interesting physics, the relaxation process post-pulsation is equally intriguing yet less frequently studied. In this work theoretical analysis and experimental quantification on the ellipsoidal relaxation of vesicles are presented, which reveal the simplicity and universal aspects of this process. The Helfrich formula, which is derived only for equilibrated shapes, is shown to be applicable to dynamic situations such as in relaxation. A closed-form solution is derived which predicts the vesicle aspect ratio as a function of time. Scattered data are unified by a timescale, 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'' regime. The bending rigidity and the initial membrane tension can be simultaneously extracted from the data/model analysis, posing the current approach as an effective means for the mechanical analysis of biomembranes.

  5. Dry Dock No. 3 general overview. Looking toward head of ...

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

    Dry Dock No. 3 general overview. Looking toward head of dock. View facing southwest - U.S. Naval Base, Pearl Harbor, Dry Dock No. 3, On northern shoreline of shipyard, west of Dry Dock Nos. 1 & 2, near the intersection of Avenue G and Sixth Street, Pearl City, Honolulu County, HI

  6. Dry Dock No. 3. Detail view of sidewall, near center ...

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

    Dry Dock No. 3. Detail view of sidewall, near center of dock with stair leading to grade level. View facing east-southeast - U.S. Naval Base, Pearl Harbor, Dry Dock No. 3, On northern shoreline of shipyard, west of Dry Dock Nos. 1 & 2, near the intersection of Avenue G and Sixth Street, Pearl City, Honolulu County, HI

  7. 1. Full SW side of dock as viewed from shore ...

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

    1. Full SW side of dock as viewed from shore at the Oil/Creosote Unloading Dock. This view formed a panorama with photo WA-131-H-5, which shows the Oil/Creosote Unloading Dock. - Pacific Creosoting Plant, West Dock, 5350 Creosote Place, Northeast, Bremerton, Kitsap County, WA

  8. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    SciTech Connect

    Sato, Mai; Kitaguchi, Tetsuya; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  9. Ciliary vesicle formation: a prelude to ciliogenesis.

    PubMed

    Yee, Laura E; Reiter, Jeremy F

    2015-03-23

    Reporting recently in Nature Cell Biology, Lu et al. (2015) identify two Eps15-homology-domain-containing proteins as critical effectors of ciliary vesicle formation, an early event in ciliogenesis. Functional dissection reveals that one of them works to convert small vesicles associated with mother centriole distal appendages into a larger ciliary vesicle. PMID:25805133

  10. Design and Preliminary Testing of the International Docking Adapter's Peripheral Docking Target

    NASA Technical Reports Server (NTRS)

    Foster, Christopher W.; Blaschak, Johnathan; Eldridge, Erin A.; Brazzel, Jack P.; Spehar, Peter T.

    2015-01-01

    The International Docking Adapter's Peripheral Docking Target (PDT) was designed to allow a docking spacecraft to judge its alignment relative to the docking system. The PDT was designed to be compatible with relative sensors using visible cameras, thermal imagers, or Light Detection and Ranging (LIDAR) technologies. The conceptual design team tested prototype designs and materials to determine the contrast requirements for the features. This paper will discuss the design of the PDT, the methodology and results of the tests, and the conclusions pertaining to PDT design that were drawn from testing.

  11. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

    PubMed Central

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S.; Olson, Arthur J.; Sanner, Michel F.

    2015-01-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that

  12. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility.

    PubMed

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S; Olson, Arthur J; Sanner, Michel F

    2015-12-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR-AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 -a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 -a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down

  13. GPU.proton.DOCK: Genuine Protein Ultrafast proton equilibria consistent DOCKing.

    PubMed

    Kantardjiev, Alexander A

    2011-07-01

    GPU.proton.DOCK (Genuine Protein Ultrafast proton equilibria consistent DOCKing) is a state of the art service for in silico prediction of protein-protein interactions via rigorous and ultrafast docking code. It is unique in providing stringent account of electrostatic interactions self-consistency and proton equilibria mutual effects of docking partners. GPU.proton.DOCK is the first server offering such a crucial supplement to protein docking algorithms--a step toward more reliable and high accuracy docking results. The code (especially the Fast Fourier Transform bottleneck and electrostatic fields computation) is parallelized to run on a GPU supercomputer. The high performance will be of use for large-scale structural bioinformatics and systems biology projects, thus bridging physics of the interactions with analysis of molecular networks. We propose workflows for exploring in silico charge mutagenesis effects. Special emphasis is given to the interface-intuitive and user-friendly. The input is comprised of the atomic coordinate files in PDB format. The advanced user is provided with a special input section for addition of non-polypeptide charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. The output is comprised of docked complexes in PDB format as well as interactive visualization in a molecular viewer. GPU.proton.DOCK server can be accessed at http://gpudock.orgchm.bas.bg/. PMID:21666258

  14. CovalentDock: automated covalent docking with parameterized covalent linkage energy estimation and molecular geometry constraints.

    PubMed

    Ouyang, Xuchang; Zhou, Shuo; Su, Chinh Tran To; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

    2013-02-01

    Covalent linkage formation is a very important mechanism for many covalent drugs to work. However, partly due to the limitations of proper computational tools for covalent docking, most covalent drugs are not discovered systematically. In this article, we present a new covalent docking package, the CovalentDock, built on the top of the source code of Autodock. We developed an empirical model of free energy change estimation for covalent linkage formation, which is compatible with existing scoring functions used in docking, while handling the molecular geometry constrains of the covalent linkage with special atom types and directional grid maps. Integrated preparation scripts are also written for the automation of the whole covalent docking workflow. The result tested on existing crystal structures with covalent linkage shows that CovalentDock can reproduce the native covalent complexes with significant improved accuracy when compared with the default covalent docking method in Autodock. Experiments also suggest that CovalentDock is capable of covalent virtual screening with satisfactory enrichment performance. In addition, the investigation on the results also shows that the chirality and target selectivity along with the molecular geometry constrains are well preserved by CovalentDock, showing great capability of this method in the application for covalent drug discovery.

  15. NMR-Assisted Molecular Docking Methodologies.

    PubMed

    Sturlese, Mattia; Bellanda, Massimo; Moro, Stefano

    2015-08-01

    Nuclear magnetic resonance (NMR) spectroscopy and molecular docking are regularly being employed as helpful tools of drug discovery research. Molecular docking is an extremely rapid method to evaluate possible binders from a large chemical library in a fast and cheap manner. NMR techniques can directly detect a protein-ligand interaction, can determine the corresponding association constant, and can consistently identify the ligand binding cavity. Consequently, molecular docking and NMR techniques are naturally complementary techniques where the combination of the two has the potential to improve the overall efficiency of drug discovery process. In this review, we would like to summarize the state of the art of docking methods which have been recently bridged to NMR experiments to identify novel and effective therapeutic drug candidates.

  16. Progress Resupply Craft Docks to Space Station

    NASA Video Gallery

    The 39th ISS Progress resupply vehicle automatically docked to the aft port of the Zvezda service module of the International Space Station at 7:58 a.m. EDT on September 12 using the Kurs automated...

  17. Fast-Tracked Soyuz Docks to Station

    NASA Video Gallery

    The Soyuz TMA-08M spacecraft carrying three new Expedition 35 crew members docks with the International Space Station at 10:28 p.m. EDT Thursday, completing its accelerated journey to the orbiting ...

  18. Evaluation of OMV ranging and docking systems

    NASA Technical Reports Server (NTRS)

    Mcdonald, M. W.

    1985-01-01

    The Orbital Maneuvering Vehicle (OMV) will serve as a shuttle-based or permanent space station-based vehicle designed to rendezvous and soft dock with various other free-flying space vehicles for purposes of inspection, support, and retrieval. This study is concerned primarily with the eventual need for the OMV to rendezvous and dock softly with the Edwin P. Hubble Space Telescope (ST). Utilizing the available capabilities of the large microwave anechoic chamber facility at Marshall Space Flight Center for simulating docking target vehicle motions in a free-space environment, a program is being devised for benchmark testing of rendezvous and docking sensor systems proposed for use on the OMV. A testing regimen suitable for evaluating the accuracy and tracking agility in sensing range, range rate, and angle information at close ranges (0 R 30m) has been developed.

  19. New Expedition 27 Trio Docks to Station

    NASA Video Gallery

    The International Space Station welcomed three new flight engineers when they docked Wednesday April 6, 2011 at 7:09 p.m. EDT in the Soyuz TMA-21 spacecraft. Flight Engineers Ron Garan, Alexander S...

  20. Expedition 26 Docks to the Station

    NASA Video Gallery

    New Expedition 26 crew members Dmitry Kondratyev, Catherine Coleman and Paolo Nespoli docked to the Rassvet mini-research module Friday, Dec. 15, 2010, at 3:11 p.m. EST. They launched Wednesday on ...

  1. NMR-Assisted Molecular Docking Methodologies.

    PubMed

    Sturlese, Mattia; Bellanda, Massimo; Moro, Stefano

    2015-08-01

    Nuclear magnetic resonance (NMR) spectroscopy and molecular docking are regularly being employed as helpful tools of drug discovery research. Molecular docking is an extremely rapid method to evaluate possible binders from a large chemical library in a fast and cheap manner. NMR techniques can directly detect a protein-ligand interaction, can determine the corresponding association constant, and can consistently identify the ligand binding cavity. Consequently, molecular docking and NMR techniques are naturally complementary techniques where the combination of the two has the potential to improve the overall efficiency of drug discovery process. In this review, we would like to summarize the state of the art of docking methods which have been recently bridged to NMR experiments to identify novel and effective therapeutic drug candidates. PMID:27490497

  2. Protein Flexibility in Docking and Surface Mapping

    PubMed Central

    Lexa, Katrina W.; Carlson, Heather A.

    2012-01-01

    Structure-based drug design has become an essential tool for rapid lead discovery and optimization. As available structural information has increased, researchers have become increasingly aware of the importance of protein flexibility for accurate description of the native state. Typical protein–ligand docking efforts still rely on a single rigid receptor, which is an incomplete representation of potential binding conformations of the protein. These rigid docking efforts typically show the best performance rates between 50 and 75%, while fully flexible docking methods can enhance pose prediction up to 80–95%. This review examines the current toolbox for flexible protein–ligand docking and receptor surface mapping. Present limitations and possibilities for future development are discussed. PMID:22569329

  3. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1.

    PubMed

    Huang, Ping; Altshuller, Yelena M; Hou, June Chunqiu; Pessin, Jeffrey E; Frohman, Michael A

    2005-06-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. PMID:15772157

  4. Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion.

    PubMed

    Marra, Vincenzo; Burden, Jemima J; Crawford, Freya; Staras, Kevin

    2014-01-01

    Fast activity-driven turnover of neurotransmitter-filled vesicles at presynaptic terminals is a crucial step in information transfer in the CNS. Characterization of the relationship between the nanoscale organization of synaptic vesicles and their functional properties during transmission is currently of interest. Here we outline a procedure for ultrastructural investigation of functional vesicles in synapses from native mammalian brain tissue. FM dye is injected into the target region of a brain slice and upstream axons are electrically activated to stimulate vesicle turnover and dye uptake. In the presence of diaminobenzidine (DAB), photoactivation of dye-filled vesicles yields an osmiophilic precipitate that is visible in electron micrographs. When combined with serial-section electron microscopy, fundamental ultrastructure-function relationships of presynaptic terminals in native circuits are revealed. We outline the utility of this protocol for the 3D reconstruction of a recycling vesicle pool in CA3-CA1 synapses from an acute hippocampal slice and for the characterization of its anatomically defined docked pool. This protocol requires 6-7 d.

  5. Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling.

    PubMed

    Frank, Thomas; Rutherford, Mark A; Strenzke, Nicola; Neef, Andreas; Pangršič, Tina; Khimich, Darina; Fejtova, Anna; Fetjova, Anna; Gundelfinger, Eckart D; Liberman, M Charles; Harke, Benjamin; Bryan, Keith E; Lee, Amy; Egner, Alexander; Riedel, Dietmar; Moser, Tobias

    2010-11-18

    At the presynaptic active zone, Ca²+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²+ channels and vesicles, and (2) promote vesicle replenishment.

  6. Ovarian serous carcinogenesis from tubal secretory cells.

    PubMed

    Zhang, Wenjing; Wei, Linxuan; Li, Lingmin; Yang, Binlie; Kong, Beihua; Yao, Guang; Zheng, Wenxin

    2015-11-01

    Due to a poor understanding of tumorigenesis, ovarian cancers remain the most lethal gynecologic malignancy and cause horrific deaths. In the last decade, a new dualistic model for ovarian cancer was proposed, wherein ovarian serous cancers are classified as either high-grade or low-grade, with each having different tumorigenic processes, and pathologic and clinical features. Surprisingly, both high- and low-grade ovarian serous cancers were recently found to originate not in the ovaries, but rather from the secretory cells of the fallopian tube, mostly from the tubal fimbriated ends. In this article, we review the evidentiary basis for the aforementioned paradigm shift in the cell origin of ovarian serous cancers, as well as its potential clinical implications. PMID:26174492

  7. Some features of secretory systems in plants.

    PubMed

    Juniper, B E; Gilchrist, A J; Robins, R J

    1977-09-01

    Recent work on secretion in plants is reviewed, with emphasis on the anatomy and physiology of root cap cells in higher plants, the stalked glands of Drosera capensis, and the secretory mechanism of Dionaea muscipula. Cells of the root cap of higher plants switch from a geo-perceptive role to one of mucilage secretion at maturation. Features of this process, the role of the Golgi and the pathway for mucilage distribution are reviewed. In contrast, the stalked glands of the leaves of Drosera capensis are much longer lived and have a complex anatomy. The mechanisms for mucilage secretion, protein absorption and the role of the cell membranes in the internal secretion of the protein are described, using data from X-ray microscopv. The secretion of fluid and protein by Dionaea is stimulated by various nitrogen-containing compounds. Uric acid, often excreted by captured insects, is particularly effective in this respect.

  8. Secretory component: a glandular epithelial cell marker.

    PubMed Central

    Harris, J. P.; South, M. A.

    1981-01-01

    Secretory component (SC) has been demonstrated to be produced by both normal and malignantly transformed glandular epithelial cells. By an indirect immunofluorescent technique, this study surveys tumors of varied cellular origin in order to determine the reliability of SC as a marker for tumor cells derived from glandular epithelium. Both primary and metastatic tumors of glandular epithelial origin demonstrated SC fluorescence, while nonglandular epithelial tumors did not. This observation was extended to live single-cell preparations, which demonstrated intense cell-surface fluorescence only when glandular epithelial tumors cells were examined. Additionally, fixed, cytocentrifuged, single-cell preparations of glandular epithelial tumors demonstrated cytoplasmic SC fluorescence. When breast carcinoma was examined, all cases demonstrated SC, regardless of the degree of differentiation. This assay appears to have useful clinical application in that the finding of SC provides indication of the glandular epithelial origin of a malignantly transformed cell. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6271014

  9. Effect of colchicine on the transport of precursor enamel protein in secretory ameloblasts studied by /sup 3/H-proline radioautography in vitro

    SciTech Connect

    Matsuo, S.; Takano, Y.; Wakisaka, S.; Ichikawa, H.; Nishikawa, S.; Akai, M.

    1988-08-01

    The incorporation of 3H-proline into the secretory ameloblasts of rat molar tooth germs cultured with or without colchicine was studied by light and electron microscope radioautography to determine the function of microtubules in the transport of precursor enamel protein from the rough-surfaced endoplasmic reticulum (rER) to the Golgi cisternae. The grain counts over the transitional vesicles, which accumulated in various cellular regions with colchicine treatment, continued to increase with chase time, unlike in controls. At 30 and 90 min chase, these counts were significantly higher than in controls. Moreover, the total grain count over the organelles (rER, pale granules, and transitional vesicles), which are positioned before the Golgi cisternae in the synthetic pathway, maintained a significantly higher level at 90 min chase in colchicine-treated tooth germs than in controls. The transport of synthesized protein to the Golgi cisternae via transitional vesicles was suppressed in colchicine-treated tooth germs. Some grains appeared with time over pale granular materials that appeared in the intercellular spaces of secretory ameloblasts with colchicine treatment. However, at each chase period, the grain count over pale granular materials was not so high as the count over the enamel in control. The present results indicate that colchicine affects the transport of newly synthesized protein from the rER to the Golgi cisterna via transitional vesicles, probably by interfering with the oriented transport related to microtubular function. It is suggested that the microtubular system may be concerned with the movement of the transitional vesicles.

  10. Triangulation methods for automated docking

    NASA Technical Reports Server (NTRS)

    Bales, John W.

    1996-01-01

    An automated docking system must have a reliable method for determining range and orientation of the passive (target) vehicle with respect to the active vehicle. This method must also provide accurate information on the rates of change of range to and orientation of the passive vehicle. The method must be accurate within required tolerances and capable of operating in real time. The method being developed at Marshall Space Flight Center employs a single TV camera, a laser illumination system and a target consisting, in its minimal configuration, of three retro-reflectors. Two of the retro-reflectors are mounted flush to the same surface, with the third retro-reflector mounted to a post fixed midway between the other two and jutting at a right angle from the surface. For redundancy, two additional retroreflectors are mounted on the surface on a line at right angles to the line containing the first two retro-reflectors, and equally spaced on either side of the post. The target vehicle will contain a large target for initial acquisition and several smaller targets for close range.

  11. Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway.

    PubMed

    Ibrahim, Hala; Pertl, Heidi; Pittertschatscher, Klaus; Fadl-Allah, Ezzat; el-Shahed, Ahmed; Bentrup, Friedrich-Wilhelm; Obermeyer, Gerhard

    2002-05-01

    An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth stopped. Brefeldin A (1 microM) and cytochalasin D (1 microM), which block the production and transport of secretory vesicles, respectively, inhibited the acPase secretion. The Ca2+ channel blocker gadolinium (100 microM Gd3+) also inhibited acPase secretion and tube growth, whereas 3 mM caffeine, another Ca2+ uptake inhibitor, stimulated the acPase release, while tube growth was inhibited. The Yariv reagent (beta-D-glucosyl)3 Yariv phenylglycoside stopped tube growth by binding to arabinogalactan proteins of the tube tip cell wall but did not affect acPase secretion. A strong correlation between tube growth and acPase release was detected. The secreted acPase activity had a pH optimum at pH 5.5, a KM of 0.4 mM for p-nitrophenyl phosphate, and was inhibited by zinc, molybdate, phosphate, and fluoride ions, but not by tartrate. In electrophoresis gels the main acPase activity was detected at 32 kDa. The conspicuous correlation between activity of the secretory pathway and acPase secretion during tube elongation strongly indicates an important role of the acPase during pollen tube growth and the secreted acPase activity may serve as a useful marker enzyme assay for secretory activity in pollen tubes.

