ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

PubMed

Diao, Jiajie; Liu, Rong; Rong, Yueguang; Zhao, Minglei; Zhang, Jing; Lai, Ying; Zhou, Qiangjun; Wilz, Livia M; Li, Jianxu; Vivona, Sandro; Pfuetzner, Richard A; Brunger, Axel T; Zhong, Qing

2015-04-23

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

Transport According to GARP: Receiving Retrograde Cargo at the Trans-Golgi Network

PubMed Central

Bonifacino, Juan S.; Hierro, Aitor

2010-01-01

Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, COG and Dsl1, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, underscoring the essential nature of GARP-mediated retrograde transport. PMID:21183348

Creating Knock-outs of Conserved Oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function

PubMed Central

Blackburn, Jessica Bailey; Lupashin, Vladimir V.

2017-01-01

Summary The Conserved Oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex’s role in glycosylation homeostasis to obtain COG complex subunit knock-outs (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis. PMID:27632008

Sorting of amphiphile membrane components in curvature and composition gradients

NASA Astrophysics Data System (ADS)

Tian, Aiwei

Phase and shape heterogeneities in biomembranes are of functional importance. However, it is difficult to elucidate the roles membrane heterogeneities play in maintaining cellular function due to the complexity of biomembranes. Therefore, investigations of phase behavior and composition/curvature coupling in lipid and polymer model membranes offer some advantages. In this thesis, phase properties in lipid and polymer giant vesicles were studied. Line tension at the fluid/fluid phase boundary of giant lipid unilamellar vesicles was determined directly by micropipette aspiration, and found to be composition-dependent. Dynamics of calcium-induced domains within polyanionic vesicles subject to chemical stimuli were investigated, which revealed the strength of molecular interaction and suggested applications in triggered delivery. In addition, curvature sorting of lipids and proteins was examined. Lipid membrane tethers were pulled from giant unilamellar vesicles using two micropipettes and a bead. Tether radius can be controlled and measured in this system. By examining fluorescence intensity of labeled molecules as a function of curvature, we found that DiI dyes (lipid analogues with spontaneous curvatures) had no curvature preference down to radii of 10 nm. Theoretical calculation predicted that the distribution of small lipids was dominated by entropy instead of bending energy. However protein Cholera toxin subunit B was efficiently sorted away from the high positive curvature due to its negative spontaneous curvature. Bending stiffness was determined to decrease as curvature increased in homogeneous membranes with ternary lipid mixtures near a critical consulate point, revealing the strong preferential intermolecular interactions of such mixtures. In addition, diffusion controlled domain growth was observed in tethers pulled from phase-separated vesicles, which provides a new dynamic sorting principle for lipids and proteins in curvature gradients.

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein (GARP) Complex with the t-SNARE Syntaxin 6

PubMed Central

Abascal-Palacios, Guillermo; Schindler, Christina; Rojas, Adriana L; Bonifacino, Juan S.; Hierro, Aitor

2016-01-01

Summary The Golgi-Associated Retrograde Protein (GARP) is a tethering complex involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein we report the structural basis for the interaction of the human Ang2 subunit of GARP with Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a novel binding mode in which a di-tyrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction. The same binding determinants are found in other tethering proteins and syntaxins, suggesting a general interaction mechanism. PMID:23932592

A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sano, Hiroyuki; Peck, Grantley R.; Blachon, Stephanie

Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the twomore » highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation. - Highlights: • Insulin stimulates the fusion of vesicles containing GLUT4 with the plasma membrane. • This requires vesicular Rab10-GTP and the exocyst plasma membrane tethering complex. • We find that Rab10-GTP associates with the Exoc6 subunit of the exocyst. • We find that knockdown of Exoc6 inhibits fusion of GLUT4 vesicles with the membrane. • The interaction of Rab10-GTP with Exoc6 potentially links signaling to exocytosis.« less

Colloidosomes formed by nonpolar/polar/nonpolar nanoball amphiphiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Hung-Yu; Sheng, Yu-Jane, E-mail: yjsheng@ntu.edu.tw, E-mail: hktsao@cc.ncu.edu.tw; Tu, Sheng-Hung