  12. Readily releasable pool of synaptic vesicles measured at single synaptic contacts.

    PubMed

    Trigo, Federico F; Sakaba, Takeshi; Ogden, David; Marty, Alain

    2012-10-30

    To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

  13. Readily releasable pool of synaptic vesicles measured at single synaptic contacts

    PubMed Central

    Trigo, Federico F.; Sakaba, Takeshi; Ogden, David; Marty, Alain

    2012-01-01

    To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7. PMID:23074252

  14. Tail docking in pigs: acute physiological and behavioural responses.

    PubMed

    Sutherland, M A; Bryer, P J; Krebs, N; McGlone, J J

    2008-02-01

    Tail docking of piglets is a routine procedure on farms to control tail-biting behaviour; however, docking can cause an acute stress response. The objectives of this research were to determine the stress responses to tail docking in piglets and to compare two methods of tail docking; cautery iron (CAUT) and the more commonly used blunt trauma cutters (BT). At approximately 6 days of age, piglets were tail docked using CAUT (n = 20), BT (n = 20) or sham tail docked with their tails remaining intact (CON; n = 40). Blood samples were taken prior to tail docking and at 30, 60 and 90 min after tail docking to evaluate the effect of tail docking on white blood cell (WBC) measures and cortisol concentrations. The above experiment was repeated to observe behaviour without the periodic blood sampling, so as not to confound the effects of blood sampling on piglet behaviour. Piglet behaviour was recorded in the farrowing crate using 1 min scan-samples via live observations for 60 min prior to and 90 min after tail docking. Total WBC counts were reduced (P > 0.05) among BT and CAUT compared with CON piglets 30 min after tail docking. Cortisol concentrations were higher (P < 0.01) among BT compared with CON and CAUT piglets 60 min after tail docking. Cautery and BT-docked piglets spent more (P < 0.05) time posterior scooting compared with CON piglets between 0 and 15 min, and 31 and 45 min after tail docking. Piglets tail docked using CAUT and BT tended to spend more (P < 0.07) time sitting than CON piglets between 0 and 15 min post tail docking. Elevated blood cortisol can be reduced by the use of the CAUT rather than the BT method of tail docking. Although the tail docking-induced rise in cortisol was prevented by using CAUT, the behavioural response to BT and CAUT docking methods was similar. PMID:22445023

  15. Scanning electron microscopy of the endometrium during the secretory phase.

    PubMed Central

    Motta, P M; Andrews, P M

    1976-01-01

    Scanning electron microscopy was used to study the surface morphology of the rabbit endometrium during the secretory phase of the oestrous cycle. The free surfaces of ciliated and of inactive active secretory cells are described. Changes in secretory cell surface morphology resulting from accumulation and secretion of material involve the apparent retraction of microvilli and the formation of one or more bulbous protrusions of the cell's apical surface. These protrusions may be relatively smooth surfaced or exhibit long slender micro-extensions. The protrusions grow in size and are eventually pinched off. Loss of the bulbous protrusions often leaves behind crater-like invaginations of the cell's surface. Secretory cells adjacent to the endometrial glands are the first to exhibit signs of mucin accumulation and secretion. The single cilium of a secretory cell is not apparently affected by the secretory process. Signs of ciliated and secretory cell degeneration, and possible sloughing, are also described. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:1033932

  16. dockYard--a repository to assist modeling of protein-protein docking.

    PubMed

    Mitra, Pralay; Pal, Debnath

    2011-03-01

    In the absence of interlogs, building docking models is a time intensive task, involving generation of a large pool of docking decoys followed by refinement and screening to identify near native docking solutions. This limits the researcher interested in building docking methods with the choice of benchmarking only a limited number of protein complexes. We have created a repository called dockYard ( http://pallab.serc.iisc.ernet.in/dockYard ), that allows modelers interested in protein-protein interaction to access large volume of information on protein dimers and their interlogs, and also download decoys for their work if they are interested in building modeling methods. dockYard currently offers four categories of docking decoys derived from: Bound (native dimer co-crystallized), Unbound (individual subunits are crystallized, as well as the target dimer), Variants (match the previous two categories in at least one subunit with 100% sequence identity), and Interlogs (match the previous categories in at least one subunit with ≥ 90% or ≥ 50% sequence identity). The web service offers options for full or selective download based on search parameters. Our portal also serves as a repository to modelers who may want to share their decoy sets with the community.

  17. SwissDock, a protein-small molecule docking web service based on EADock DSS

    PubMed Central

    Grosdidier, Aurélien; Zoete, Vincent; Michielin, Olivier

    2011-01-01

    Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community. PMID:21624888

  18. Presynaptic Calcium Channel Localization and Calcium Dependent Synaptic Vesicle Exocytosis Regulated by the Fuseless Protein

    PubMed Central

    Long, A. Ashleigh; Kim, Eunju; Leung, Hung-Tat; Woodruff, Elvin; An, Lingling; Doerge, R. W.; Pak, William L.; Broadie, Kendal

    2009-01-01

    Summary A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted 8-pass transmembrane protein in the presynaptic membrane. Null fusl mutants display >75% reduction in evoked synaptic transmission but, conversely, a ~3-fold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wildtype fusl transgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca2+ concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca2+ channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca2+ channel domains required for efficient coupling of the Ca2+ influx and synaptic vesicle exocytosis during neurotransmission. PMID:18385325

  19. Membrane docking mode of the C2 domain of PKCε: an infrared spectroscopy and FRET study.

    PubMed

    Ausili, Alessio; Berglin, Mattias; Elwing, Hans; Egea-Jiménez, Antonio L; Corbalán-García, Senena; Gómez-Fernández, Juan C

    2013-02-01

    The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the β-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the β-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45° to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of β-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the β-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39°) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49°).

  20. Crusader Automated Docking System Phase 3 report

    SciTech Connect

    Jatko, W.B.; Goddard, J.S.; Ferrell, R.K.; Gleason, S.S.; Hicks, J.S.; Varma, V.K.

    1996-03-01

    The US Army is developing the next generation of battlefield artillery vehicles, including an advanced, self-propelled howitzer and a companion resupply vehicle. The resupply vehicle is intended to rendezvous with the howitzer near the battlefront and to upload ammunition to the howitzer. The Army has recommended that the vehicles incorporate robotics to increase safety, by allowing the crew to remain inside their vehicles during resupply operations. Oak Ridge National Laboratory has developed an autonomous docking system for a 6-D.F. robotic, ammunition transfer arm. The docking system augments the operator`s abilities by determining the position and orientation (pose) of a docking port. The pose is the location of the x, y, and z reference axes in 3-D space; and the orientation is the rotations--roll, pitch, and yaw--about those axes. Bye precisely determining the pose of the docking port, the robot can be instructed to move to the docking position without operator intervention. The system uses a video camera and frame grabber to digitize images of the special docking port. Custom algorithms were developed to recognize the port in the camera image, to determine the pose from its image features, and to distribute the results to the robot control computer. The system is loosely coupled to the robot and can be easily adapted to different mechanical configurations. The system has successfully demonstrated autonomous docking on a 24-in. tabletop robot and a 12-ft ammunition resupply robot. The update rate, measurement accuracy, continuous operation, and accuracy with obstructed view have been determined experimentally.

  1. Docking screens: right for the right reasons?

    PubMed Central

    Kolb, Peter; Irwin, John J.

    2012-01-01

    Whereas docking screens have emerged as the most practical way to use protein structure for ligand discovery, an inconsistent track record raises questions about how well docking actually works. In its favor, a growing number of publications report the successful discovery of new ligands, often supported by experimental affinity data and controls for artifacts. Few reports, however, actually test the underlying structural hypotheses that docking makes. To be successful and not just lucky, prospective docking must not only rank a true ligand among the top scoring compounds, it must also correctly orient the ligand so the score it receives is biophysically sound. If the correct binding pose is not predicted, a skeptic might well infer that the discovery was serendipitous. Surveying over 15 years of the docking literature, we were surprised to discover how rarely sufficient evidence is presented to establish whether docking actually worked for the right reasons. The paucity of experimental tests of theoretically predicted poses undermines confidence in a technique that has otherwise become widely accepted. Of course, solving a crystal structure is not always possible, and even when it is, it can be a lot of work, and is not readily accessible to all groups. Even when a structure can be determined, investigators may prefer to gloss over an erroneous structural prediction to better focus on their discovery. Still, the absence of a direct test of theory by experiment is a loss for method developers seeking to understand and improve docking methods. We hope this review will motivate investigators to solve structures and compare them with their predictions whenever possible, to advance the field. PMID:19754393

  2. DockQ: A Quality Measure for Protein-Protein Docking Models.

    PubMed

    Basu, Sankar; Wallner, Björn

    2016-01-01

    The state-of-the-art to assess the structural quality of docking models is currently based on three related yet independent quality measures: Fnat, LRMS, and iRMS as proposed and standardized by CAPRI. These quality measures quantify different aspects of the quality of a particular docking model and need to be viewed together to reveal the true quality, e.g. a model with relatively poor LRMS (>10Å) might still qualify as 'acceptable' with a descent Fnat (>0.50) and iRMS (<3.0Å). This is also the reason why the so called CAPRI criteria for assessing the quality of docking models is defined by applying various ad-hoc cutoffs on these measures to classify a docking model into the four classes: Incorrect, Acceptable, Medium, or High quality. This classification has been useful in CAPRI, but since models are grouped in only four bins it is also rather limiting, making it difficult to rank models, correlate with scoring functions or use it as target function in machine learning algorithms. Here, we present DockQ, a continuous protein-protein docking model quality measure derived by combining Fnat, LRMS, and iRMS to a single score in the range [0, 1] that can be used to assess the quality of protein docking models. By using DockQ on CAPRI models it is possible to almost completely reproduce the original CAPRI classification into Incorrect, Acceptable, Medium and High quality. An average PPV of 94% at 90% Recall demonstrating that there is no need to apply predefined ad-hoc cutoffs to classify docking models. Since DockQ recapitulates the CAPRI classification almost perfectly, it can be viewed as a higher resolution version of the CAPRI classification, making it possible to estimate model quality in a more quantitative way using Z-scores or sum of top ranked models, which has been so valuable for the CASP community. The possibility to directly correlate a quality measure to a scoring function has been crucial for the development of scoring functions for protein structure

  3. DockQ: A Quality Measure for Protein-Protein Docking Models

    PubMed Central

    Basu, Sankar

    2016-01-01

    The state-of-the-art to assess the structural quality of docking models is currently based on three related yet independent quality measures: Fnat, LRMS, and iRMS as proposed and standardized by CAPRI. These quality measures quantify different aspects of the quality of a particular docking model and need to be viewed together to reveal the true quality, e.g. a model with relatively poor LRMS (>10Å) might still qualify as 'acceptable' with a descent Fnat (>0.50) and iRMS (<3.0Å). This is also the reason why the so called CAPRI criteria for assessing the quality of docking models is defined by applying various ad-hoc cutoffs on these measures to classify a docking model into the four classes: Incorrect, Acceptable, Medium, or High quality. This classification has been useful in CAPRI, but since models are grouped in only four bins it is also rather limiting, making it difficult to rank models, correlate with scoring functions or use it as target function in machine learning algorithms. Here, we present DockQ, a continuous protein-protein docking model quality measure derived by combining Fnat, LRMS, and iRMS to a single score in the range [0, 1] that can be used to assess the quality of protein docking models. By using DockQ on CAPRI models it is possible to almost completely reproduce the original CAPRI classification into Incorrect, Acceptable, Medium and High quality. An average PPV of 94% at 90% Recall demonstrating that there is no need to apply predefined ad-hoc cutoffs to classify docking models. Since DockQ recapitulates the CAPRI classification almost perfectly, it can be viewed as a higher resolution version of the CAPRI classification, making it possible to estimate model quality in a more quantitative way using Z-scores or sum of top ranked models, which has been so valuable for the CASP community. The possibility to directly correlate a quality measure to a scoring function has been crucial for the development of scoring functions for protein structure

  4. Distinct stages in the recognition, sorting, and packaging of proTGFα into COPII-coated transport vesicles

    PubMed Central

    Zhang, Pengcheng; Schekman, Randy

    2016-01-01

    In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles. PMID:27122606

  5. Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size

    PubMed Central

    Graham, Margaret E.; O'Callaghan, Dermott W.; McMahon, Harvey T.; Burgoyne, Robert D.

    2002-01-01

    Accumulating evidence suggests that the kinetics of release from single secretory vesicles can be regulated and that quantal size can be modified during fast kiss-and-run fusion. Multiple pathways for vesicle retrieval have been identified involving clathrin and dynamin. It has been unclear whether dynamin could participate in a fast kiss-and-run process to reclose a transient fusion pore and thereby limit vesicle release. We have disrupted dynamin function in adrenal chromaffin cells by expression of the amphiphysin Src-homology domain 3 (SH3) or by application of guanosine 5′-[γ-thio]triphosphate (GTPγS), and have monitored single vesicle release events, evoked by digitonin and Ca2+, by using carbon-fiber amperometry. Under both conditions, there was an increase in mean quantal size accompanying an increase in the half-width of amperometric spikes and a slowing of the fall time. These data suggest the existence of a dynamin-dependent process that can terminate vesicle release under basal conditions. Protein kinase C activation changed release kinetics and decreased quantal size by shortening the release period. The effects of phorbol ester treatment were not prevented by expression of the amphiphysin SH3 domain or by GTPγS suggesting the existence of alternative dynamin-independent process underlying fast kiss-and-run exocytosis. PMID:11997474

  6. Spontaneous vesicle recycling in the synaptic bouton.

    PubMed

    Truckenbrodt, Sven; Rizzoli, Silvio O

    2014-01-01

    The trigger for synaptic vesicle exocytosis is Ca(2+), which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca(2+) levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca(2+) sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca(2+). The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs) rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs) responding to Ca(2+) fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

  7. Golgi- and trans-Golgi network-mediated vesicle trafficking is required for wax secretion from epidermal cells.

    PubMed

    McFarlane, Heather E; Watanabe, Yoichiro; Yang, Weili; Huang, Yan; Ohlrogge, John; Samuels, A Lacey

    2014-03-01

    Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters. PMID:24468625

  8. Flexible ligand docking using conformational ensembles.

    PubMed Central

    Lorber, D. M.; Shoichet, B. K.

    1998-01-01

    Molecular docking algorithms suggest possible structures for molecular complexes. They are used to model biological function and to discover potential ligands. A present challenge for docking algorithms is the treatment of molecular flexibility. Here, the rigid body program, DOCK, is modified to allow it to rapidly fit multiple conformations of ligands. Conformations of a given molecule are pre-calculated in the same frame of reference, so that each conformer shares a common rigid fragment with all other conformations. The ligand conformers are then docked together, as an ensemble, into a receptor binding site. This takes advantage of the redundancy present in differing conformers of the same molecule. The algorithm was tested using three organic ligand protein systems and two protein-protein systems. Both the bound and unbound conformations of the receptors were used. The ligand ensemble method found conformations that resembled those determined in X-ray crystal structures (RMS values typically less than 1.5 A). To test the method's usefulness for inhibitor discovery, multi-compound and multi-conformer databases were screened for compounds known to bind to dihydrofolate reductase and compounds known to bind to thymidylate synthase. In both cases, known inhibitors and substrates were identified in conformations resembling those observed experimentally. The ligand ensemble method was 100-fold faster than docking a single conformation at a time and was able to screen a database of over 34 million conformations from 117,000 molecules in one to four CPU days on a workstation. PMID:9568900

  9. Protein docking using case-based reasoning.

    PubMed

    Ghoorah, Anisah W; Devignes, Marie-Dominique; Smaïl-Tabbone, Malika; Ritchie, David W

    2013-12-01

    Protein docking algorithms aim to calculate the three-dimensional (3D) structure of a protein complex starting from its unbound components. Although ab initio docking algorithms are improving, there is a growing need to use homology modeling techniques to exploit the rapidly increasing volumes of structural information that now exist. However, most current homology modeling approaches involve finding a pair of complete single-chain structures in a homologous protein complex to use as a 3D template, despite the fact that protein complexes are often formed from one or more domain-domain interactions (DDIs). To model 3D protein complexes by domain-domain homology, we have developed a case-based reasoning approach called KBDOCK which systematically identifies and reuses domain family binding sites from our database of nonredundant DDIs. When tested on 54 protein complexes from the Protein Docking Benchmark, our approach provides a near-perfect way to model single-domain protein complexes when full-homology templates are available, and it extends our ability to model more difficult cases when only partial or incomplete templates exist. These promising early results highlight the need for a new and diverse docking benchmark set, specifically designed to assess homology docking approaches. PMID:24123156

  10. Udock, the interactive docking entertainment system.

    PubMed

    Levieux, Guillaume; Tiger, Guillaume; Mader, Stéphanie; Zagury, Jean-François; Natkin, Stéphane; Montes, Matthieu

    2014-01-01

    Protein-protein interactions play a crucial role in biological processes. Protein docking calculations' goal is to predict, given two proteins of known structures, the associate conformation of the corresponding complex. Here, we present a new interactive protein docking system, Udock, that makes use of users' cognitive capabilities added up. In Udock, the users tackle simplified representations of protein structures and explore protein-protein interfaces' conformational space using a gamified interactive docking system with on the fly scoring. We assumed that if given appropriate tools, a naïve user's cognitive capabilities could provide relevant data for (1) the prediction of correct interfaces in binary protein complexes and (2) the identification of the experimental partner in interaction among a set of decoys. To explore this approach experimentally, we conducted a preliminary two week long playtest where the registered users could perform a cross-docking on a dataset comprising 4 binary protein complexes. The users explored almost all the surface of the proteins that were available in the dataset but favored certain regions that seemed more attractive as potential docking spots. These favored regions were located inside or nearby the experimental binding interface for 5 out of the 8 proteins in the dataset. For most of them, the best scores were obtained with the experimental partner. The alpha version of Udock is freely accessible at http://udock.fr.

  11. Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis

    PubMed Central

    Stinchcombe, Jane C.; Randzavola, Lyra O.; Angus, Karen L.; Mantell, Judith M.; Verkade, Paul; Griffiths, Gillian M.

    2015-01-01

    Summary Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1, 2, 3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4, 5, 6, 7, 8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome. PMID:26670998

  12. Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

    PubMed

    Stinchcombe, Jane C; Randzavola, Lyra O; Angus, Karen L; Mantell, Judith M; Verkade, Paul; Griffiths, Gillian M

    2015-12-21

    Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome.

  13. Relationship between vesicle size and steric hindrance influences vesicle rupture on solid supports.

    PubMed

    Jackman, Joshua A; Kim, Min Chul; Zhdanov, Vladimir P; Cho, Nam-Joon

    2016-01-28

    Phospholipid assemblies on solid supports mimic the cell membrane, and provide a platform to study membrane biology. Among the different types of model membranes, the planar bilayer is a two-dimensional lipid bilayer sheet that can be formed by the adsorption and spontaneous rupture of vesicles. The formation process is influenced by the interactions between vesicles and the solid support as well as between vesicles. On silicon oxide, which is a commonly used solid support, vesicles typically adsorb until reaching a critical coverage and then spontaneous rupture begins. Although it is generally understood that spontaneous rupture leads to planar bilayer formation, oversaturation of vesicles at the critical coverage can hinder the whole process due to a steric factor. To date, the role of this factor has been scrutinized only in relation to temperature, and the influence of additional parameters remains to be elucidated. In this work, we have investigated how vesicle size and corresponding steric constraints influence the kinetics of vesicle adsorption and rupture and, more specifically, how the state of adsorbed vesicles after fusion depends on the vesicle size. Using quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP), we characterized the adsorption kinetics of vesicles onto silicon oxide and the lateral mobility of solid-supported lipid assemblies. While the vesicle adsorption kinetics were diffusion-limited up to the onset of vesicle rupture, the extent of rupture depended on vesicle size and it was observed that larger vesicles are more prone to steric effects than smaller vesicles. We discuss this finding in terms of the structural transformation from adsorbed vesicles to a planar bilayer, including how the interplay of thermodynamic, kinetic and steric factors can affect vesicle rupture on solid supports. PMID:26739602

  14. Relationship between vesicle size and steric hindrance influences vesicle rupture on solid supports.

    PubMed

    Jackman, Joshua A; Kim, Min Chul; Zhdanov, Vladimir P; Cho, Nam-Joon

    2016-01-28

    Phospholipid assemblies on solid supports mimic the cell membrane, and provide a platform to study membrane biology. Among the different types of model membranes, the planar bilayer is a two-dimensional lipid bilayer sheet that can be formed by the adsorption and spontaneous rupture of vesicles. The formation process is influenced by the interactions between vesicles and the solid support as well as between vesicles. On silicon oxide, which is a commonly used solid support, vesicles typically adsorb until reaching a critical coverage and then spontaneous rupture begins. Although it is generally understood that spontaneous rupture leads to planar bilayer formation, oversaturation of vesicles at the critical coverage can hinder the whole process due to a steric factor. To date, the role of this factor has been scrutinized only in relation to temperature, and the influence of additional parameters remains to be elucidated. In this work, we have investigated how vesicle size and corresponding steric constraints influence the kinetics of vesicle adsorption and rupture and, more specifically, how the state of adsorbed vesicles after fusion depends on the vesicle size. Using quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP), we characterized the adsorption kinetics of vesicles onto silicon oxide and the lateral mobility of solid-supported lipid assemblies. While the vesicle adsorption kinetics were diffusion-limited up to the onset of vesicle rupture, the extent of rupture depended on vesicle size and it was observed that larger vesicles are more prone to steric effects than smaller vesicles. We discuss this finding in terms of the structural transformation from adsorbed vesicles to a planar bilayer, including how the interplay of thermodynamic, kinetic and steric factors can affect vesicle rupture on solid supports.

  15. A Novel Mutation of DAX-1 Associated with Secretory Azoospermia

    PubMed Central

    Yang, Lihua; Liu, Yuchen; Diao, Ruiying; Cai, Zhiming; Li, Honggang; Gui, Yaoting

    2015-01-01

    Secretory azoospermia is a severe form of male infertility caused by unknown factors. DAX-1 is predominantly expressed in mammalian reproductive tissues and plays an important role in spermatogenesis because Dax-1 knockout male mice show spermatogenesis defects. To examine whether DAX-1 is involved in the pathogenesis of secretory azoospermia in humans, we sequenced all of the exons of DAX-1 in 776 patients diagnosed with secretory azoospermia and 709 proven fertile men. A number of coding mutations unique to the patient group, including two synonymous mutations and six missense mutations, were identified. Of the missense mutations, our functional assay demonstrated that the V385L mutation caused the reduced functioning of DAX-1. This novel mutation (p. V385L) of DAX-1 is the first to be identified in association with secretory azoospermia, thereby highlighting the important role of DAX-1 in spermatogenesis. PMID:26207377

  16. A Novel Mutation of DAX-1 Associated with Secretory Azoospermia.

    PubMed

    Mou, Lisha; Xie, Nie; Yang, Lihua; Liu, Yuchen; Diao, Ruiying; Cai, Zhiming; Li, Honggang; Gui, Yaoting

    2015-01-01

    Secretory azoospermia is a severe form of male infertility caused by unknown factors. DAX-1 is predominantly expressed in mammalian reproductive tissues and plays an important role in spermatogenesis because Dax-1 knockout male mice show spermatogenesis defects. To examine whether DAX-1 is involved in the pathogenesis of secretory azoospermia in humans, we sequenced all of the exons of DAX-1 in 776 patients diagnosed with secretory azoospermia and 709 proven fertile men. A number of coding mutations unique to the patient group, including two synonymous mutations and six missense mutations, were identified. Of the missense mutations, our functional assay demonstrated that the V385L mutation caused the reduced functioning of DAX-1. This novel mutation (p. V385L) of DAX-1 is the first to be identified in association with secretory azoospermia, thereby highlighting the important role of DAX-1 in spermatogenesis. PMID:26207377

  17. Mammary analogue secretory carcinoma mimicking salivary adenoma.

    PubMed

    Williams, Lindsay; Chiosea, Simion I

    2013-12-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor characterized by ETV6 translocation. It appears that prior studies have identified MASC by reviewing salivary gland carcinomas, such as acinic cell carcinoma and adenocarcinoma, not otherwise specified. To address the possibility of MASC mimicking benign salivary neoplasms we reviewed 12 salivary gland (cyst)adenomas diagnosed prior to the discovery of MASC. One encapsulated (cyst)adenoma of the parotid gland demonstrated features of MASC. The diagnosis was confirmed by fluorescence in situ hybridization with an ETV6 break-apart probe. An unusual complex pattern of ETV6 rearrangement with duplication of the telomeric/distal ETV6 probe was identified. This case illustrates that MASC may mimic salivary (cyst)adenomas. To more accurately assess true clinical and morphologic spectrum of MASC, future studies may have to include review of salivary (cyst)adenomas. The differential diagnosis of MASC may have to be expanded to include cases resembling salivary (cyst)adenomas.

  18. Synaptic Vesicle Proteins and Active Zone Plasticity.

    PubMed

    Kittel, Robert J; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention.

  19. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  20. Methods to Purify and Assay Secretory Pathway Kinases.

    PubMed

    Tagliabracci, Vincent S; Wen, Jianzhong; Xiao, Junyu

    2016-01-01

    Members of the four-jointed and VLK families of secretory pathway kinases appear to be responsible for the phosphorylation of secreted proteins and proteoglycans. These enzymes have been implicated in many biological processes and mutations in several of these kinases cause human diseases. Here, we describe methods to purify and assay two members of the four-jointed family of secretory kinases: the Fam20C protein kinase and the Fam20B proteoglycan kinase. PMID:27632012

  1. Overview of LIDS Docking Seals Development

    NASA Technical Reports Server (NTRS)

    Dunlap, Pat; Steinetz, Bruce; Daniels, Chris

    2008-01-01

    NASA is developing a new docking system to support future space exploration missions to low-Earth orbit, the Moon, and Mars. This mechanism, called the Low Impact Docking System (LIDS), is designed to connect pressurized space vehicles and structures including the Crew Exploration Vehicle, International Space Station, and lunar lander. NASA Glenn Research Center (GRC) is playing a key role in developing the main interface seal for this new docking system. These seals will be approximately 147 cm (58 in.) in diameter. GRC is evaluating the performance of candidate seal designs under simulated operating conditions at both sub-scale and full-scale levels. GRC is ultimately responsible for delivering flight hardware seals to NASA Johnson Space Center around 2013 for integration into LIDS flight units.

  2. Electro-optical rendezvous and docking sensors

    NASA Technical Reports Server (NTRS)

    Tubbs, David J.; Kesler, Lynn O.; Sirko, Robert J.

    1991-01-01

    Electro-optical sensors provide unique and critical functionality for space missions requiring rendezvous, docking, and berthing. McDonnell Douglas is developing a complete rendezvous and docking system for both manned and unmanned missions. This paper examines our sensor development and the systems and missions which benefit from rendezvous and docking sensors. Simulation results quantifying system performance improvements in key areas are given, with associated sensor performance requirements. A brief review of NASA-funded development activities and the current performance of electro-optical sensors for space applications is given. We will also describe current activities at McDonnell Douglas for a fully functional demonstration to address specific NASA mission needs.

  3. Multiple exposure of Rendezvous Docking Simulator

    NASA Technical Reports Server (NTRS)

    1964-01-01

    Multiple exposure of Rendezvous Docking Simulator. Francis B. Smith, described the simmulator as follows: '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.'

  4. The Complexity of Vesicle Transport Factors in Plants Examined by Orthology Search

    PubMed Central

    Mirus, Oliver; Scharf, Klaus-Dieter; Fragkostefanakis, Sotirios; Schleiff, Enrico

    2014-01-01

    Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the ‘core-set’ of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed. PMID:24844592

  5. Secretory pathway of cellulase: a mini-review

    PubMed Central

    2013-01-01

    Cellulase plays an important role in modern industry and holds great potential in biofuel production. Many different types of organisms produce cellulase, which go through secretory pathways to reach the extracellular space, where enzymatic reactions take place. Secretory pathways in various cells have been the focus of many research fields; however, there are few studies on secretory pathways of cellulases in the literature. It is therefore necessary and important to review the current knowledge on the secretory pathways of cellulases. In this mini-review, we address the subcellular locations of cellulases in different organisms, discuss the secretory pathways of cellulases in different organisms, and examine the secretory mechanisms of cellulases. These sections start with a description of general secreted proteins, advance to the situation of cellulases, and end with the knowledge of cellulases, as documented in UniProt Knowledgebase (UniProtKB). Finally, gaps in existing knowledge are highlighted, which may shed light on future studies for biofuel engineering. PMID:24295495

  6. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    PubMed

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html.

  7. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    PubMed

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html. PMID:22669908

  8. Proximity Operations and Docking Sensor Development

    NASA Technical Reports Server (NTRS)

    Howard, Richard T.; Bryan, Thomas C.; Brewster, Linda L.; Lee, James E.

    2009-01-01

    The Next Generation Advanced Video Guidance Sensor (NGAVGS) has been under development for the last three years as a long-range proximity operations and docking sensor for use in an Automated Rendezvous and Docking (AR&D) system. The first autonomous rendezvous and docking in the history of the U.S. Space Program was successfully accomplished by Orbital Express, using the Advanced Video Guidance Sensor (AVGS) as the primary docking sensor. That flight proved that the United States now has a mature and flight proven sensor technology for supporting Crew Exploration Vehicles (CEV) and Commercial Orbital Transport Systems (COTS) Automated Rendezvous and Docking (AR&D). NASA video sensors have worked well in the past: the AVGS used on the Demonstration of Autonomous Rendezvous Technology (DART) mission operated successfully in spot mode out to 2 km, and the first generation rendezvous and docking sensor, the Video Guidance Sensor (VGS), was developed and successfully flown on Space Shuttle flights in 1997 and 1998. 12 Parts obsolescence issues prevent the construction of more AVGS units, and the next generation sensor was updated to allow it to support the CEV and COTS programs. The flight proven AR&D sensor has been redesigned to update parts and add additional capabilities for CEV and COTS with the development of the Next Generation AVGS at the Marshall Space Flight Center. The obsolete imager and processor are being replaced with new radiation tolerant parts. In addition, new capabilities include greater sensor range, auto ranging capability, and real-time video output. This paper presents some sensor hardware trades, use of highly integrated laser components, and addresses the needs of future vehicles that may rendezvous and dock with the International Space Station (ISS) and other Constellation vehicles. It also discusses approaches for upgrading AVGS to address parts obsolescence, and concepts for minimizing the sensor footprint, weight, and power requirements

  9. Apollo-Soyuz test project docking system

    NASA Technical Reports Server (NTRS)

    Swan, W. L., Jr.

    1976-01-01

    The United States and Soviet Union in July 1975 successfully completed a joint space mission utilizing each country's spacecraft and the compatible docking system designed and fabricated by each country. The compatible docking system is described, along with the extensive research, development, and testing leading up to the successful mission. It also describes the formulation and implementation of methods for breaking the language barrier, bridging the extensive distances for communication and travel, and adjusting to each country's different culture during the three-year development program.

  10. Autonomous movement of a chemically powered vesicle

    NASA Astrophysics Data System (ADS)

    Gupta, Shivam; Sreeja, K. K.; Thakur, Snigdha

    2015-10-01

    We investigate the diffusio-phoretic motion of a deformable vesicle. A vesicle is built from the linked catalytic and noncatalytic vertices that consumes fuel in the environment and utilize the resulting self-generated concentration gradient to exhibit propulsive motion. Under nonequilibrium conditions it is found that the self-propulsion velocity of the vesicle depends on its shape, which in turn is controlled by the bending rigidity of the membrane and solvent density around it. The self-propulsion velocity of the vesicle for different shapes has been calculated and the factors which affect the velocity are identified.

  11. Autonomous movement of a chemically powered vesicle.

    PubMed

    Gupta, Shivam; Sreeja, K K; Thakur, Snigdha

    2015-10-01

    We investigate the diffusio-phoretic motion of a deformable vesicle. A vesicle is built from the linked catalytic and noncatalytic vertices that consumes fuel in the environment and utilize the resulting self-generated concentration gradient to exhibit propulsive motion. Under nonequilibrium conditions it is found that the self-propulsion velocity of the vesicle depends on its shape, which in turn is controlled by the bending rigidity of the membrane and solvent density around it. The self-propulsion velocity of the vesicle for different shapes has been calculated and the factors which affect the velocity are identified. PMID:26565268

  12. SwarmDock and the Use of Normal Modes in Protein-Protein Docking

    PubMed Central

    Moal, Iain H.; Bates, Paul A.

    2010-01-01

    Here is presented an investigation of the use of normal modes in protein-protein docking, both in theory and in practice. Upper limits of the ability of normal modes to capture the unbound to bound conformational change are calculated on a large test set, with particular focus on the binding interface, the subset of residues from which the binding energy is calculated. Further, the SwarmDock algorithm is presented, to demonstrate that the modelling of conformational change as a linear combination of normal modes is an effective method of modelling flexibility in protein-protein docking. PMID:21152290

  13. 11. View from dock of "Keku Canning Co" office and ...

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

    11. View from dock of "Keku Canning Co" office and stores and the cooling building, Seine storage building looking down the cannery dock - Kake Salmon Cannery, 540 Keku Road, Kake, Wrangell-Petersburg Census Area, AK

  14. 38. View from dock of "Keku Canning Co" office and ...

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

    38. View from dock of "Keku Canning Co" office and stores and the cooling building, Sein storage building looking down cannery dock - Kake Salmon Cannery, 540 Keku Road, Kake, Wrangell-Petersburg Census Area, AK

  15. 11. Northeast front, dock no. 493. View to west. ...

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

    11. Northeast front, dock no. 493. 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

  16. 5. Southwest front, dock no. 492. View to east. ...

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

    5. Southwest front, dock no. 492. View to east. - 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

  17. 22. GENERAL VIEW OF DOCK, TAKEN FROM THE ROOF OF ...

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

    22. GENERAL VIEW OF DOCK, TAKEN FROM THE ROOF OF THE POWER HOUSE, LOOKING NORTH. - Pennsylvania Railway Ore Dock, Lake Erie at Whiskey Island, approximately 1.5 miles west of Public Square, Cleveland, Cuyahoga County, OH

  18. View south, wharf B, showing western docking structure, decking detail ...

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

    View south, wharf B, showing western docking structure, decking detail - U.S. Coast Guard Sandy Hook Station, Western Docking Structure, West of intersection of Canfield Road & Hartshorne Drive, Highlands, Monmouth County, NJ

  19. 20. VIEW OF EAST END OF SPERRY OCEAN DOCK, SHOWING ...

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

    20. VIEW OF EAST END OF SPERRY OCEAN DOCK, SHOWING SOUTH AND EAST ELEVATION, INCLUDING PORTION OF THE SPERRY OCEAN DOCK DECK, LOOKING NORTH - Puget Sound Flouring Mills, 611 Schuster Parkway, Tacoma, Pierce County, WA

  20. DETAIL OF NOSE DOCK DOOR ON NORTH (FRONT) ELEVATION OF ...

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

    DETAIL OF NOSE DOCK DOOR ON NORTH (FRONT) ELEVATION OF BUILDING. VIEW TO SOUTH. - Plattsburgh Air Force Base, Aircraft Maintenance Dock, Alabama Avenue at Arkansas Street, Plattsburgh, Clinton County, NY

  1. 19. View of initiation of docking for the USS ENTERPRISE, ...

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

    19. View of initiation of docking for the USS ENTERPRISE, showing vessel approaching entrance to dock (11/28/85). - Hunters Point Naval Shipyard, Drydock No. 4, East terminus of Palou Avenue, San Francisco, San Francisco County, CA

  2. 23. View, looking east, of USS ENTERPRISE in Dry Dock ...

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

    23. View, looking east, of USS ENTERPRISE in Dry Dock No. 4 with dock pumped down (11/86). - Hunters Point Naval Shipyard, Drydock No. 4, East terminus of Palou Avenue, San Francisco, San Francisco County, CA

  3. 4. South (shore) end of dock as viewed from shore ...