2014-08-07

Fullerene-based amphiphiles are able to form bilayer vesicles in aqueous solution. In this study, the self-assembly behavior of polymer-tethered nanoballs (NBs) with nonpolar/polar/nonpolar (n-p-n{sup ′}) motif in a selective solvent is investigated by dissipative particle dynamics. A model NB bears two hydrophobic polymeric arms (n{sup ′}-part) tethered on an extremely hydrophobic NB (n-part) with hydrophilic patch (p-part) patterned on its surface. Dependent on the hydrophobicity and length of tethered arms, three types of aggregates are exhibited, including NB vesicle, core-shell micelle, and segmented-worm. NB vesicles are developed for a wide range of hydrophobic arm lengths. The presence of tethered armsmore » perturbs the bilayer structure formed by NBs. The structural properties including the order parameter, membrane thickness, and area density of the inner leaflet decrease with increasing the arm length. These results indicate that for NBs with longer arms, the extent of interdigitation in the membrane rises so that the overcrowded arms in the inner corona are relaxed. The transport and mechanical properties are evaluated as well. As the arm length grows, the permeability increases significantly because the steric bulk of tethered arms loosens the packing of NBs. By contrast, the membrane tension decreases owing to the reduction of NB/solvent contacts by the polymer corona. Although fusion can reduce membrane tension, NB vesicles show strong resistance to fusion. Moreover, the size-dependent behavior observed in small liposomes is not significant for NB vesicles due to isotropic geometry of NB. Our simulation results are consistent with the experimental findings.« less

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1.

PubMed

Morgera, Francesca; Sallah, Margaret R; Dubuke, Michelle L; Gandhi, Pallavi; Brewer, Daniel N; Carr, Chavela M; Munson, Mary

2012-01-01

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

TRAPPC9 mediates the interaction between p150 and COPII vesicles at the target membrane.

PubMed

Zong, Min; Satoh, Ayano; Yu, Mei Kuen; Siu, Ka Yu; Ng, Wing Yan; Chan, Hsiao Chang; Tanner, Julian A; Yu, Sidney

2012-01-01

The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150(Glued), a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown. We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150(Glued) away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150(Glued) via the same carboxyl terminal domain of p150(Glued) that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150(Glued) and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150(Glued) from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane. These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.

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

PubMed

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

2016-02-15

The Ca(2+)-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 micro-scopy. 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. © 2016 Kabachinski, Kielar-Grevstad, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Solid-Supported Lipid Membranes: Formation, Stability and Applications

NASA Astrophysics Data System (ADS)

Goh, Haw Zan

This thesis presents a comprehensive investigation of the formation of supported lipid membranes with vesicle hemifusion, their stability under detergents and organic solvents and their applications in molecular biology. In Chapter 3, we describe how isolated patches of DOPC bilayers supported on glass surfaces are dissolved by various detergents (decyl maltoside, dodecyl maltoside, CHAPS, CTAB, SDS, TritonX-100 and Tween20) at their CMC, as investigated by fluorescence video microscopy. In general, detergents partition into distal leaflets of bilayers and lead to the expansion of the bilayers through a rolling motion of the distal over the proximal leaflets, in agreement with the first stage of the established 3-stage model of lipid vesicle solubilization by detergents. Subsequently, we study the partitioning of organic solvents (methanol, ethanol, isopropanol, propanol, acetone and chloroform) into isolated bilayer patches on glass in Chapter 4 with fluorescence microscopy. The area expansion of bilayers due to the partitioning of organic solvents is measured. From the titration of organic solvents, we measured the rate of area expansion as a function of the volume fraction of organic solvents, which is proposed to be a measure of strength of interactions between solvents and membranes. From the same experiments, we also measure the maximum expansion of bilayers (or the maximum binding stoichiometry between organic solvents and lipids) before structural breakdown, which depends on the depth of penetration of solvents to the membranes. In Chapter 5, we investigate the formation of sparsely-tethered bilayer lipid membranes (stBLMs) with vesicle hemifusion. In vesicle hemifusion, lipid vesicles in contact with a hydrophobic alkyl-terminated self-assembled monolayer (SAM) deposit a lipid monolayer to the SAM surface, thus completing the bilayer. Electrical Impedance Spectroscopy and Neutron Reflectivity are used to probe the integrity of stBLMs in terms of their insulating and structural properties. Preparation conditions are screened for those that are optimal for stBLM formation. Concentrations of lipid vesicles, hydrophobicity of SAMs, the presence of calcium and high concentrations of salt are identified as the key parameters. We show that stBLMs can be formed with vesicles of different compositions. Vesicle hemifusion opens up a new route in preserving the chemical compositions of stBLMs and facilitating membrane proteins incorporation. In Chapter 6, we visualize the hemifusion pathway of giant unilamellar vesicles (GUVs) with planar hydrophobic surfaces at the single vesicle level with fluorescence video microscopy. When a GUV hemifuses to a surface, its outer leaflet breaks apart and remains connected to the surface presumably through a hemifusion diaphragm. Lipids from the outer leaflet are transferred to the surface as a lipid monolayer that expands radially outward from the hemifusion diaphragm, thereby forming the loosely packed outer hemifusion zone. In Chapter 7, we develop an in vitro assay employing stBLMs and lipid vesicles to examine the functionality of GRASP in membrane tethering. Membrane-bound GRASP on opposing membranes dimerizes and tethers fluorescently-labeled vesicles to stBLMs. The fluorescence intensity of images taken at stBLM surfaces is used to quantify the tethering activity. Both wild type and mutant proteins were studied to shed light on the molecular mechanism of tethering. We show that the GRASP domain is sufficient and necessary for membrane tethering. In addition, the tethering capability of GRASP is impaired when the internal ligands and the binding pockets participating in dimerization are deleted and mutated. Membrane anchors, sizes of vesicles and membrane compositions are explored for their influence on the outcomes of the assay. Furthermore, preliminary analysis from neutron reflectivity measurements shows that both the internal ligands and binding pockets are exposed instead of buried toward the membrane surface. In summary, we establish a functional assay for studying GRASP activity in vitro. (Abstract shortened by UMI.)