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

    4. South (shore) end of dock as viewed from shore looking north includes section of creosote pipe as it leaves the shore. - Pacific Creosoting Plant, Oil-Creosote Unloading Dock, 5350 Creosote Place, Northeast, Bremerton, Kitsap County, WA

  4. 12. View to north along recovery dock along east side ...

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

    12. View to north along recovery dock along east side of firing pier. Steel brackets originally supported a sheltering canopy over the dock. - Naval Torpedo Station, Firing Pier, North end of Gould Island in Narragansett Bay, Newport, Newport County, RI

  5. AutoDock4(Zn): an improved AutoDock force field for small-molecule docking to zinc metalloproteins.

    PubMed

    Santos-Martins, Diogo; Forli, Stefano; Ramos, Maria João; Olson, Arthur J

    2014-08-25

    Zinc is present in a wide variety of proteins and is important in the metabolism of most organisms. Zinc metalloenzymes are therapeutically relevant targets in diseases such as cancer, heart disease, bacterial infection, and Alzheimer's disease. In most cases a drug molecule targeting such enzymes establishes an interaction that coordinates with the zinc ion. Thus, accurate prediction of the interaction of ligands with zinc is an important aspect of computational docking and virtual screening against zinc containing proteins. We have extended the AutoDock force field to include a specialized potential describing the interactions of zinc-coordinating ligands. This potential describes both the energetic and geometric components of the interaction. The new force field, named AutoDock4Zn, was calibrated on a data set of 292 crystal complexes containing zinc. Redocking experiments show that the force field provides significant improvement in performance in both free energy of binding estimation as well as in root-mean-square deviation from the crystal structure pose. The new force field has been implemented in AutoDock without modification to the source code.

  6. A New Scoring Function for Molecular Docking Based on AutoDock and AutoDock Vina.

    PubMed

    Tanchuk, Vsevolod Yu; Tanin, Volodymyr O; Vovk, Andriy I; Poda, Gennady

    2015-01-01

    Molecular docking of small molecules in the protein binding sites is the most widely used computational technique in modern structure-based drug discovery. Although accurate prediction of binding modes of small molecules can be achieved in most cases, estimation of their binding affinities remains mediocre at best. As an attempt to improve the correlation between the inhibitory constants, pKi, and scoring, we created a new, hybrid scoring function. The new function is a linear combination of the terms of the scoring functions of AutoDock and AutoDock Vina. It was trained on 2,412 protein-ligand complexes from the PDBbind database (www.pdbbind.org.cn, version 2012) and validated on a set of 313 complexes released in the 2013 version as a test set. The new function was included in a modified version of AutoDock. The hybrid scoring function showed a statistically significant improvement in both training and test sets in terms of correlation with and root mean square and mean absolute errors in prediction of pKi values. It was also tested on the CSAR 2014 Benchmark Exercise dataset (team T) and produced reasonably good results.

  7. Construction of macroscopic cytomimetic vesicle aggregates based on click chemistry: controllable vesicle fusion and phase separation.

    PubMed

    Jin, Haibao; Huang, Wei; Zheng, Yongli; Zhou, Yongfeng; Yan, Deyue

    2012-07-01

    Vesicle-vesicle aggregation to mimic cell-cell aggregation has attracted much attention. Here, hyperbranched polymer vesicles (branched-polymersomes, BPs) with a cell-like size were selected as model membranes, and the vesicle aggregation process, triggered by click chemistry of the copper-catalysed azide-alkyne cycloaddition reaction, was systematically studied. For this purpose, azide and alkynyl groups were loaded on the membranes of BPs through the co-assembly method to obtain N(3)-BPs and Alk-BPs, respectively. Subsequently, macroscopic vesicle aggregates were obtained when these two kinds of functional BPs were mixed together with the ratio of azide to alkynyl groups of about 1:1. Both the vesicle fusion events and lateral phase separation on the vesicle membrane occurred during such a vesicle aggregation process, and the fusion rate and phase-separation degree could be controlled by adjusting the clickable group content. The vesicle aggregation process with N(3) -micelles as desmosome mimics to connect with Alk-BPs through click-chemistry reaction was also studied, and large-scale vesicle aggregates without vesicle fusion were obtained in this process. The present work has extended the controllable cytomimetic vesicle aggregation process with the use of covalent bonds, instead of noncovalent bonds, as the driving force.

  8. Endothelial Cell-Surface Gp60 Activates Vesicle Formation and Trafficking via Gi-Coupled Src Kinase Signaling Pathway

    PubMed Central

    Minshall, Richard D.; Tiruppathi, Chinnaswamy; Vogel, Stephen M.; Niles, Walter D.; Gilchrist, Annette; Hamm, Heidi E.; Malik, Asrar B.

    2000-01-01

    We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial 125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11–amino acid Gαi carboxyl-terminal peptide inhibited endothelial 125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of Gαi with the caveolin-1, whereas dn-Src inhibited Gαi binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60

  9. TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

    PubMed

    Ohara-Imaizumi, Mica; Nishiwaki, Chiyono; Kikuta, Toshiteru; Nagai, Shintaro; Nakamichi, Yoko; Nagamatsu, Shinya

    2004-07-01

    We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat beta-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat beta-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat beta-cells.

  10. Electrical synapse formation disrupts calcium-dependent exocytosis, but not vesicle mobilization.

    PubMed

    Neunuebel, Joshua P; Zoran, Mark J

    2005-06-01

    Electrical coupling exists prior to the onset of chemical connectivity at many developing and regenerating synapses. At cholinergic synapses in vitro, trophic factors facilitated the formation of electrical synapses and interfered with functional neurotransmitter release in response to photolytic elevations of intracellular calcium. In contrast, neurons lacking trophic factor induction and electrical coupling possessed flash-evoked transmitter release. Changes in cytosolic calcium and postsynaptic responsiveness to acetylcholine were not affected by electrical coupling. These data indicate that transient electrical synapse formation delayed chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and synchronized, vesicular release. Despite the inability to release neurotransmitter, neurons that had possessed strong electrical coupling recruited secretory vesicles to sites of synaptic contact. These results suggest that the mechanism by which neurotransmission is disrupted during electrical synapse formation is downstream of both calcium influx and synaptic vesicle mobilization. Therefore, electrical synaptogenesis may inhibit synaptic vesicles from acquiring a readily releasable state. We hypothesize that gap junctions might negatively interact with exocytotic processes, thereby diminishing chemical neurotransmission. PMID:15765535

  11. Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

    PubMed Central

    1995-01-01

    At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures. PMID:8522582

  12. Proteomics of extracellular vesicles: Exosomes and ectosomes.

    PubMed

    Choi, Dong-Sic; Kim, Dae-Kyum; Kim, Yoon-Keun; Gho, Yong Song

    2015-01-01

    Almost all bacteria, archaea, and eukaryotic cells shed extracellular vesicles either constitutively or in a regulated manner. These nanosized membrane vesicles are spherical, bilayered proteolipids that harbor specific subsets of proteins, DNAs, RNAs, and lipids. Recent research has facilitated conceptual advancements in this emerging field that indicate that extracellular vesicles act as intercellular communicasomes by transferring signals to their target cell via surface ligands and delivering receptors and functional molecules. Recent progress in mass spectrometry-based proteomic analyses of mammalian extracellular vesicles derived from diverse cell types and body fluids has resulted in the identification of several thousand vesicular proteins that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of extracellular vesicles and contribute to the discovery of diagnostic and therapeutic target proteins. This review focuses on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), EVpedia (a free web-based integrated database of high-throughput data for systematic analyses of extracellular vesicles; http://evpedia.info), and the intravesicular protein-protein interaction network analyses of mammalian extracellular vesicles. The goal of this article is to encourage further studies to construct a comprehensive proteome database for extracellular vesicles that will help us to not only decode the biogenesis and cargo-sorting mechanisms during vesicle formation but also elucidate the pathophysiological roles of these complex extracellular organelles.

  13. 4. Southwest fronts, dock nos. 491 and 492. Southeast end, ...

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

    4. Southwest fronts, dock nos. 491 and 492. Southeast end, dock no. 492. View to north. - 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

  14. 17. ORE DOCK, LOOKING EAST FROM HULETT NO. 1. WHEN ...

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

    17. ORE DOCK, LOOKING EAST FROM HULETT NO. 1. WHEN BUILT IN 1911-1912, THIS WAS THE LARGEST ORE-UNLOADING DOCK ON THE GREAT LAKES. - Pennsylvania Railway Ore Dock, Lake Erie at Whiskey Island, approximately 1.5 miles west of Public Square, Cleveland, Cuyahoga County, OH

  15. 34. Yards & Docks (Y & D) Drawing 216244 (1943), ...

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

    34. Yards & Docks (Y & D) Drawing 216244 (1943), 'Dry Dock Bilge Block And Drainage Layout'; showing basic dry dock floor plan and sectional view. - Hunters Point Naval Shipyard, Drydock No. 4, East terminus of Palou Avenue, San Francisco, San Francisco County, CA

  16. Dry Dock No. 3 general overview. Looking toward caisson end. ...

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

    Dry Dock No. 3 general overview. Looking toward caisson end. View facing north - U.S. Naval Base, Pearl Harbor, Dry Dock No. 3, On northern shoreline of shipyard, west of Dry Dock Nos. 1 & 2, near the intersection of Avenue G and Sixth Street, Pearl City, Honolulu County, HI

  17. Dry Dock No. 3 general overview. Looking toward caisson end ...

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

    Dry Dock No. 3 general overview. Looking toward caisson end with crane tracks in view. View facing north-northeast - U.S. Naval Base, Pearl Harbor, Dry Dock No. 3, On northern shoreline of shipyard, west of Dry Dock Nos. 1 & 2, near the intersection of Avenue G and Sixth Street, Pearl City, Honolulu County, HI

  18. Service building. Cross section thru dry dock nos. 4 & ...

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

    Service building. Cross section thru dry dock nos. 4 & 5 showing service bldg & 20-75-150 ton cranes (dry dock associates, May 23, 1941). In files of Cushman & Wakefield, building no. 501, Philadelphia Naval Business Center. - Naval Base Philadelphia-Philadelphia Naval Shipyard, Service Building, Dry Docks No. 4 & 5, League Island, Philadelphia, Philadelphia County, PA

  19. View of head of dry dock with stair to left ...

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

    View of head of dry dock with stair to left of shot. View facing south - U.S. Naval Base, Pearl Harbor, Dry Dock No. 2, On northern shoreline of Shipyard, between Dry Dock Nos. 1 & 3, Pearl City, Honolulu County, HI

  20. 6. Looking west showing top of dock: steaming frozen ore ...

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

    6. Looking west showing top of dock: steaming frozen ore which had been put in pockets in December 1959, May 6, 1990. Photographer: unknown - Marquette Ore Dock No. 6, Ore Dock, On pilings in Marquette City Lower Harbor, Marquette, Marquette County, MI

  1. 1. Aerial view looking south at Dry Docks 2 and ...

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

    1. Aerial view looking south at Dry Docks 2 and 3. A battleship (possibly the USS PENNSYLVANIA or USS ARIZONA) is in Dock 3. Future Dry Dock 4 site and Point Avisadero is in the upper right corner of photo (1935). Photographer unknown. - Hunters Point Naval Shipyard, Drydock No. 4, East terminus of Palou Avenue, San Francisco, San Francisco County, CA

  2. CovalentDock Cloud: a web server for automated covalent docking

    PubMed Central

    Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

    2013-01-01

    Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/. PMID:23677616

  3. STALK : an interactive virtual molecular docking system.

    SciTech Connect

    Levine, D.; Facello, M.; Hallstrom, P.; Reeder, G.; Walenz, B.; Stevens, F.; Univ. of Illinois

    1997-04-01

    Several recent technologies-genetic algorithms, parallel and distributed computing, virtual reality, and high-speed networking-underlie a new approach to the computational study of how biomolecules interact or 'dock' together. With the Stalk system, a user in a virtual reality environment can interact with a genetic algorithm running on a parallel computer to help in the search for likely geometric configurations.

  4. Pharmacophore-Based Similarity Scoring for DOCK

    PubMed Central

    2015-01-01

    Pharmacophore modeling incorporates geometric and chemical features of known inhibitors and/or targeted binding sites to rationally identify and design new drug leads. In this study, we have encoded a three-dimensional pharmacophore matching similarity (FMS) scoring function into the structure-based design program DOCK. Validation and characterization of the method are presented through pose reproduction, crossdocking, and enrichment studies. When used alone, FMS scoring dramatically improves pose reproduction success to 93.5% (∼20% increase) and reduces sampling failures to 3.7% (∼6% drop) compared to the standard energy score (SGE) across 1043 protein–ligand complexes. The combined FMS+SGE function further improves success to 98.3%. Crossdocking experiments using FMS and FMS+SGE scoring, for six diverse protein families, similarly showed improvements in success, provided proper pharmacophore references are employed. For enrichment, incorporating pharmacophores during sampling and scoring, in most cases, also yield improved outcomes when docking and rank-ordering libraries of known actives and decoys to 15 systems. Retrospective analyses of virtual screenings to three clinical drug targets (EGFR, IGF-1R, and HIVgp41) using X-ray structures of known inhibitors as pharmacophore references are also reported, including a customized FMS scoring protocol to bias on selected regions in the reference. Overall, the results and fundamental insights gained from this study should benefit the docking community in general, particularly researchers using the new FMS method to guide computational drug discovery with DOCK. PMID:25229837

  5. Closed-loop autonomous docking system

    NASA Technical Reports Server (NTRS)

    Dabney, Richard W. (Inventor); Howard, Richard T. (Inventor)

    1992-01-01

    An autonomous docking system is provided which produces commands for the steering and propulsion system of a chase vehicle used in the docking of that chase vehicle with a target vehicle. The docking system comprises a passive optical target affixed to the target vehicle and comprising three reflective areas including a central area mounted on a short post, and tracking sensor and process controller apparatus carried by the chase vehicle. The latter apparatus comprises a laser diode array for illuminating the target so as to cause light to be reflected from the reflective areas of the target; a sensor for detecting the light reflected from the target and for producing an electrical output signal in accordance with an image of the reflected light; a signal processor for processing the electrical output signal in accordance with an image of the reflected light; a signal processor for processing the electrical output signal and for producing, based thereon, output signals relating to the relative range, roll, pitch, yaw, azimuth, and elevation of the chase and target vehicles; and a docking process controller, responsive to the output signals produced by the signal processor, for producing command signals for controlling the steering and propulsion system of the chase vehicle.

  6. New Crew Docks to Poisk Module

    NASA Video Gallery

    Three new Expedition 31 crew members Gennady Padalka, Joe Acaba and Sergei Revin docked to the International Space Station’s Poisk module Thursday May 17, at 12:36 a.m. EDT. They began their jour...

  7. Potential and limitations of ensemble docking.

    PubMed

    Korb, Oliver; Olsson, Tjelvar S G; Bowden, Simon J; Hall, Richard J; Verdonk, Marcel L; Liebeschuetz, John W; Cole, Jason C

    2012-05-25

    A major problem in structure-based virtual screening applications is the appropriate selection of a single or even multiple protein structures to be used in the virtual screening process. A priori it is unknown which protein structure(s) will perform best in a virtual screening experiment. We investigated the performance of ensemble docking, as a function of ensemble size, for eight targets of pharmaceutical interest. Starting from single protein structure docking results, for each ensemble size up to 500,000 combinations of protein structures were generated, and, for each ensemble, pose prediction and virtual screening results were derived. Comparison of single to multiple protein structure results suggests improvements when looking at the performance of the worst and the average over all single protein structures to the performance of the worst and average over all protein ensembles of size two or greater, respectively. We identified several key factors affecting ensemble docking performance, including the sampling accuracy of the docking algorithm, the choice of the scoring function, and the similarity of database ligands to the cocrystallized ligands of ligand-bound protein structures in an ensemble. Due to these factors, the prospective selection of optimum ensembles is a challenging task, shown by a reassessment of published ensemble selection protocols. PMID:22482774

  8. Vision-guided heterogeneous mobile robot docking

    NASA Astrophysics Data System (ADS)

    Spofford, John R.; Blitch, John; Klarquist, William N.; Murphy, Robin R.

    1999-08-01

    Teams of heterogeneous mobile robots are a key aspect of future unmanned system for operations in complex and dynamic urban environments, such as that envisioned by DARPA's Tactical Mobile Robotics program. One examples of an interaction among such team members is the docking of small robot of limited sensory and processing capability with a larger, more capable robot. Applications for such docking include the transfer of power, data, and materia, as well as physically combined maneuver or manipulation. A two-robot system is considered in this paper. The smaller 'throwable' robot contains a video camera capable of imaging the larger 'packable' robot and transmitting the imagery. The packable robot can both sense the throwable robot through an onboard camera, as well as sense itself through the throwable robot's transmitted video, and is capable of processing imagery from either source. This paper describes recent results in the development of control and sensing strategies for automatic mid-range docking of these two robots. Decisions addressed include the selection of which robot's image sensor to use and which robot to maneuver. Initial experimental results are presented for docking using sensor data from each robot.

  9. DOCK8 deficiency in six Iranian patients.

    PubMed

    Saghafi, Shiva; Pourpak, Zahra; Nussbaumer, Franziska; Fazlollahi, Mohammad Reza; Houshmand, Massoud; Hamidieh, Amir Ali; Bemanian, Mohammad Hassan; Nabavi, Mohammad; Parvaneh, Nima; Grimbacher, Bodo; Moin, Mostafa; Glocker, Cristina

    2016-06-01

    DOCK8 deficiency is a rare autosomal recessive combined immunodeficiency with high IgE level, eosinophilia, severe eczema, extensive cutaneous viral, and respiratory bacterial infections, mostly in populations with higher prevalence of consanguinity. Molecular diagnosis of this gene is a useful approach for early diagnosis and timely HSCT due to deleterious consequences. PMID:27398204

  10. Induced fit docking, and the use of QM/MM methods in docking.

    PubMed

    Xu, Mengang; Lill, Markus A

    2013-09-01

    Docking methods are popular computational techniques in drug discovery to identify new active molecules that bind to a given biological target. Although widely used, the predictive reliability of docking methods is often limited by the inability to accurately and efficiently model protein flexibility and quantify binding strength. We highlight several emerging concepts that address those methodological issues including a discussion on the incorporation of QM/MM methodologies in the scoring process.

  11. Building macromolecular assemblies by information-driven docking: introducing the HADDOCK multibody docking server.

    PubMed

    Karaca, Ezgi; Melquiond, Adrien S J; de Vries, Sjoerd J; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

    2010-08-01

    Over the last years, large scale proteomics studies have generated a wealth of information of biomolecular complexes. Adding the structural dimension to the resulting interactomes represents a major challenge that classical structural experimental methods alone will have difficulties to confront. To meet this challenge, complementary modeling techniques such as docking are thus needed. Among the current docking methods, HADDOCK (High Ambiguity-Driven DOCKing) distinguishes itself from others by the use of experimental and/or bioinformatics data to drive the modeling process and has shown a strong performance in the critical assessment of prediction of interactions (CAPRI), a blind experiment for the prediction of interactions. Although most docking programs are limited to binary complexes, HADDOCK can deal with multiple molecules (up to six), a capability that will be required to build large macromolecular assemblies. We present here a novel web interface of HADDOCK that allows the user to dock up to six biomolecules simultaneously. This interface allows the inclusion of a large variety of both experimental and/or bioinformatics data and supports several types of cyclic and dihedral symmetries in the docking of multibody assemblies. The server was tested on a benchmark of six cases, containing five symmetric homo-oligomeric protein complexes and one symmetric protein-DNA complex. Our results reveal that, in the presence of either bioinformatics and/or experimental data, HADDOCK shows an excellent performance: in all cases, HADDOCK was able to generate good to high quality solutions and ranked them at the top, demonstrating its ability to model symmetric multicomponent assemblies. Docking methods can thus play an important role in adding the structural dimension to interactomes. However, although the current docking methodologies were successful for a vast range of cases, considering the variety and complexity of macromolecular assemblies, inclusion of some kind of

  12. Leiomyoma of the seminal vesicles: laparoscopic excision.

    PubMed

    Casado Varela, Javier; Hermida Gutiérrez, Juan Francisco; Castillón Vela, Ignacio T; León Rueda, Maria Eugenia; Ortega Medina, Luis; Moreno Sierra, Jesús

    2014-01-01

    Leiomyoma of the seminal vesicles is an extremely rare type of benign tumor of the genitourinary system and can cause lower urinary tract symptoms. Despite their low incidence, these tumors can be identified with transrectal ultrasound of the seminal vesicles during prostate examination. The removal of these tumors is facilitated by a laparoscopic approach.

  13. Dynamical simulations of vesicle growth and division

    NASA Astrophysics Data System (ADS)

    Ruiz-Herrero, Teresa; Mahadevan, L.

    2015-03-01

    Prebiotic cells constitute a beautiful and intriguing example of self-replicating vesicles. How these cells managed to grow and divide without sophisticated machinery is still an open question. The properties of these primitive vesicles can shed light on the ways modern cells have evolved by exploiting those characteristics to develop their replication mechanisms. The equilibrium configurations of elastic shells are well understood, however the dynamical behavior during growth still lacks of a deep theoretical understanding. To study vesicle growth from a general perspective, we have developed a minimal generic model where vesicles are represented by a 2D spring network and characterized by a minimum set of magnitudes: growth rate, permeability, bending stiffness, viscosity and temperature. We have performed hybrid molecuar dynamic simulations as a function of a reduced set of dimensionless parameters. Three main outcomes were observed: vesicles that grow without division, vesicles that divide symmetrically, and vesicles that act as generators of daughter vesicles. The type of outcome depends on the system parameters and specifically on its dynamics via two timescales. Furthermore, we found sets of parameters where the system shows size homeostasis. TRH was supported by Ramon Areces Foundation.

  14. Synaptic vesicle distribution by conveyor belt.

    PubMed

    Moughamian, Armen J; Holzbaur, Erika L F

    2012-03-01

    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. PMID:22385955

  15. Molecular underpinnings of synaptic vesicle pool heterogeneity.

    PubMed

    Crawford, Devon C; Kavalali, Ege T

    2015-04-01

    Neuronal communication relies on chemical synaptic transmission for information transfer and processing. Chemical neurotransmission is initiated by synaptic vesicle fusion with the presynaptic active zone resulting in release of neurotransmitters. Classical models have assumed that all synaptic vesicles within a synapse have the same potential to fuse under different functional contexts. In this model, functional differences among synaptic vesicle populations are ascribed to their spatial distribution in the synapse with respect to the active zone. Emerging evidence suggests, however, that synaptic vesicles are not a homogenous population of organelles, and they possess intrinsic molecular differences and differential interaction partners. Recent studies have reported a diverse array of synaptic molecules that selectively regulate synaptic vesicles' ability to fuse synchronously and asynchronously in response to action potentials or spontaneously irrespective of action potentials. Here we discuss these molecular mediators of vesicle pool heterogeneity that are found on the synaptic vesicle membrane, on the presynaptic plasma membrane, or within the cytosol and consider some of the functional consequences of this diversity. This emerging molecular framework presents novel avenues to probe synaptic function and uncover how synaptic vesicle pools impact neuronal signaling.

  16. Nanoplasmonic ruler to measure lipid vesicle deformation.

    PubMed

    Jackman, Joshua A; Špačková, Barbora; Linardy, Eric; Kim, Min Chul; Yoon, Bo Kyeong; Homola, Jiří; Cho, Nam-Joon

    2016-01-01

    A nanoplasmonic ruler method is presented in order to measure the deformation of adsorbed, nm-scale lipid vesicles on solid supports. It is demonstrated that single adsorbed vesicles undergo greater deformation on silicon oxide over titanium oxide, offering direct experimental evidence to support membrane tension-based theoretical models of supported lipid bilayer formation.

  17. Docking and scoring protein interactions: CAPRI 2009.

    PubMed

    Lensink, Marc F; Wodak, Shoshana J

    2010-11-15

    Protein docking algorithms are assessed by evaluating blind predictions performed during 2007-2009 in Rounds 13-19 of the community-wide experiment on critical assessment of predicted interactions (CAPRI). We evaluated the ability of these algorithms to sample docking poses and to single out specific association modes in 14 targets, representing 11 distinct protein complexes. These complexes play important biological roles in RNA maturation, G-protein signal processing, and enzyme inhibition and function. One target involved protein-RNA interactions not previously considered in CAPRI, several others were hetero-oligomers, or featured multiple interfaces between the same protein pair. For most targets, predictions started from the experimentally determined structures of the free (unbound) components, or from models built from known structures of related or similar proteins. To succeed they therefore needed to account for conformational changes and model inaccuracies. In total, 64 groups and 12 web-servers submitted docking predictions of which 4420 were evaluated. Overall our assessment reveals that 67% of the groups, more than ever before, produced acceptable models or better for at least one target, with many groups submitting multiple high- and medium-accuracy models for two to six targets. Forty-one groups including four web-servers participated in the scoring experiment with 1296 evaluated models. Scoring predictions also show signs of progress evidenced from the large proportion of correct models submitted. But singling out the best models remains a challenge, which also adversely affects the ability to correctly rank docking models. With the increased interest in translating abstract protein interaction networks into realistic models of protein assemblies, the growing CAPRI community is actively developing more efficient and reliable docking and scoring methods for everyone to use.

  18. Protein-RNA Complexes and Efficient Automatic Docking: Expanding RosettaDock Possibilities

    PubMed Central

    Guilhot-Gaudeffroy, Adrien; Froidevaux, Christine; Azé, Jérôme; Bernauer, Julie

    2014-01-01

    Protein-RNA complexes provide a wide range of essential functions in the cell. Their atomic experimental structure solving, despite essential to the understanding of these functions, is often difficult and expensive. Docking approaches that have been developed for proteins are often challenging to adapt for RNA because of its inherent flexibility and the structural data available being relatively scarce. In this study we adapted the RosettaDock protocol for protein-RNA complexes both at the nucleotide and atomic levels. Using a genetic algorithm-based strategy, and a non-redundant protein-RNA dataset, we derived a RosettaDock scoring scheme able not only to discriminate but also score efficiently docking decoys. The approach proved to be both efficient and robust for generating and identifying suitable structures when applied to two protein-RNA docking benchmarks in both bound and unbound settings. It also compares well to existing strategies. This is the first approach that currently offers a multi-level optimized scoring approach integrated in a full docking suite, leading the way to adaptive fully flexible strategies. PMID:25268579

  19. Protein-RNA complexes and efficient automatic docking: expanding RosettaDock possibilities.

    PubMed

    Guilhot-Gaudeffroy, Adrien; Froidevaux, Christine; Azé, Jérôme; Bernauer, Julie

    2014-01-01

    Protein-RNA complexes provide a wide range of essential functions in the cell. Their atomic experimental structure solving, despite essential to the understanding of these functions, is often difficult and expensive. Docking approaches that have been developed for proteins are often challenging to adapt for RNA because of its inherent flexibility and the structural data available being relatively scarce. In this study we adapted the RosettaDock protocol for protein-RNA complexes both at the nucleotide and atomic levels. Using a genetic algorithm-based strategy, and a non-redundant protein-RNA dataset, we derived a RosettaDock scoring scheme able not only to discriminate but also score efficiently docking decoys. The approach proved to be both efficient and robust for generating and identifying suitable structures when applied to two protein-RNA docking benchmarks in both bound and unbound settings. It also compares well to existing strategies. This is the first approach that currently offers a multi-level optimized scoring approach integrated in a full docking suite, leading the way to adaptive fully flexible strategies.

  20. VORFFIP-driven dock: V-D2OCK, a fast and accurate protein docking strategy.

    PubMed

    Segura, Joan; Marín-López, Manuel Alejandro; Jones, Pamela F; Oliva, Baldo; Fernandez-Fuentes, Narcis

    2015-01-01

    The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D2OCK). This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D2OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD2OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D2OCK predictions from the convenience of their web-browsers.

  1. Protein-RNA complexes and efficient automatic docking: expanding RosettaDock possibilities.

    PubMed

    Guilhot-Gaudeffroy, Adrien; Froidevaux, Christine; Azé, Jérôme; Bernauer, Julie

    2014-01-01

    Protein-RNA complexes provide a wide range of essential functions in the cell. Their atomic experimental structure solving, despite essential to the understanding of these functions, is often difficult and expensive. Docking approaches that have been developed for proteins are often challenging to adapt for RNA because of its inherent flexibility and the structural data available being relatively scarce. In this study we adapted the RosettaDock protocol for protein-RNA complexes both at the nucleotide and atomic levels. Using a genetic algorithm-based strategy, and a non-redundant protein-RNA dataset, we derived a RosettaDock scoring scheme able not only to discriminate but also score efficiently docking decoys. The approach proved to be both efficient and robust for generating and identifying suitable structures when applied to two protein-RNA docking benchmarks in both bound and unbound settings. It also compares well to existing strategies. This is the first approach that currently offers a multi-level optimized scoring approach integrated in a full docking suite, leading the way to adaptive fully flexible strategies. PMID:25268579

  2. Emergence and stability of intermediate open vesicles in disk-to-vesicle transitions.

    PubMed

    Li, Jianfeng; Zhang, Hongdong; Qiu, Feng; Shi, An-Chang

    2013-07-01

    The transition between two basic structures, a disk and an enclosed vesicle, of a finite membrane is studied by examining the minimum energy path (MEP) connecting these two states. The MEP is constructed using the string method applied to continuum elastic membrane models. The results reveal that, besides the commonly observed disk and vesicle, open vesicles (bowl-shaped vesicles or vesicles with a pore) can become stable or metastable shapes. The emergence, stability, and probability distribution of these open vesicles are analyzed. It is demonstrated that open vesicles can be stabilized by higher-order elastic energies. The estimated probability distribution of the different structures is in good agreement with available experiments. PMID:23944502

  3. [Transvesical Removal of Seminal Vesicle Cystadenoma].

    PubMed

    Takayasu, Kenta; Harada, Jiro; Kawa, Gen; Ota, Syuichi; Sakurai, Takanori

    2015-07-01

    Primary tumors of the seminal vesicles are extremely rare. There have been 25 reports of this tumor from overseas and most cases are cystadenoma. We report a case of seminal vesicle cystadenoma in a 70-year-old man who presented with lower abdominal pain and urinary frequency. A digital rectal examination detected a projecting and hard mass in the right side of the prostate. Magnetic resonance imaging (MRI) showed a 15 cm multiple cystic mass continuous with the right seminal vesicle. A transrectal needle biopsy revealed benign tissue. The tumor was resected using an open transvesical approach that enabled full exposure of the seminal vesicle without damaging the nerves and blood supply of the bladder. Pathology was consistent with a benign seminal vesicle cystadenoma. We describe the natural history, pathology,and surgical approach in this case.

  4. Bicarbonate transport in sheep parotid secretory cells.

    PubMed Central

    Steward, M C; Poronnik, P; Cook, D I

    1996-01-01

    1. Intracellular pH (pH1) was measured by microfluorimetry in secretory endpieces isolated from sheep parotid glands and loaded with the pH-sensitive fluoroprobe 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). 2. Stimulation with 1 microM acetylcholine (ACh) caused a large, transient decrease in pH1 of 0.37 +/- 0.02 pH units followed by a slower recovery. The transient, which was reduced by 60% in the absence of HCO3-, could be attributed mainly to HCO3- efflux. During sustained stimulation, pH1 increased to a value that exceeded the resting value by 0.083 +/- 0.023 pH units after 20 min. 3. The anion channel blocker NPPB (0.1 mM) reduced the transient acidification in response to ACh by 48% and raised pH1 during sustained stimulation. Simultaneous application of NPPB and ACh accelerated the re-alkalinization following the initial acidification, indicating that NPPB inhibits HCO3- efflux. 4. The stilbene derivative H2DIDS (0.5 mM) reduced the transient acidification in response to ACh by 76% but caused a marked decrease in pH1 during sustained stimulation. Simultaneous application of H2DIDS and ACh slowed the re-alkalinization following the initial acidification, indicating that the main effect of H2DIDS was to inhibit HCO3- accumulation. 5. In the absence of HCO3-, the recovery from an acid load was unaffected by ACh stimulation. Acid extrusion, although dependent on Na+, was not inhibited by amiloride (1 mM), clonidine (1 mM) or H2DIDS (0.5 mM) and was therefore provisionally attributed to a Na(+)-H+ exchanger isoform other than NHE1 or NHE2. 6. In the presence of HCO3-, the rate of recovery from an acid load was reduced during ACh stimulation, probably as a result of the increased efflux of HCO3-. Acid extrusion was dependent on Na+ and was significantly inhibited by H2DIDS. 7. We conclude that ACh-evoked HCO3- secretion in the sheep parotid gland differs from that in many other salivary glands by being driven predominantly by basolateral Na(+)-HCO3

  5. 13. ORE DOCK, LOOKING EAST FROM HULETT NO. 1. WHEN ...

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

    13. ORE DOCK, LOOKING EAST FROM HULETT NO. 1. WHEN BUILT IN 1911-1912, THIS WAS THE LARGEST ORE-UNLOADING DOCK ON THE GREAT LAKES. THE DOCK FEATURED FOUR HULETT UNLOADERS, EACH WITH A BUCKET CAPACITY OF 17 TONS; A 15-TON CAPACITY ORE STOCKING AND REHANDLING BRIDGE; AND A ONE-MILLION-TON CAPACITY ORE STORAGE YARD. THE WILLIAM-SEAVER-MORGAN COMPANY OF CLEVELAND BUILT THE DOCK EQUIPMENT. - Pennsylvania Railway Ore Dock, Lake Erie at Whiskey Island, approximately 1.5 miles west of Public Square, Cleveland, Cuyahoga County, OH

  6. Analysis of Yeast Extracellular Vesicles.

    PubMed

    Rodrigues, Marcio L; Oliveira, Debora L; Vargas, Gabriele; Girard-Dias, Wendell; Franzen, Anderson J; Frasés, Susana; Miranda, Kildare; Nimrichter, Leonardo

    2016-01-01

    Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering. PMID:27665559

  7. Analysis of Yeast Extracellular Vesicles.

    PubMed

    Rodrigues, Marcio L; Oliveira, Debora L; Vargas, Gabriele; Girard-Dias, Wendell; Franzen, Anderson J; Frasés, Susana; Miranda, Kildare; Nimrichter, Leonardo

    2016-01-01

    Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering.

  8. Synaptic vesicles in electromotoneurones. I. Axonal transport, site of transmitter uptake and processing of a core proteoglycan during maturation.

    PubMed Central

    Kiene, M L; Stadler, H

    1987-01-01

    We were able by using an in vivo pulse-label technique to trace part of the life cycle of a secretory organelle, the acetylcholine-storing synaptic vesicle from electromotoneurones of Torpedo marmorata. This technique uses [35S]sulphate incorporation into the cell bodies of the electromotoneurones which results in radioactive labelling of a synaptic vesicle heparansulphate proteoglycan--a major core component. Vesicles are anterogradely transported in the axons at a fast rate as 'empty' organelles (VP0 population). In the nerve terminal, maturation of the granule to a population (VP1) fully charged with acetylcholine and ATP occurs. Finally after a longer time interval a change to a third population (VP2) is observed. This population is reduced in diameter as compared to VP0 and VP1 suggesting, in agreement with earlier reports, that it has undergone exo-endocytosis. The changes from VP0 to VP1 and VP2 are accompanied by a degradation of the core proteoglycan as measured by gel filtration of the 35S-labelled compound. The results show that vesicles are axonally transported as preformed organelles, exist in the neurone at least in three different populations and that the nerve terminal is the major site of transmitter uptake. Images Fig. 1. Fig. 4. Fig. 6. PMID:2444433

  9. Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

    PubMed

    Schuster, Martin; Martin-Urdiroz, Magdalena; Higuchi, Yujiro; Hacker, Christian; Kilaru, Sreedhar; Gurr, Sarah J; Steinberg, Gero

    2016-01-01

    Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation. PMID:27563844

  10. Effects of chronic ethanol ingestion on the vasoactive intestinal peptide receptor-effector system from rat seminal vesicle membranes.