Mammalian TRAPPIII Complex positively modulates the recruitment of Sec13/31 onto COPII vesicles

PubMed Central

Zhao, Shan; Li, Chun Man; Luo, Xiao Min; Siu, Gavin Ka Yu; Gan, Wen Jia; Zhang, Lin; Wu, William K. K.; Chan, Hsiao Chang; Yu, Sidney

2017-01-01

The Transport protein particle (TRAPP) complex is a tethering factor for COPII vesicle. Of three forms of TRAPP (TRAPPI, II and III) complexes identified so far, TRAPPIII has been largely considered to play a role in autophagy. While depletion of TRAPPIII specific subunits caused defects in the early secretory pathway and TRAPPIII might interact with components of the COPII vesicle coat, its exact role remains to be determined. In this study, we studied the function of TRAPPIII in early secretory pathway using a TRAPPIII-specific subunit, TRAPPC12, as starting point. We found that TRAPPC12 was localized to the ER exit sites and ERGIC. In cells deleted with TRAPPC12, ERGIC and to a lesser extent, the Golgi became dispersed. ER-to-Golgi transport was also delayed. TRAPPC12, but not TRAPPC8, bound to Sec13/Sec31A tetramer but each Sec protein alone could not interact with TRAPPC12. TRAPPIII positively modulated the assembly of COPII outer layer during COPII vesicle formation. These results identified a novel function of TRAPPIII as a positive modulator of the outer layer of the COPII coat. PMID:28240221

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

Integrated self-organization of transitional ER and early Golgi compartments.

PubMed

Glick, Benjamin S

2014-02-01

COPII coated vesicles bud from an ER domain termed the transitional ER (tER), but the mechanism that clusters COPII vesicles at tER sites is unknown. tER sites are closely associated with early Golgi or pre-Golgi structures, suggesting that the clustering of nascent COPII vesicles could be achieved by tethering to adjacent membranes. This model challenges the prevailing view that COPII vesicles are clustered by a scaffolding protein at the ER surface. Although Sec16 was proposed to serve as such a scaffolding protein, recent data suggest that rather than organizing COPII into higher-order structures, Sec16 acts at the level of individual COPII vesicles to regulate COPII turnover. A plausible synthesis is that tER sites are created by tethering to Golgi membranes and are regulated by Sec16. Meanwhile, the COPII vesicles that bud from tER sites are thought to nucleate new Golgi cisternae. Thus, an integrated self-organization process may generate tER-Golgi units. © 2014 WILEY Periodicals, Inc.