    PubMed

    Juarranz, M G; Marinero, M J; Bodega, G; Prieto, J C; Guijarro, L G

    1999-02-01

    We studied the modifications of the vasoactive intestinal peptide (VIP) receptor/effector system from the rat seminal vesicle after chronic ethanol ingestion. Ethanol treatment resulted in a decreased height of the secretory epithelium of seminal vesicle as well as in a weight loss of this gland. These morphological changes were accompanied by an increase of immunoreactive vasoactive intestinal peptide (VIP) levels and a decrease of the stimulatory effect of VIP adenylate cyclase activity in the seminal vesicle. The loss of sensitivity of the enzyme to VIP was conceivably related to a decrease in the affinity of VIP receptors rather than to a decrease in their number. The changes in the affinity of the VIP receptors were accompanied with a lower sensitivity of VIP binding to GTP, which suggest an uncoupling between the receptor and the transductor molecules. However, chronic exposure to ethanol did not modify either the levels of G-protein subunits (alpha(s) and alpha(i1/2)) or the GTPase activity from seminal vesicle membranes. Moreover, ethanol feeding did not affect adenylate cyclase activity stimulated by forskolin or by Gpp(NH)p. Thus, ethanol-induced changes in the sensitivity of adenylate cyclase to VIP appear to be attributed to an alteration in the VIP-receptor/G-protein interphase rather than in the G-protein/adenylate cyclase connection. PMID:10069562

  11. Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

    PubMed

    Schuster, Martin; Martin-Urdiroz, Magdalena; Higuchi, Yujiro; Hacker, Christian; Kilaru, Sreedhar; Gurr, Sarah J; Steinberg, Gero

    2016-01-01

    Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation.

  12. Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

    PubMed Central

    Lagow, Robert D; Bao, Hong; Cohen, Evan N; Daniels, Richard W; Zuzek, Aleksej; Williams, Wade H; Macleod, Gregory T; Sutton, R. Bryan; Zhang, Bing

    2007-01-01

    Both constitutive secretion and Ca2+-regulated exocytosis require the assembly of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex. PMID:17341138

  13. Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli▿ †

    PubMed Central

    Fisher, Adam C.; Haitjema, Charles H.; Guarino, Cassandra; Çelik, Eda; Endicott, Christine E.; Reading, Craig A.; Merritt, Judith H.; Ptak, A. Celeste; Zhang, Sheng; DeLisa, Matthew P.

    2011-01-01

    The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates. PMID:21131519

  14. Tubal function in chronic secretory otitis media in children.

    PubMed

    Poulsen, G; Tos, M

    1977-01-01

    In 100 children (150 ears) with chronic secretory otitis media the function of the Eustachian tube during treatment with grommet was investigated by air equalisation methods. Tubal function proved poor in the great majority at the beginning of the treatment, but towards its completion there was some improvement. After extrusion of the grommet, tubal function was investigated on the same material by tympanometry. 34% had normal middle-ear pressure initially, and 43% 12-18 months after closure of the perforation. There was no relation between tubal function shown by air equalisation methods and by tympanometry, and the air equalisation methods proved of less value than tympanometry in assessing the course and prognosis of secretory otitis. The pathogenetic theories - the ex vacuo and the secretory theory - are discussed in relation to the chronic tubal dysfunction found to be the most common direct cause of the disease.

  15. On the Computing Potential of Intracellular Vesicles

    PubMed Central

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal ‘circuitry’ and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a ‘vesicle modification’ of the archetypal CBC ‘billiard ball model’ of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle ‘programming’ in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  16. Optical Manipulation of Vesicles for Optofluidic Applications

    SciTech Connect

    Vasdekis, Andreas E.; Scott, E. A.; O'Neil, C. P.; Psaltis, D.; Hubbell, J. A.

    2013-09-12

    In this report, we review our recent results in the optical micromanipulation of vesicles. Traditionally, vesicle manipulation has been possible by employing photon momentum and optical trapping, giving rise to unique observations of vesicle shape changes and soft matter mechanics. Contrary to these attempts, we employ photon energy rather than momentum, by sensitizing vesicles with an oxidizing moiety. The later converts incident photons to reactive oxygen species, which in turn attack and compromise the stability of the vesicle membrane. Both coherent and incoherent radiation was employed. Polymersome re-organization into smaller diameter vesicles was possible by focusing the excitation beam in the vicinity of the polymersomes. Extended vesicle illumination with a collimated beam lead to their complete destabilization and micelle formation. Single particle analysis revealed that payload release takes place within seconds of illumination in an explosive burst. We will discuss the destabilization and payload release kinetics, as revealed by high resolution microscopy at the single particle level, as well as potential applications in single cell biomodulation.

  17. Optical manipulation of vesicles for optofluidic applications

    NASA Astrophysics Data System (ADS)

    Vasdekis, A. E.; Scott, E. A.; O'Neil, C. P.; Psaltis, D.; Hubbell, J. A.

    2013-09-01

    In this report, we review our recent results in the optical micromanipulation of vesicles. Traditionally, vesicle manipulation has been possible by employing photon momentum and optical trapping, giving rise to unique observations of vesicle shape changes and soft matter mechanics. Contrary to these attempts, we employ photon energy rather than momentum, by sensitizing vesicles with an oxidizing moiety. The later converts incident photons to reactive oxygen species, which in turn attack and compromise the stability of the vesicle membrane. Both coherent and incoherent radiation was employed. Polymersome re-organization into smaller diameter vesicles was possible by focusing the excitation beam in the vicinity of the polymersomes. Extended vesicle illumination with a collimated beam lead to their complete destabilization and micelle formation. Single particle analysis revealed that payload release takes place within seconds of illumination in an explosive burst. We will discuss the destabilization and payload release kinetics, as revealed by high resolution microscopy at the single particle level, as well as potential applications in single cell biomodulation.

  18. On the Computing Potential of Intracellular Vesicles.

    PubMed

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  19. Telerobotic rendezvous and docking vision system architecture

    NASA Technical Reports Server (NTRS)

    Gravely, Ben; Myers, Donald; Moody, David

    1992-01-01

    This research program has successfully demonstrated a new target label architecture that allows a microcomputer to determine the position, orientation, and identity of an object. It contains a CAD-like database with specific geometric information about the object for approach, grasping, and docking maneuvers. Successful demonstrations were performed selecting and docking an ORU box with either of two ORU receptacles. Small, but significant differences were seen in the two camera types used in the program, and camera sensitive program elements have been identified. The software has been formatted into a new co-autonomy system which provides various levels of operator interaction and promises to allow effective application of telerobotic systems while code improvements are continuing.

  20. MSFC Three Point Docking Mechanism design review

    NASA Technical Reports Server (NTRS)

    Schaefer, Otto; Ambrosio, Anthony

    1992-01-01

    In the next few decades, we will be launching expensive satellites and space platforms that will require recovery for economic reasons, because of initial malfunction, servicing, repairs, or out of a concern for post lifetime debris removal. The planned availability of a Three Point Docking Mechanism (TPDM) is a positive step towards an operational satellite retrieval infrastructure. This study effort supports NASA/MSFC engineering work in developing an automated docking capability. The work was performed by the Grumman Space & Electronics Group as a concept evaluation/test for the Tumbling Satellite Retrieval Kit. Simulation of a TPDM capture was performed in Grumman's Large Amplitude Space Simulator (LASS) using mockups of both parts (the mechanism and payload). Similar TPDM simulation activities and more extensive hardware testing was performed at NASA/MSFC in the Flight Robotics Laboratory and Space Station/Space Operations Mechanism Test Bed (6-DOF Facility).

  1. Laser space rendezvous and docking tradeoff

    NASA Technical Reports Server (NTRS)

    Adelman, S.; Levinson, S.; Raber, P.; Weindling, F.

    1974-01-01

    A spaceborne laser radar (LADAR) was configured to meet the requirements for rendezvous and docking with a cooperative object in synchronous orbit. The LADAR, configurated using existing pulsed CO2 laser technology and a 1980 system technology baseline, is well suited for the envisioned space tug missions. The performance of a family of candidate LADARS was analyzed. Tradeoff studies as a function of size, weight, and power consumption were carried out for maximum ranges of 50, 100, 200, and 300 nautical miles. The investigation supports the original contention that a rendezvous and docking LADAR can be constructed to offer a cost effective and reliable solution to the envisioned space missions. In fact, the CO2 ladar system offers distinct advantages over other candidate systems.

  2. The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    PubMed Central

    Stalder, Danièle; Novick, Peter J.

    2016-01-01

    Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

  3. BP-Dock: A Flexible Docking Scheme for Exploring Protein–Ligand Interactions Based on Unbound Structures

    PubMed Central

    Bolia, Ashini; Gerek, Z. Nevin; Ozkan, S. Banu

    2016-01-01

    Molecular docking serves as an important tool in modeling protein–ligand interactions. However, it is still challenging to incorporate overall receptor flexibility, especially backbone flexibility, in docking due to the large conformational space that needs to be sampled. To overcome this problem, we developed a novel flexible docking approach, BP-Dock (Backbone Perturbation-Dock) that can integrate both backbone and side chain conformational changes induced by ligand binding through a multi-scale approach. In the BP-Dock method, we mimic the nature of binding-induced events as a first-order approximation by perturbing the residues along the protein chain with a small Brownian kick one at a time. The response fluctuation profile of the chain upon these perturbations is computed using the perturbation response scanning method. These response fluctuation profiles are then used to generate binding-induced multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, we applied our approach on a large and diverse data set using unbound structures as receptors. We also compared the BP-Dock results with bound and unbound docking, where overall receptor flexibility was not taken into account. Our results highlight the importance of modeling backbone flexibility in docking for recapitulating the experimental binding affinities, especially when an unbound structure is used. With BP-Dock, we can generate a wide range of binding site conformations realized in nature even in the absence of a ligand that can help us to improve the accuracy of unbound docking. We expect that our fast and efficient flexible docking approach may further aid in our understanding of protein–ligand interactions as well as virtual screening of novel targets for rational drug design. PMID:24380381

  4. Expression of familial Alzheimer disease presenilin 1 gene attenuates vesicle traffic and reduces peptide secretion in cultured astrocytes devoid of pathologic tissue environment.

    PubMed

    Stenovec, Matjaž; Trkov, Saša; Lasič, Eva; Terzieva, Slavica; Kreft, Marko; Rodríguez Arellano, José Julio; Parpura, Vladimir; Verkhratsky, Alexei; Zorec, Robert

    2016-02-01

    In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild-type (wt) and 3xTg-AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored with Fluo-2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP- and LysoTracker-labeled vesicles was diminished in 3xTg-AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP-evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1M146V). The ATP-evoked ANP discharge from single vesicles was less efficient in 3xTg-AD and PS1M146V-expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca(2+)]i responses; the latter were less frequent (P < 0.001) in 3xTg-AD astrocytes. Expression of PS1M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD.

  5. Extracellular vesicles: Exosomes, microvesicles, and friends

    PubMed Central

    Stoorvogel, Willem

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function. PMID:23420871

  6. Molecular docking using the molecular lipophilicity potential as hydrophobic descriptor: impact on GOLD docking performance.

    PubMed

    Nurisso, Alessandra; Bravo, Juan; Carrupt, Pierre-Alain; Daina, Antoine

    2012-05-25

    GOLD is a molecular docking software widely used in drug design. In the initial steps of docking, it creates a list of hydrophobic fitting points inside protein cavities that steer the positioning of ligand hydrophobic moieties. These points are generated based on the Lennard-Jones potential between a carbon probe and each atom of the residues delimitating the binding site. To thoroughly describe hydrophobic regions in protein pockets and properly guide ligand hydrophobic moieties toward favorable areas, an in-house tool, the MLP filter, was developed and herein applied. This strategy only retains GOLD hydrophobic fitting points that match the rigorous definition of hydrophobicity given by the molecular lipophilicity potential (MLP), a molecular interaction field that relies on an atomic fragmental system based on 1-octanol/water experimental partition coefficients (log P(oct)). MLP computations in the binding sites of crystallographic protein structures revealed that a significant number of points considered hydrophobic by GOLD were actually polar according to the MLP definition of hydrophobicity. To examine the impact of this new tool, ligand-protein complexes from the Astex Diverse Set and the PDB bind core database were redocked with and without the use of the MLP filter. Reliable docking results were obtained by using the MLP filter that increased the quality of docking in nonpolar cavities and outperformed the standard GOLD docking approach.

  7. Multiple exposure of Rendezvous Docking Simulator

    NASA Technical Reports Server (NTRS)

    1964-01-01

    Multiple exposure of Rendezvous Docking Simulator. Francis B. Smith, described the simulator as follows: '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.' Published in Barton C. Hacker and James M. Grimwood, On the Shoulders of Titans: A History of Project Gemini, NASA SP-4203; Francis B. Smith, 'Simulators for Manned Space Research,' Paper presented at the 1966 IEEE International convention, March 21-25, 1966.

  8. Combined Immunodeficiency Associated with DOCK8 Mutations

    PubMed Central

    Zhang, Qian; Davis, Jeremiah C.; Lamborn, Ian T.; Freeman, Alexandra F.; Jing, Huie; Favreau, Amanda J.; Matthews, Helen F.; Davis, Joie; Turner, Maria L.; Uzel, Gulbu; Holland, Steven M.; Su, Helen C.

    2010-01-01

    BACKGROUND Recurrent sinopulmonary and cutaneous viral infections with elevated serum levels of IgE are features of some variants of combined immunodeficiency. The genetic causes of these variants are unknown. METHODS We collected longitudinal clinical data on 11 patients from eight families who had recurrent sinopulmonary and cutaneous viral infections. We performed comparative genomic hybridization arrays and targeted gene sequencing. Variants with predicted loss-of-expression mutations were confirmed by means of a quantitative reverse-transcriptase –polymerase-chain-reaction assay and immunoblotting. We evaluated the number and function of lymphocytes with the use of in vitro assays and flow cytometry. RESULTS Patients had recurrent otitis media, sinusitis, and pneumonias; recurrent Staphylococcus aureus skin infections with otitis externa; recurrent, severe herpes simplex virus or herpes zoster infections; extensive and persistent infections with molluscum contagiosum; and human papillomavirus infections. Most patients had severe atopy with anaphylaxis; several had squamous-cell carcinomas, and one had T-cell lymphoma –leukemia. Elevated serum IgE levels, hypereosinophilia, low numbers of T cells and B cells, low serum IgM levels, and variable IgG antibody responses were common. Expansion in vitro of activated CD8 T cells was impaired. Novel homozygous or compound heterozygous deletions and point mutations in the gene encoding the dedicator of cytokinesis 8 protein (DOCK8) led to the absence of DOCK8 protein in lymphocytes. CONCLUSIONS Autosomal recessive DOCK8 deficiency is associated with a novel variant of combined immunodeficiency. PMID:19776401

  9. Structural templates for comparative protein docking

    PubMed Central

    Anishchenko, Ivan; Kundrotas, Petras J.; Tuzikov, Alexander V.; Vakser, Ilya A.

    2014-01-01

    Structural characterization of protein-protein interactions is important for understanding life processes. Because of the inherent limitations of experimental techniques, such characterization requires computational approaches. Along with the traditional protein-protein docking (free search for a match between two proteins), comparative (template-based) modeling of protein-protein complexes has been gaining popularity. Its development puts an emphasis on full and partial structural similarity between the target protein monomers and the protein-protein complexes previously determined by experimental techniques (templates). The template-based docking relies on the quality and diversity of the template set. We present a carefully curated, non-redundant library of templates containing 4,950 full structures of binary complexes and 5,936 protein-protein interfaces extracted from the full structures at 12Å distance cut-off. Redundancy in the libraries was removed by clustering the PDB structures based on structural similarity. The value of the clustering threshold was determined from the analysis of the clusters and the docking performance on a benchmark set. High structural quality of the interfaces in the template and validation sets was achieved by automated procedures and manual curation. The library is included in the Dockground resource for molecular recognition studies at http://dockground.bioinformatics.ku.edu. PMID:25488330

  10. Structural templates for comparative protein docking.

    PubMed

    Anishchenko, Ivan; Kundrotas, Petras J; Tuzikov, Alexander V; Vakser, Ilya A

    2015-09-01

    Structural characterization of protein-protein interactions is important for understanding life processes. Because of the inherent limitations of experimental techniques, such characterization requires computational approaches. Along with the traditional protein-protein docking (free search for a match between two proteins), comparative (template-based) modeling of protein-protein complexes has been gaining popularity. Its development puts an emphasis on full and partial structural similarity between the target protein monomers and the protein-protein complexes previously determined by experimental techniques (templates). The template-based docking relies on the quality and diversity of the template set. We present a carefully curated, nonredundant library of templates containing 4950 full structures of binary complexes and 5936 protein-protein interfaces extracted from the full structures at 12 Å distance cut-off. Redundancy in the libraries was removed by clustering the PDB structures based on structural similarity. The value of the clustering threshold was determined from the analysis of the clusters and the docking performance on a benchmark set. High structural quality of the interfaces in the template and validation sets was achieved by automated procedures and manual curation. The library is included in the Dockground resource for molecular recognition studies at http://dockground.bioinformatics.ku.edu.

  11. The targeting, docking and anti-proteolysis functions of the secretin chaperone PulS.

    PubMed

    Collin, Séverine; Krehenbrink, Martin; Guilvout, Ingrid; Pugsley, Anthony P

    2013-06-01

    The Klebsiella oxytoca lipoprotein PulS might function as either or both a pilot and a docking factor in the outer membrane targeting and assembly of the Type II secretion system secretin PulD. In the piloting model, PulS binds to PulD monomers and targets them to the outer membrane via the lipoprotein sorting pathway components LolA and LolB. In this model, PulS also protects the PulD monomers from proteolysis during transit through the periplasm. In the docking model, PulS is targeted alone to the outer membrane, where it acts as a receptor for PulD monomers, allowing them to accumulate and assemble specifically in this membrane. PulS was shown to dissociate from and/or re-associate freely with PulD multimers in zwitterionic detergent, making it difficult to determine whether PulS remains associated with PulD dodecamers in the outer membrane by co-purification. However, PulD protomers in the dodecamer were shown to be stable in the absence of PulS, indicating that PulS is only required to protect the protease-susceptible monomer. DegP was identified as one of the proteases that could contribute to PulD degradation in the absence of PulS. Studies on the in vitro assembly and targeting of PulD into Escherichia coli membrane vesicles demonstrated its strong preference to insert into the inner membrane, as is the case in vivo in the absence of PulS. However, PulD could be targeted to outer membrane fragments in vitro if they were preloaded with PulS, indicating the technical feasibility of the docking model. We conclude that both modes of action might contribute to efficient outer membrane targeting of PulD in vivo, although the piloting function is likely to predominate.

  12. Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection.

    PubMed

    Heard, William; Sklenář, Jan; Tomé, Daniel F A; Robatzek, Silke; Jones, Alexandra M E

    2015-07-01

    The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein

  13. Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

    PubMed Central

    Heard, William; Sklenář, Jan; Tomé, Daniel F. A.; Robatzek, Silke; Jones, Alexandra M. E.