Effect of myristoylated N-terminus of Arf1 on the bending rigidity of phospholipid membranes

NASA Astrophysics Data System (ADS)

Burrola Gabilondo, Beatriz; Zhou, Hernan; Randazzo, Paul A.; Losert, Wolfgang

2010-03-01

The protein Arf1 is part of the COPI vesicle transport process from the Golgi to the ER. It binds to membranes via a myristoylated N-terminus and it has been shown to tubulate Large Unilamellar Vesicles. The effect of the N-terminus of Arf1 on physical properties of membranes has not been studied, with the exception of curvature. We previously found that the myristoylated N-terminus increases the packing of the lipid molecules, but has no effect on the lateral mobility. We tested the hypothesis that myristoylated peptides affect the bending rigidity of phospholipid Giant Unilamellar Vesicles (GUV). We use optical tweezers to pull tethers from GUV and measure the force of pulling the tether, as well as the retraction speed of the tether once it is released. We also used flicker spectroscopy to estimate the values of the mechanical properties of GUV. We will present results of the force and tether retraction measurements, as well as mechanical properties estimates from flicker, for GUV in the presence of varying concentrations of myristoylated and non-myristoylated N-terminus of Arf1, and compare these with measurements for GUV in the absence of peptide.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Passive Diffusion as a Mechanism Underlying Ribbon Synapse Vesicle Release and Resupply

PubMed Central

Graydon, Cole W.; Zhang, Jun; Oesch, Nicholas W.; Sousa, Alioscka A.; Leapman, Richard D.

2014-01-01

Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, “analog” sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-μm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon–vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates. PMID:24990916

Passive diffusion as a mechanism underlying ribbon synapse vesicle release and resupply.

PubMed

Graydon, Cole W; Zhang, Jun; Oesch, Nicholas W; Sousa, Alioscka A; Leapman, Richard D; Diamond, Jeffrey S

2014-07-02

Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, "analog" sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-μm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon-vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates. Copyright © 2014 the authors 0270-6474/14/348948-15$15.00/0.

Fluid-membrane tethers: minimal surfaces and elastic boundary layers.

PubMed

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

2002-04-01

Thin cylindrical tethers are common lipid bilayer membrane structures, arising in situations ranging from micromanipulation experiments on artificial vesicles to the dynamic structure of the Golgi apparatus. We study the shape and formation of a tether in terms of the classical soap-film problem, which is applied to the case of a membrane disk under tension subject to a point force. A tether forms from the elastic boundary layer near the point of application of the force, for sufficiently large displacement. Analytic results for various aspects of the membrane shape are given.

Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

PubMed Central

Martin, Sophie G.

2012-01-01

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk. PMID:22768263

Volume and porosity thermal regulation in lipid mesophases by coupling mobile ligands to soft membranes

NASA Astrophysics Data System (ADS)

Parolini, Lucia; Mognetti, Bortolo M.; Kotar, Jurij; Eiser, Erika; Cicuta, Pietro; di Michele, Lorenzo

2015-01-01

Short DNA linkers are increasingly being exploited for driving-specific self-assembly of Brownian objects. DNA-functionalized colloids can assemble into ordered or amorphous materials with tailored morphology. Recently, the same approach has been applied to compliant units, including emulsion droplets and lipid vesicles. The liquid structure of these substrates introduces new degrees of freedom: the tethers can diffuse and rearrange, radically changing the physics of the interactions. Unlike droplets, vesicles are extremely deformable and DNA-mediated adhesion causes significant shape adjustments. We investigate experimentally the thermal response of pairs and networks of DNA-tethered liposomes and observe two intriguing and possibly useful collective properties: negative thermal expansion and tuneable porosity of the liposome networks. A model providing a thorough understanding of this unexpected phenomenon is developed, explaining the emergent properties out of the interplay between the temperature-dependent deformability of the vesicles and the DNA-mediated adhesive forces.

Volume and porosity thermal regulation in lipid mesophases by coupling mobile ligands to soft membranes

PubMed Central

Parolini, Lucia; Mognetti, Bortolo M.; Kotar, Jurij; Eiser, Erika; Cicuta, Pietro; Di Michele, Lorenzo

2015-01-01

Short DNA linkers are increasingly being exploited for driving-specific self-assembly of Brownian objects. DNA-functionalized colloids can assemble into ordered or amorphous materials with tailored morphology. Recently, the same approach has been applied to compliant units, including emulsion droplets and lipid vesicles. The liquid structure of these substrates introduces new degrees of freedom: the tethers can diffuse and rearrange, radically changing the physics of the interactions. Unlike droplets, vesicles are extremely deformable and DNA-mediated adhesion causes significant shape adjustments. We investigate experimentally the thermal response of pairs and networks of DNA-tethered liposomes and observe two intriguing and possibly useful collective properties: negative thermal expansion and tuneable porosity of the liposome networks. A model providing a thorough understanding of this unexpected phenomenon is developed, explaining the emergent properties out of the interplay between the temperature-dependent deformability of the vesicles and the DNA-mediated adhesive forces. PMID:25565580

Arabidopsis COG Complex Subunits COG3 and COG8 Modulate Golgi Morphology, Vesicle Trafficking Homeostasis and Are Essential for Pollen Tube Growth

PubMed Central

Li, Yingxin; Li, Pengxiang; Gao, Caiji; Ding, Yu; Lan, Zhiyi; Shi, Zhixuan; Rui, Qingchen; Feng, Yihong; Liu, Yulong; Zhao, Yanxue; Wu, Chengyun; Zhang, Qian; Li, Yan; Jiang, Liwen

2016-01-01

Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth. PMID:27448097

Exocyst and autophagy-related membrane trafficking in plants.

PubMed

Pecenková, Tamara; Markovic, Vedrana; Sabol, Peter; Kulich, Ivan; Žárský, Viktor

2017-12-18

Endomembrane traffic in eukaryotic cells functions partially as a means of communication; delivery of membrane in one direction has to be balanced with a reduction at the other end. This effect is typically the case during the defence against pathogens. To combat pathogens, cellular growth and differentiation are suppressed, while endomembrane traffic is poised towards limiting the pathogen attack. The octameric exocyst vesicle-tethering complex was originally discovered as a factor facilitating vesicle-targeting and vesicle-plasma membrane (PM) fusion during exocytosis prior to and possibly during SNARE complex formation. Interestingly, it was recently implicated both in animals and plants in autophagy membrane traffic. In animal cells, the exocyst is integrated into the mTOR-regulated energy metabolism stress/starvation pathway, participating in the formation and especially initiation of an autophagosome. In plants, the first functional link was to autophagy-related anthocyanin import to the vacuole and to starvation. In this concise review, we summarize the current knowledge of exocyst functions in autophagy and defence in plants that might involve unconventional secretion and compare it with animal conditions. Formation of different exocyst complexes during undisturbed cell growth, as opposed to periods of cellular stress reactions involving autophagy, might contribute to the coordination of endomembrane trafficking pathways. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Synaptic ribbon. Conveyor belt or safety belt?

PubMed

Parsons, T D; Sterling, P

2003-02-06

The synaptic ribbon in neurons that release transmitter via graded potentials has been considered as a conveyor belt that actively moves vesicles toward their release sites. But evidence has accumulated to the contrary, and it now seems plausible that the ribbon serves instead as a safety belt to tether vesicles stably in mutual contact and thus facilitate multivesicular release by compound exocytosis.

Diffusion rate limitations in actin-based propulsion of hard and deformable particles.

PubMed

Dickinson, Richard B; Purich, Daniel L

2006-08-15

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.

Entry and Exit Mechanisms at the cis-Face of the Golgi Complex

PubMed Central

Lorente-Rodríguez, Andrés; Barlowe, Charles

2011-01-01

Vesicular transport of protein and lipid cargo from the endoplasmic reticulum (ER) to cis-Golgi compartments depends on coat protein complexes, Rab GTPases, tethering factors, and membrane fusion catalysts. ER-derived vesicles deliver cargo to an ER-Golgi intermediate compartment (ERGIC) that then fuses with and/or matures into cis-Golgi compartments. The forward transport pathway to cis-Golgi compartments is balanced by a retrograde directed pathway that recycles transport machinery back to the ER. How trafficking through the ERGIC and cis-Golgi is coordinated to maintain organelle structure and function is poorly understood and highlights central questions regarding trafficking routes and organization of the early secretory pathway. PMID:21482742

Sculpting and fusing biomimetic vesicle networks using optical tweezers.