    2015-01-01

    The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein

  14. Kinetics of particle wrapping by a vesicle

    NASA Astrophysics Data System (ADS)

    Mirigian, Stephen; Muthukumar, Murugappan

    2013-07-01

    We present theoretical results on kinetics for the passive wrapping of a single, rigid particle by a flexible membrane. Using a simple geometric ansatz for the shape of the membrane/particle complex we first compute free energy profiles as a function of the particle size, attraction strength between the particle and vesicle, and material properties of the vesicle—bending stiffness and stretching modulus. The free energy profiles thus computed are taken as input to a stochastic model of the wrapping process, described by a Fokker-Planck equation. We compute average uptake rates of the particle into the vesicle. We find that the rate of particle uptake falls to zero outside of a thermodynamically allowed range of particle sizes. Within the thermodynamically allowed range of particle size, the rate of uptake is variable and we compute the optimal particle size and maximal uptake rate as a function of the attraction strength, the vesicle size, and vesicle material properties.

  15. Physiopathologic dynamics of vesicle traffic in astrocytes.

    PubMed

    Potokar, Maja; Stenovec, Matjaž; Kreft, Marko; Gabrijel, Mateja; Zorec, Robert

    2011-02-01

    The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.

  16. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  17. Transformation of oil droplets into giant vesicles.

    PubMed

    Sheng, Li; Kurihara, Kensuke

    2016-06-14

    We propose a protocell model in which compartments are constructed via a new process involving the formation of robust vesicles using an autocatalytic, self-reproducing oil droplet system as a 'scaffold'. PMID:27152371

  18. Monte Carlo simulations of fluid vesicles.

    PubMed

    Sreeja, K K; Ipsen, John H; Sunil Kumar, P B

    2015-07-15

    Lipid vesicles are closed two dimensional fluid surfaces that are studied extensively as model systems for understanding the physical properties of biological membranes. Here we review the recent developments in the Monte Carlo techniques for simulating fluid vesicles and discuss some of their applications. The technique, which treats the membrane as an elastic sheet, is most suitable for the study of large scale conformations of membranes. The model can be used to study vesicles with fixed and varying topologies. Here we focus on the case of multi-component membranes with the local lipid and protein composition coupled to the membrane curvature leading to a variety of shapes. The phase diagram is more intriguing in the case of fluid vesicles having an in-plane orientational order that induce anisotropic directional curvatures. Methods to explore the steady state morphological structures due to active flux of materials have also been described in the context of Monte Carlo simulations. PMID:26087479

  19. Monte Carlo simulations of fluid vesicles

    NASA Astrophysics Data System (ADS)

    Sreeja, K. K.; Ipsen, John H.; Kumar, P. B. Sunil

    2015-07-01

    Lipid vesicles are closed two dimensional fluid surfaces that are studied extensively as model systems for understanding the physical properties of biological membranes. Here we review the recent developments in the Monte Carlo techniques for simulating fluid vesicles and discuss some of their applications. The technique, which treats the membrane as an elastic sheet, is most suitable for the study of large scale conformations of membranes. The model can be used to study vesicles with fixed and varying topologies. Here we focus on the case of multi-component membranes with the local lipid and protein composition coupled to the membrane curvature leading to a variety of shapes. The phase diagram is more intriguing in the case of fluid vesicles having an in-plane orientational order that induce anisotropic directional curvatures. Methods to explore the steady state morphological structures due to active flux of materials have also been described in the context of Monte Carlo simulations.

  20. Stability of Spherical Vesicles in Electric Fields

    PubMed Central

    2010-01-01

    The stability of spherical vesicles in alternating (ac) electric fields is studied theoretically for asymmetric conductivity conditions across their membranes. The vesicle deformation is obtained from a balance between the curvature elastic energies and the work done by the Maxwell stresses. The present theory describes and clarifies the mechanisms for the four types of morphological transitions observed experimentally on vesicles exposed to ac fields in the frequency range from 500 to 2 × 107 Hz. The displacement currents across the membranes redirect the electric fields toward the membrane normal to accumulate electric charges by the Maxwell−Wagner mechanism. These accumulated electric charges provide the underlying molecular mechanism for the morphological transitions of vesicles as observed on the micrometer scale. PMID:20575588

  1. Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1-5 ms resolution.

    PubMed

    Wang, Tingting; Smith, Elizabeth A; Chapman, Edwin R; Weisshaar, James C

    2009-05-20

    A single-vesicle, fluorescence-based, SNARE-driven fusion assay enables simultaneous measurement of lipid mixing and content release with 5 ms/frame, or even 1 ms/frame, time resolution. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (<5 ms duration), intense spike of calcein fluorescence due to content release and dequenching coincides with inner-leaflet lipid mixing within 10 ms. The spike provides more sensitive detection of productive hemifusion events than do lipid labels alone. Consequently, many fast events previously thought to be prompt, full fusion events are now reclassified as productive hemifusion. Both full fusion and hemifusion occur with a time constant of 5-10 ms. At 60% phosphatidylethanolamine lipid composition, productive and dead-end hemifusion account for 65% of all fusion events. However, quantitative analysis shows that calcein is released into the space above the bilayer (vesicle bursting), rather than the thin aqueous space between the bilayer and glass. Evidently, at the instant of inner-leaflet mixing, flattening of the vesicle increases the internal pressure beyond the bursting point. This may be related to in vivo observations suggesting that membrane lysis often competes with membrane fusion.

  2. Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome.

    PubMed Central

    Webb, G C; Zhang, J; Garlow, S J; Wesp, A; Riezman, H; Jones, E W

    1997-01-01

    Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome. Images PMID:9168472

  3. Extracellular vesicles in parasitic diseases

    PubMed Central

    Marcilla, Antonio; Martin-Jaular, Lorena; Trelis, Maria; de Menezes-Neto, Armando; Osuna, Antonio; Bernal, Dolores; Fernandez-Becerra, Carmen; Almeida, Igor C.; del Portillo, Hernando A.

    2014-01-01

    Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens. PMID:25536932

  4. Clinical applications of the radioimmunoassay of secretory tuberculoprotein.

    PubMed Central

    Straus, E; Wu, N; Quraishi, M A; Levine, S

    1981-01-01

    A radioimmunoassay that measures a specific secretory tuberculoprotein was used to detect Mycobacterium tuberculosis in 9 of 30 liquid cultures of sputum. The accumulation of immunoreactive material in liquid cultures containing isoniazid was shown to reflect in vitro susceptibility of mycobacteria to the antibiotic effects of the drug. PMID:6789332

  5. Separation of rat pituitary secretory granules by continuous flow electrophoresis

    NASA Technical Reports Server (NTRS)

    Hayes, Daniel; Exton, Carrie; Salada, Thomas; Shellenberger, Kathy; Waddle, Jenny; Hymer, W. C.

    1990-01-01

    The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

  6. Medical management of secretory syndromes related to gastroenteropancreatic neuroendocrine tumours.

    PubMed

    Dimitriadis, Georgios K; Weickert, Martin O; Randeva, Harpal S; Kaltsas, Gregory; Grossman, Ashley

    2016-09-01

    Although recent epidemiological evidence indicates that the prevalence of non-functioning gastroenteropancreatic (GEP) neuroendocrine tumours (NETs) is rising, a significant number of GEP-NETs still present with symptoms related to the secretion of biologically active substances leading to the development of distinct clinical syndromes. In the past, these syndromes were associated with substantial morbidity and mortality due to the lack of specific therapies; however, since the introduction of long-acting somatostatin analogues and medications such as proton pump inhibitors, their control has been greatly improved. As a result, nowadays, the main cause of morbidity and mortality in GEP-NETs is mostly directly related to tumour growth and the extent of metastatic disease. However, in some patients with functioning tumours and extensive disease, control of the secretory syndrome still remains problematic, necessitating the employment of several cytoreductive techniques, which may not always be sufficient. Recently, new agents directed against tumour growth, or exerting increased binding activity to receptors expressed in these tumours, or interfering with the synthetic pathway of some of the compounds secreted by these tumours, have been developed. Since there are no specific guidelines addressing the totality of the management of the secretory syndromes related to GEP-NETs, this review aims at critically analysing the medical management of previously recognised secretory syndromes; it also addresses areas of uncertainty, assesses the newer therapeutic developments and also addresses recently described but poorly characterised secretory syndromes related to GEP-NETs. PMID:27461388

  7. Effects of tail docking and docking length on neuroanatomical changes in healed tail tips of pigs.

    PubMed

    Herskin, M S; Thodberg, K; Jensen, H E

    2015-04-01

    In pig production, piglets are tail docked at birth in order to prevent tail biting later in life. In order to examine the effects of tail docking and docking length on the formation of neuromas, we used 65 pigs and the following four treatments: intact tails (n=18); leaving 75% (n=17); leaving 50% (n=19); or leaving 25% (n=11) of the tail length on the pigs. The piglets were docked between day 2 and 4 after birth using a gas-heated apparatus, and were kept under conventional conditions until slaughter at 22 weeks of age, where tails were removed and examined macroscopically and histologically. The tail lengths and diameters differed at slaughter (lengths: 30.6±0.6; 24.9±0.4; 19.8±0.6; 8.7±0.6 cm; P<0.001; tail diameter: 0.5±0.03; 0.8±0.02; 1.0±0.03; 1.4±0.04 cm; P<0.001, respectively). Docking resulted in a higher proportion of tails with neuromas (64 v. 0%; P<0.001), number of neuromas per tail (1.0±0.2 v. 0; P<0.001) and size of neuromas (1023±592 v. 0 μm; P<0.001). The results show that tail docking piglets using hot-iron cautery causes formation of neuromas in the outermost part of the tail tip. The presence of neuromas might lead to altered nociceptive thresholds, which need to be confirmed in future studies. PMID:25482535

  8. InterEvDock: a docking server to predict the structure of protein-protein interactions using evolutionary information.

    PubMed

    Yu, Jinchao; Vavrusa, Marek; Andreani, Jessica; Rey, Julien; Tufféry, Pierre; Guerois, Raphaël

    2016-07-01

    The structural modeling of protein-protein interactions is key in understanding how cell machineries cross-talk with each other. Molecular docking simulations provide efficient means to explore how two unbound protein structures interact. InterEvDock is a server for protein docking based on a free rigid-body docking strategy. A systematic rigid-body docking search is performed using the FRODOCK program and the resulting models are re-scored with InterEvScore and SOAP-PP statistical potentials. The InterEvScore potential was specifically designed to integrate co-evolutionary information in the docking process. InterEvDock server is thus particularly well suited in case homologous sequences are available for both binding partners. The server returns 10 structures of the most likely consensus models together with 10 predicted residues most likely involved in the interface. In 91% of all complexes tested in the benchmark, at least one residue out of the 10 predicted is involved in the interface, providing useful guidelines for mutagenesis. InterEvDock is able to identify a correct model among the top10 models for 49% of the rigid-body cases with evolutionary information, making it a unique and efficient tool to explore structural interactomes under an evolutionary perspective. The InterEvDock web interface is available at http://bioserv.rpbs.univ-paris-diderot.fr/services/InterEvDock/.

  9. Modeling of protein-peptide interactions using the CABS-dock web server for binding site search and flexible docking.

    PubMed

    Blaszczyk, Maciej; Kurcinski, Mateusz; Kouza, Maksim; Wieteska, Lukasz; Debinski, Aleksander; Kolinski, Andrzej; Kmiecik, Sebastian

    2016-01-15

    Protein-peptide interactions play essential functional roles in living organisms and their structural characterization is a hot subject of current experimental and theoretical research. Computational modeling of the structure of protein-peptide interactions is usually divided into two stages: prediction of the binding site at a protein receptor surface, and then docking (and modeling) the peptide structure into the known binding site. This paper presents a comprehensive CABS-dock method for the simultaneous search of binding sites and flexible protein-peptide docking, available as a user's friendly web server. We present example CABS-dock results obtained in the default CABS-dock mode and using its advanced options that enable the user to increase the range of flexibility for chosen receptor fragments or to exclude user-selected binding modes from docking search. Furthermore, we demonstrate a strategy to improve CABS-dock performance by assessing the quality of models with classical molecular dynamics. Finally, we discuss the promising extensions and applications of the CABS-dock method and provide a tutorial appendix for the convenient analysis and visualization of CABS-dock results. The CABS-dock web server is freely available at http://biocomp.chem.uw.edu.pl/CABSdock/.

  10. InterEvDock: a docking server to predict the structure of protein-protein interactions using evolutionary information.

    PubMed

    Yu, Jinchao; Vavrusa, Marek; Andreani, Jessica; Rey, Julien; Tufféry, Pierre; Guerois, Raphaël

    2016-07-01

    The structural modeling of protein-protein interactions is key in understanding how cell machineries cross-talk with each other. Molecular docking simulations provide efficient means to explore how two unbound protein structures interact. InterEvDock is a server for protein docking based on a free rigid-body docking strategy. A systematic rigid-body docking search is performed using the FRODOCK program and the resulting models are re-scored with InterEvScore and SOAP-PP statistical potentials. The InterEvScore potential was specifically designed to integrate co-evolutionary information in the docking process. InterEvDock server is thus particularly well suited in case homologous sequences are available for both binding partners. The server returns 10 structures of the most likely consensus models together with 10 predicted residues most likely involved in the interface. In 91% of all complexes tested in the benchmark, at least one residue out of the 10 predicted is involved in the interface, providing useful guidelines for mutagenesis. InterEvDock is able to identify a correct model among the top10 models for 49% of the rigid-body cases with evolutionary information, making it a unique and efficient tool to explore structural interactomes under an evolutionary perspective. The InterEvDock web interface is available at http://bioserv.rpbs.univ-paris-diderot.fr/services/InterEvDock/. PMID:27131368

  11. Lipid vesicle aggregation induced by cooling.

    PubMed

    Howard, Frank B; Levin, Ira W

    2010-01-01

    Lipid bilayer fusion is a complex process requiring several intermediate steps. Initially, the two bilayers are brought into close contact following removal of intervening water layers and overcoming electrostatic repulsions between opposing bilayer head groups. In this study we monitor by light scattering the reversible aggregation of phosphatidylcholine single shell vesicles during which adhesion occurs but stops prior to a fusion process. Light scattering measurements of dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water show that lowering the temperature of about 0.14 micron single shell vesicles of DPPC (from 20 degrees C to 5 degrees C) and about 2 micron vesicles of DSPC (from 20 degrees C to 15 degrees C), but not of 1 micron vesicles of DMPC, results in extensive aggregation within 24 hours that is reversible by an increase in temperature. Aggregation of DSPC vesicles was confirmed by direct visual observation. Orientation of lipid head groups parallel to the plane of the bilayer and consequent reduction of the negative surface charge can account for the ability of DPPC and DSPC vesicles to aggregate. Retention of negatively charged phosphates on the surface and the burial of positively charged cholines within the bilayer offer an explanation for the failure of DMPC vesicles to aggregate. Lowering the temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) vesicles from 20 degrees C to 5 degrees C failed to increase aggregation within 24 hours at Mg(++)/DPPS ratios that begin to initiate aggregation and fusion.

  12. Differential detergent sensitivity of extracellular vesicle subpopulations.

    PubMed

    Osteikoetxea, Xabier; Sódar, Barbara; Németh, Andrea; Szabó-Taylor, Katalin; Pálóczi, Krisztina; Vukman, Krisztina V; Tamási, Viola; Balogh, Andrea; Kittel, Ágnes; Pállinger, Éva; Buzás, Edit Irén

    2015-10-14

    Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

  13. Sucrose induces vesicle accumulation and autophagy.

    PubMed

    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes.

  14. Vesicle-associated melanization in Cryptococcus neoformans.

    PubMed

    Eisenman, Helene C; Frases, Susana; Nicola, André M; Rodrigues, Marcio L; Casadevall, Arturo

    2009-12-01

    Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungus Cryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall of C. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursor L-3,4-dihydroxyphenylalanine (L-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelled L-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model for C. neoformans melanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall.

  15. Vesicle-associated melanization in Cryptococcus neoformans

    PubMed Central

    Eisenman, Helene C.; Frases, Susana; Nicola, André M.; Rodrigues, Marcio L.; Casadevall, Arturo

    2009-01-01

    Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungus Cryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall of C. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursor l-3,4-dihydroxyphenylalanine (l-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelled l-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model for C. neoformans melanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall. PMID:19729402

  16. Spontaneous unilamellar polymer vesicles in aqueous solution.

    PubMed

    Kim, Tae-Hwan; Song, Chaeyeon; Han, Young-Soo; Jang, Jong-Dae; Choi, Myung Chul

    2014-01-21

    A unilamellar polymeric vesicle is a self-assembled structure of a block copolymer that forms a spherical single bilayer structure with a hydrophobic interlayer and a hydrophilic surface. Due to their enhanced colloidal stability and mechanical property, controllable surface functionality, or tunable membrane thickness, polymeric vesicles are useful in nano and bio-science, providing potential applications as nanosized carriers for catalysts, drugs, and enzymes. For fabrication of a unilamellar vesicle, however, preparative procedures with a few steps are inherently required. Herein, without complicated preparative procedures, we report spontaneous unilamellar polymeric vesicles with nanometer sizes (<100 nm), which are prepared by simply mixing a triblock copolymer, Pluronic P85 (PEO26PPO40PEO26), and an organic derivative, 5-methyl salicylic acid (5mS), in aqueous solution. Depending on the 5mS concentration and the temperature, the P85-5mS mixtures presented various self-assembled nanostructures such as spherical and cylindrical micelles or vesicles, which were characterized by small angle neutron scattering and cryo-TEM, resulting in a phase diagram drawn as a function of temperature and the 5mS concentration. Interestingly the critical temperature for the micelle-to-vesicle phase transition was easily controlled by varying the 5mS concentration, i.e. it was decreased with increasing the 5mS concentration. PMID:24652418

  17. Sucrose induces vesicle accumulation and autophagy.

    PubMed

    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes. PMID:25389129

  18. The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes.

    PubMed

    Kim, Hyeran; O'Connell, Richard; Maekawa-Yoshikawa, Makoto; Uemura, Tomohiro; Neumann, Ulla; Schulze-Lefert, Paul

    2014-09-01

    Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM. PMID:24941879

  19. A Comparison of Candidate Seal Designs for Future Docking Systems

    NASA Technical Reports Server (NTRS)

    Dunlap, Patrick, H., Jr.; Steinetz, Bruce, M.