PubMed

Bolognesi, Guido; Friddin, Mark S; Salehi-Reyhani, Ali; Barlow, Nathan E; Brooks, Nicholas J; Ces, Oscar; Elani, Yuval

2018-05-14

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

PubMed

Quon, Evan; Sere, Yves Y; Chauhan, Neha; Johansen, Jesper; Sullivan, David P; Dittman, Jeremy S; Rice, William J; Chan, Robin B; Di Paolo, Gilbert; Beh, Christopher T; Menon, Anant K

2018-05-01

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

The zebrafish mutant vps18 as a model for vesicle-traffic related hypopigmentation diseases.

PubMed

Maldonado, Ernesto; Hernandez, Fabiola; Lozano, Carlos; Castro, Marta E; Navarro, Rosa E

2006-08-01

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky-Pudlak, Chediak-Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18(hi2499A), which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18(hi2499A) the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18(hi2499A) larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18(hi2499A) can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.

Multisubunit tethering complexes in higher plants.

PubMed

Ravikumar, Raksha; Steiner, Alexander; Assaad, Farhah F

2017-12-01

Tethering complexes mediate the initial, specific contact between donor and acceptor membranes. This review focuses on the modularity and function of multisubunit tethering complexes (MTCs) in higher plants. One emphasis is on molecular interactions of plant MTCs. Here, a number of insights have been gained concerning interactions between different tethering complexes, and between tethers and microtubule-associated proteins. The roles of tethering complexes in abiotic stress responses appear indirect, but in the context of biotic stress responses it has been suggested that some tethers are direct targets of pathogen effectors or virulence factors. In light of the central roles tethering complexes play in plant development, an emerging concept is that tethers may be co-opted for plant adaptive responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. PMID:26041286

Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites.

PubMed

Yu, Haijia; Liu, Yinghui; Gulbranson, Daniel R; Paine, Alex; Rathore, Shailendra S; Shen, Jingshi

2016-04-19

Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation.

"An estimate of the probability of vesicle exocytosis in a Monte Carlo model of buffered diffusion of calcium channel currents"

NASA Astrophysics Data System (ADS)

Dimcovic, Z. M.; Eagan, T. P.; Kidane, T. K.; Brown, R. W.; Petschek, R. G.; McEnery, M. W.

2001-10-01

The opening of voltage-dependent calcium channels results in an influx of calcium ions promoting the fusion of synaptic vesicles. The fusion leads to release of neurotransmitters, which in turn allow the propagation of nerve impulses. A Monte Carlo model of the diffusion of calcium following its surge into the cell is used to estimate the probability for exocytosis. Besides the calcium absorption by fixed and mobile buffers, key ingredients are the physical size and position of the tethered vesicle and a sensing model for the interaction of the vesicle and calcium. The release probability is compared to previously published studies where the finite vesicle size was not considered. (Supported by NIH MH55747, AHA 96001250, NSF0086643, and a CWRU Presidential Research Initiative grant.)

ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.

PubMed

Manford, Andrew G; Stefan, Christopher J; Yuan, Helen L; Macgurn, Jason A; Emr, Scott D

2012-12-11

Endoplasmic reticulum-plasma membrane (ER-PM) junctions are conserved structures defined as regions of the ER that tightly associate with the plasma membrane. However, little is known about the mechanisms that tether these organelles together and why such connections are maintained. Using a quantitative proteomic approach, we identified three families of ER-PM tethering proteins in yeast: Ist2 (related to mammalian TMEM16 ion channels), the tricalbins (Tcb1/2/3, orthologs of the extended synaptotagmins), and Scs2 and Scs22 (vesicle-associated membrane protein-associated proteins). Loss of all six tethering proteins results in the separation of the ER from the PM and the accumulation of cytoplasmic ER. Importantly, we find that phosphoinositide signaling is misregulated at the PM, and the unfolded protein response is constitutively activated in the ER in cells lacking ER-PM tether proteins. These results reveal critical roles for ER-PM contacts in cell signaling, organelle morphology, and ER function. Copyright © 2012 Elsevier Inc. All rights reserved.

EXO70A1-mediated vesicle trafficking is critical for tracheary element development in Arabidopsis.