    2012-01-01

    NASA is developing a new docking system to support future space exploration missions to low Earth orbit, the Moon, and other destinations. A key component of this system is the seal at the main docking interface which inhibits the loss of cabin air once docking is complete. Depending on the mission, the seal must be able to dock in either a seal-on-flange or seal-on-seal configuration. Seal-on-flange mating would occur when a docking system equipped with a seal docks to a system with a flat metal flange. This would occur when a vehicle docks to a node on the International Space Station. Seal-on-seal mating would occur when two docking systems equipped with seals dock to each other. Two types of seal designs were identified for this application: Gask-O-seals and multi-piece seals. Both types of seals had a pair of seal bulbs to satisfy the redundancy requirement. A series of performance assessments and comparisons were made between the candidate seal designs indicating that they meet the requirements for leak rate and compression and adhesion loads under a range of operating conditions. Other design factors such as part count, integration into the docking system tunnel, seal-on-seal mating, and cost were also considered leading to the selection of the multi-piece seal design for the new docking system. The results of this study can be used by designers of future docking systems and other habitable volumes to select the seal design best-suited for their particular application.

  20. Magnet-Based System for Docking of Miniature Spacecraft

    NASA Technical Reports Server (NTRS)

    Howard, Nathan; Nguyen, Hai D.

    2007-01-01

    A prototype system for docking a miniature spacecraft with a larger spacecraft has been developed by engineers at the Johnson Space Center. Engineers working on Mini AERCam, a free-flying robotic camera, needed to find a way to successfully dock and undock their miniature spacecraft to refuel the propulsion and recharge the batteries. The subsystems developed (see figure) include (1) a docking port, designed for the larger spacecraft, which contains an electromagnet, a ball lock mechanism, and a service probe; and (2) a docking cluster, designed for the smaller spacecraft, which contains either a permanent magnet or an electromagnet. A typical docking operation begins with the docking spacecraft maneuvering into position near the docking port on the parent vehicle. The electromagnet( s) are then turned on, and, if necessary, the docking spacecraft is then maneuvered within the capture envelope of the docking port. The capture envelope for this system is approximated by a 5-in. (12.7-cm) cube centered on the front of the docking-port electromagnet and within an angular misalignment of <30 . Thereafter, the magnetic forces draw the smaller spacecraft toward the larger one and this brings the spacecraft into approximate alignment prior to contact. Mechanical alignment guides provide the final rotational alignment into one of 12 positions. Once the docking vehicle has been captured magnetically in the docking port, the ball-lock mechanism is activated, which locks the two spacecraft together. At this point the electromagnet( s) are turned off, and the service probe extended if recharge and refueling are to be performed. Additionally, during undocking, the polarity of one electromagnet can be reversed to provide a gentle push to separate the two spacecraft. This system is currently being incorporated into the design of Mini AERCam vehicle.

  1. 21. ORE DOCK, LOOKING SOUTHWEST. THIS VIEW SHOWS THE WEST ...

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

    21. ORE DOCK, LOOKING SOUTHWEST. THIS VIEW SHOWS THE WEST END OF THE DOCK. EMPTY CARS ARE MOVED IN FROM THE WEST BY 'SHUNT CARS,' PUT INTO PLACE AS NEEDED BENEATH THE HULETTS, FILLED, THEN SHUNTED TO THE EAST END OF THE YARD WHERE THEY ARE MADE UP INTO TRAINS. THE POWER HOUSE (WITH TALL ARCHED WINDOWS) AND THE TWO-STORY DOCK OFFICE CAN BE SEEN HERE. - Pennsylvania Railway Ore Dock, Lake Erie at Whiskey Island, approximately 1.5 miles west of Public Square, Cleveland, Cuyahoga County, OH

  2. 9. North Plant, View of Canopied Loading Dock with Powerhouse ...

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

    9. North Plant, View of Canopied Loading Dock with Powerhouse to Left, Looking Northwest - Atwater Kent Manufacturing Company, North Plant, 5000 Wissahickon Avenue, Philadelphia, Philadelphia County, PA

  3. Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane.

    PubMed

    Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R

    2015-03-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

  4. Optoelectronic Sensor System for Guidance in Docking

    NASA Technical Reports Server (NTRS)

    Howard, Richard T.; Bryan, Thomas C.; Book, Michael L.; Jackson, John L.

    2004-01-01

    The Video Guidance Sensor (VGS) system is an optoelectronic sensor that provides automated guidance between two vehicles. In the original intended application, the two vehicles would be spacecraft docking together, but the basic principles of design and operation of the sensor are applicable to aircraft, robots, vehicles, or other objects that may be required to be aligned for docking, assembly, resupply, or precise separation. The system includes a sensor head containing a monochrome charge-coupled- device video camera and pulsed laser diodes mounted on the tracking vehicle, and passive reflective targets on the tracked vehicle. The lasers illuminate the targets, and the resulting video images of the targets are digitized. Then, from the positions of the digitized target images and known geometric relationships among the targets, the relative position and orientation of the vehicles are computed. As described thus far, the VGS system is based on the same principles as those of the system described in "Improved Video Sensor System for Guidance in Docking" (MFS-31150), NASA Tech Briefs, Vol. 21, No. 4 (April 1997), page 9a. However, the two systems differ in the details of design and operation. The VGS system is designed to operate with the target completely visible within a relative-azimuth range of +/-10.5deg and a relative-elevation range of +/-8deg. The VGS acquires and tracks the target within that field of view at any distance from 1.0 to 110 m and at any relative roll, pitch, and/or yaw angle within +/-10deg. The VGS produces sets of distance and relative-orientation data at a repetition rate of 5 Hz. The software of this system also accommodates the simultaneous operation of two sensors for redundancy

  5. Dynamic Docking Test System (DDTS) active table computer program NASA Advanced Docking System (NADS)

    NASA Technical Reports Server (NTRS)

    Gates, R. M.; Jantz, R. E.

    1974-01-01

    A computer program was developed to describe the three-dimensional motion of the Dynamic Docking Test System active table. The input consists of inertia and geometry data, actuator structural data, forcing function data, hydraulics data, servo electronics data, and integration control data. The output consists of table responses, actuator bending responses, and actuator responses.

  6. Digital simulation for post-docking response

    NASA Technical Reports Server (NTRS)

    Roberts, J. R.; Todd, R. S.

    1974-01-01

    The digital program, 2BODY, which simulates the translational and rotational motion of two connected rigid bodies and provides both digital and plot output is described. Relative rotation of the bodies at the connection is allowed, thereby providing a model suitable for studying system stability and response during a soft-dock regime. A users manual for the program is given as well as all the details and background pertaining to the equations of motion and mathematical models, integration scheme, and input/output routines.

  7. Autonomous Rendezvous and Docking Conference, volume 2

    NASA Technical Reports Server (NTRS)

    1990-01-01

    Autonomous Rendezvous and Docking (ARD) will be a requirement for future space programs. Clear examples include satellite servicing, repair, recovery, and reboost in the near term, and the longer range lunar and planetary exploration programs. ARD will permit more aggressive unmanned space activities, while providing a valuable operational capability for manned missions. The purpose of the conference is to identify the technologies required for an on-orbit demonstration of ARD, assess the maturity of those technologies, and provide the necessary insight for a quality assessment of programmatic management, technical, schedule, and cost risks.

  8. Rendezvous and docking with remote piloted vehicles

    NASA Technical Reports Server (NTRS)

    Micheal, J. D.

    1984-01-01

    The man-in-the-loop control system requirements for the Orbital Maneuvering Vehicle (OMV) are examined. Since many similarities exist between the Teleoperator Retrieval System (TRS) and the unfolding OMV concept, a review of the TRS control system baseline along with selected design trades which led to that baseline are discussed. TRS program issues relevant to the man-in-the-loop control system design include thruster size, communication delays and TV bandwidth compression, range/range rate radar, tumbling targets, shimmed docking interface, and control system definition. A TRS vs. OMV simulation comparative study is summarized, and the major issues currently facing the control system designer on OMV are discussed.

  9. cAMP- but not Ca(2+)-regulated Cl- conductance is lacking in cystic fibrosis mice epididymides and seminal vesicles.

    PubMed

    Leung, A Y; Wong, P Y; Yankaskas, J R; Boucher, R C

    1996-07-01

    Cystic fibrosis (CF) reflects the loss of adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- secretion consequent to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In humans, but not mice, with CF, the disease is associated with male infertility. The present study investigated the relative magnitudes of the cAMP pathways and an alternative Ca(2+)-regulated Cl- secretory pathway in primary cultures of the epididymides and the seminal vesicles of normal and CF mice. The basal equivalent short-circuit currents (Ieq) of cultures derived from the epididymides and the seminal vesicles from the CF mice were lower (6.0 +/- 0.6 and 4.0 +/- 1.0 muA/cm2, respectively) than those from normal mice (11.1 +/- 1.0 and 6.6 +/- 0.6 muA/cm2, respectively). Forskolin induced significant Ieq responses in both the epididymis (8.0 +/- 0.7 muA/cm2) and seminal vesicles (4.0 +/- 0.5 muA/cm2) from normal mice, whereas forskolin-induced changes in Ieq in CF mouse epididymis and seminal vesicles were absent, consistent with defective cAMP-CFTR-mediated Cl- secretion in CF mice. Ieq responses to agonists (ionomycin, ATP) that raise intracellular Ca2+ (Ca2+i) were larger than forskolin responses in normal animals (6.6 +/- 0.9 and 13.4 +/- 1.8 muA/cm2, respectively) and were preserved in CF (6.5 +/- 0.9 and 17.1 +/- 1.0 muA/cm2, respectively). We speculate that the fertility of male CF mice is maintained by persistent expression of the predominant alternative Ca(2+)-mediated Cl- transport system in the epididymides and seminal vesicles. PMID:8760045

  10. CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site

    PubMed Central

    Kurcinski, Mateusz; Jamroz, Michal; Blaszczyk, Maciej; Kolinski, Andrzej; Kmiecik, Sebastian

    2015-01-01

    Protein–peptide interactions play a key role in cell functions. Their structural characterization, though challenging, is important for the discovery of new drugs. The CABS-dock web server provides an interface for modeling protein–peptide interactions using a highly efficient protocol for the flexible docking of peptides to proteins. While other docking algorithms require pre-defined localization of the binding site, CABS-dock does not require such knowledge. Given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), CABS-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone. This protocol was extensively tested over the largest dataset of non-redundant protein–peptide interactions available to date (including bound and unbound docking cases). For over 80% of bound and unbound dataset cases, we obtained models with high or medium accuracy (sufficient for practical applications). Additionally, as optional features, CABS-dock can exclude user-selected binding modes from docking search or to increase the level of flexibility for chosen receptor fragments. CABS-dock is freely available as a web server at http://biocomp.chem.uw.edu.pl/CABSdock. PMID:25943545

  11. In vitro reconstitution of Rab GTPase-dependent vesicle clustering by the yeast lethal giant larvae/tomosyn homolog, Sro7.

    PubMed

    Rossi, Guendalina; Watson, Kelly; Demonch, Mallory; Temple, Brenda; Brennwald, Patrick

    2015-01-01

    Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.

  12. SCAMP5 plays a critical role in synaptic vesicle endocytosis during high neuronal activity.

    PubMed

    Zhao, Haiyan; Kim, Yoonju; Park, Joohyun; Park, Daehun; Lee, Sang-Eun; Chang, Iree; Chang, Sunghoe

    2014-07-23

    Secretory carrier membrane protein 5 (SCAMP5), a recently identified candidate gene for autism, is brain specific and highly abundant in synaptic vesicles (SVs), but its function is currently unknown. Here, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs in cultured rat hippocampal neurons resulted in a reduction in total vesicle pool size as well as in recycling pool size, but the recycling/resting pool ratio was significantly increased. SCAMP5 KD slowed endocytosis after stimulation, but impaired it severely during strong stimulation. We also found that KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during a stimulus. Reintroducing shRNA-resistant SCAMP5 reversed these endocytic defects. Therefore, our results suggest that SCAMP5 functions during high neuronal activity when a heavy load is imposed on endocytosis. Our data also raise the possibility that the reduction in expression of SCAMP5 in autistic patients may be related to the synaptic dysfunction observed in autism.

  13. A pre-docking source for the power-law behavior of spontaneous quantal release: application to the analysis of LTP

    PubMed Central

    Lamanna, Jacopo; Signorini, Maria G.; Cerutti, Sergio; Malgaroli, Antonio

    2015-01-01

    In neurons, power-law behavior with different scaling exponents has been reported at many different levels, including fluctuations in membrane potentials, synaptic transmission up to neuronal network dynamics. Unfortunately in most cases the source of this non-linear feature remains controversial. Here we have analyzed the dynamics of spontaneous quantal release at hippocampal synapses and characterized their power-law behavior. While in control conditions a fractal exponent greater than zero was rarely observed, its value was greatly increased by α-latrotoxin (α-LTX), a potent stimulator of spontaneous release, known to act at the very last step of vesicle fusion. Based on computer modeling, we confirmed that at an increase in fusion probability would unmask a pre-docking phenomenon with 1/f structure, where α estimated from the release series appears to sense the increase in release probability independently from the number of active sites. In the simplest scenario the pre-docking 1/f process could coincide with the Brownian diffusion of synaptic vesicles. Interestingly, when the effect of long-term potentiation (LTP) was tested, a ~200% long-lasting increase in quantal frequency was accompanied by a significant increase in the scaling exponent. The similarity between the action of LTP and of α-LTX suggests an increased contribution of high release probability sites following the induction of LTP. In conclusion, our results indicate that the source of the synaptic power-law behavior arises before synaptic vesicles dock to the active zone and that the fractal exponent α is capable of sensing a change in release probability independently from the number of active sites or synapses. PMID:25741239

  14. Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

    PubMed Central

    Coorssen, Jens R.; Blank, Paul S.; Tahara, Masahiro; Zimmerberg, Joshua

    1998-01-01

    Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion. PMID:9864359

  15. Activation of calcineurin by phosphotidylserine containing vesicles

    SciTech Connect

    Politino, M.; King, M.M.

    1986-05-01

    Calcineurin (CaN) is a Ca/sup 2 +/- and calmodulin-regulated phosphatase. Recent findings suggested an association of CaN with biological membranes and prompted the present investigation into the interactions of the phosphatase with phospholipids in vitro. In the absence of calmodulin, sonicated preparations of phosphatidylserine (PS) provided a five-fold activation of the Ni- and Mn-supported activities of CaN towards (/sup 32/P) histone Hl; activation in the presence of calmodulin was much less pronounced. Half-maximal activation in the absence of calmodulin required approximately 0.1 mg/ml of PS. Activation of CaN was also observed with mixed vesicles of phosphatidylcholine (PC) containing 20% PS but not with PC alone, or with phosphatidylethanolamine (PE). Molecular sieve chromatography on Ultrogel AcA 34 provided further evidence that CaN associates with phospholipid vesicles composed of PS, or PC containing 20% PS, but not with vesicles of PC or PE. Complete association with medium sized vesicles of PS and PC/PS required Ca/sup 2 +/ ions; in the absence of the metal ion at least 60% of the enzyme failed to interact with the lipids while the remainder preferentially migrated with larger vesicles. These results suggest a role for Ca/sup 2 +/ in regulating CaN's interaction with phospholipids.

  16. Intramembrane electrostatic interactions destabilize lipid vesicles.

    PubMed Central

    Shoemaker, Scott D; Vanderlick, T Kyle

    2002-01-01

    Membrane stability is of central concern in many biology and biotechnology processes. It has been suggested that intramembrane electrostatic interactions play a key role in membrane stability. However, due primarily to a lack of supporting experimental evidence, they are not commonly considered in mechanical analyses of lipid membranes. In this paper, we use the micropipette aspiration technique to characterize the elastic moduli and critical tensions of lipid vesicles with varying surface charge. Charge was induced by doping neutral phosphatidylcholine vesicles with anionic lipids phosphatidylglycerol and phosphatidic acid. Measurements were taken in potassium chloride (moderate ion-lipid binding) and tetramethylammonium chloride (low ion-lipid binding) solutions. We show that inclusion of anionic lipid does not appreciably alter the areal dilation elasticity of lipid vesicles. However, the tension required for vesicle rupture decreases with increasing anionic lipid fraction and is a function of electrolyte composition. Using vesicles with 30% charged (i.e., unbound) anionic lipid, we measured critical tension reductions of 75%, demonstrating the important role of electrostatic interactions in membrane stability. PMID:12324419

  17. Astrocytic vesicle mobility in health and disease.

    PubMed

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  18. Astrocytic Vesicle Mobility in Health and Disease

    PubMed Central

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions. PMID:23712361

  19. Polypeptide vesicles with densely packed multilayer membranes.

    PubMed

    Song, Ziyuan; Kim, Hojun; Ba, Xiaochu; Baumgartner, Ryan; Lee, Jung Seok; Tang, Haoyu; Leal, Cecilia; Cheng, Jianjun

    2015-05-28

    Multilamellar membranes are important building blocks for constructing self-assembled structures with improved barrier properties, such as multilamellar lipid vesicles. Polymeric vesicles (polymersomes) have attracted growing interest, but multilamellar polymersomes are much less explored. Here, we report the formation of polypeptide vesicles with unprecedented densely packed multilayer membrane structures with poly(ethylene glycol)-block-poly(γ-(4,5-dimethoxy-2-nitrobenzyl)-l-glutamate) (PEG-b-PL), an amphiphilic diblock rod-coil copolymer containing a short PEG block and a short hydrophobic rod-like polypeptide segment. The polypeptide rods undergo smectic ordering with PEG buried between the hydrophobic polypeptide layers. The size of both blocks and the rigidity of the hydrophobic polypeptide block are critical in determining the membrane structures. Increase of the PEG length in PEG-b-PL results in the formation of bilayer sheets, while using random-coil polypeptide block leads to the formation of large compound micelles. UV treatment causes ester bond cleavage of the polypeptide side chain, which induces helix-to-coil transition, change of copolymer amphiphilicity, and eventual disassembly of vesicles. These polypeptide vesicles with unique membrane structures provide a new insight into self-assembly structure control by precisely tuning the composition and conformation of polymeric amphiphiles.

  20. Synaptic vesicle recycling: steps and principles

    PubMed Central

    Rizzoli, Silvio O

    2014-01-01

    Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle. PMID:24596248