PubMed

Li, Shipeng; Chen, Min; Yu, Dali; Ren, Shichao; Sun, Shufeng; Liu, Linde; Ketelaar, Tijs; Emons, Anne-Mie C; Liu, Chun-Ming

2013-05-01

Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type- or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12.

PubMed

Brzezinski, Jonathon D; Modi, Apexa; Liu, Mengdan; Roth, Monica J

2016-12-15

Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60 PSPMA 65 ), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25 DLLTEDPPPY 34 , which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70 RREPP 74 ) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70 AAAAA 74 ) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p12 70-74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61 SPIASRLRGRR 71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25 DLLTEDPPPY 34 motif in the N terminus suppresses the ability to tether. This study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p12 61-71 Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

PubMed

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

A Monte Carlo Simulation of Vesicle Exocytosis in the Buffered Diffusion of Calcium Channel Currents

NASA Astrophysics Data System (ADS)

Dimcovic, Z.; Eagan, T. P.; Brown, R. W.; Petschek, R. G.; Eppell, S. J.; Yunker, A. M. R.; Sharp, A. H.; McEnery, M. W.

2001-04-01

The voltage-dependent opening of calcium channels results in an influx of calcium ions that leads to the fusion of synaptic vesicles with the cell membrane, resulting in the release of neurotransmitters. This allows nerve impulses to be transmitted from one neuron to another. A Monte Carlo model of the three-dimensional diffusion of calcium following a channel opening is employed to estimate the space and time dependence of the calcium density. The effects of fixed and mobile calcium buffers are included, and a tethered nearby vesicle is considered. The importance of the size and location of the vesicle is studied. When the vesicle is ignored, these results are compared with the analytical calculations of Naraghi and Neher and the Monte Carlo calculations of Bennett et al. The finite-vesicle-size analysis offers new insights into the process of neurosecretion. Support: NIH MH55747, AHA 96001250, NSF 0086643, and CWRU Presidential Research Initiative grants.

The Role of Ribbons at Sensory Synapses

PubMed Central

LoGiudice, Lisamarie; Matthews, Gary

2009-01-01

Synaptic ribbons are organelles that tether vesicles at the presynaptic active zones of sensory neurons in the visual, auditory and vestibular systems. These neurons generate sustained, graded electrical signals in response to sensory stimuli, and fidelity of transmission therefore requires their synapses to release neurotransmitter continuously at high rates. It has long been thought that the ribbons at the active zones of sensory synapses accomplish this task by enhancing the size and accessibility of the readily releasable pool of synaptic vesicles, which may represent the vesicles attached to the ribbon. Recent evidence suggests that synaptic ribbons immobilize vesicles in the resting cell and coordinate the transient, synchronous release of vesicles in response to stimulation, but it is not yet clear how the ribbon can efficiently mobilize and coordinate multiple vesicles for release. However, detailed anatomical, electrophysiological and optical studies have begun to reveal the mechanics of release at ribbon synapses, and this multidisciplinary approach promises to reconcile structure, function, and mechanism at these important sensory synapses. PMID:19264728

Eukaryotic membrane tethers revisited using magnetic tweezers.

PubMed

Hosu, Basarab G; Sun, Mingzhai; Marga, Françoise; Grandbois, Michel; Forgacs, Gabor

2007-04-19

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.

Eukaryotic membrane tethers revisited using magnetic tweezers

NASA Astrophysics Data System (ADS)

Hosu, Basarab G.; Sun, Mingzhai; Marga, Françoise; Grandbois, Michel; Forgacs, Gabor

2007-06-01

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.

Single Molecule Measurements of Interaction Free Energies Between the Proteins Within Binary and Ternary SNARE Complexes

PubMed Central

Liu, W.; Montana, Vedrana; Parpura, Vladimir; Mohideen, U.

2010-01-01

We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with target membranes. The dissociation rate of the binary syntaxin-synaptobrevin and the ternary syntaxin-SNAP25B-synaptobrevin complex was measured from the rupture force distribution as a function of the rate of applied force. The temperature dependence of the spontaneous dissociation rate was used to obtain the activation energy to the transition state of 19.8 ± 3.5 kcal/mol = 33 ± 6 kBT and 25.7 ± 3.0 kcal/mol = 43 ± 5 kBT for the binary and ternary complex, respectively. They are consistent with those measured previously for the ternary complex in lipid membranes and are of order expected for bilayer fusion and pore formation. The ΔG was 12.4–16.6 kcal/mol = 21–28 kBT and 13.8–18.0 kcal/mol = 23–30 kBT for the binary and ternary complex, respectively. The ternary complex was more stable by 1.4 kcal/mol = 2.3 kBT, consistent with the spontaneous dissociation rates. The higher adhesion energies and smaller molecular extensions measured with SNAP25B point to its possible unique and important physiological role in tethering/docking the vesicle in closer proximity to the plasma membrane and increasing the probability for fusion completion. PMID:20107522

CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode.

PubMed

Taneva, Svetla G; Patty, Philipus J; Frisken, Barbara J; Cornell, Rosemary B

2005-07-05

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.

ELKS active zone proteins as multitasking scaffolds for secretion

PubMed Central

Held, Richard G.

2018-01-01

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca2+ channels and specific lipids at the future site of fusion. This enables rapid and efficient neurotransmitter secretion during presynaptic depolarization by an action potential. Extensive research has revealed that this tethering is mediated by an active zone, a protein dense structure that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Although roles of individual active zone proteins in exocytosis are in part understood, the molecular mechanisms that hold the protein scaffold at the active zone together and link it to the presynaptic plasma membrane have remained unknown. This is largely due to redundancy within and across scaffolding protein families at the active zone. Recent studies, however, have uncovered that ELKS proteins, also called ERC, Rab6IP2 or CAST, act as active zone scaffolds redundant with RIMs. This redundancy has led to diverse synaptic phenotypes in studies of ELKS knockout mice, perhaps because different synapses rely to a variable extent on scaffolding redundancy. In this review, we first evaluate the need for presynaptic scaffolding, and we then discuss how the diverse synaptic and non-synaptic functional roles of ELKS support the hypothesis that ELKS provides molecular scaffolding for organizing vesicle traffic at the presynaptic active zone and in other cellular compartments. PMID:29491150

Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles

NASA Astrophysics Data System (ADS)

Yang, Boqian; He, Tao; Grauffel, Cédric; Reuter, Nathalie; Roberts, Mary; Gershenson, Anne

2013-03-01

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that Bacillus thuringiensis PI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. This work was supported by the National Institute of General Medical Science of the National Institutes of Health (R01GM060418).

Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells.

PubMed

Ji, Chen; Fan, Fan; Lou, Xuelin

2017-08-08

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) signaling is transient and spatially confined in live cells. How this pattern of signaling regulates transmitter release and hormone secretion has not been addressed. We devised an optogenetic approach to control PI(4,5)P 2 levels in time and space in insulin-secreting cells. Combining this approach with total internal reflection fluorescence microscopy, we examined individual vesicle-trafficking steps. Unlike long-term PI(4,5)P 2 perturbations, rapid and cell-wide PI(4,5)P 2 reduction in the plasma membrane (PM) strongly inhibits secretion and intracellular Ca 2+ concentration ([Ca 2+ ] i ) responses, but not sytaxin1a clustering. Interestingly, local PI(4,5)P 2 reduction selectively at vesicle docking sites causes remarkable vesicle undocking from the PM without affecting [Ca 2+ ] i . These results highlight a key role of local PI(4,5)P 2 in vesicle tethering and docking, coordinated with its role in priming and fusion. Thus, different spatiotemporal PI(4,5)P 2 signaling regulates distinct steps of vesicle trafficking, and vesicle docking may be a key target of local PI(4,5)P 2 signaling in vivo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Regulation of dendrite growth and maintenance by exocytosis

PubMed Central

Peng, Yun; Lee, Jiae; Rowland, Kimberly; Wen, Yuhui; Hua, Hope; Carlson, Nicole; Lavania, Shweta; Parrish, Jay Z.; Kim, Michael D.

2015-01-01

ABSTRACT Dendrites lengthen by several orders of magnitude during neuronal development, but how membrane is allocated in dendrites to facilitate this growth remains unclear. Here, we report that Ras opposite (Rop), the Drosophila ortholog of the key exocytosis regulator Munc18-1 (also known as STXBP1), is an essential factor mediating dendrite growth. Neurons with depleted Rop function exhibit reduced terminal dendrite outgrowth followed by primary dendrite degeneration, suggestive of differential requirements for exocytosis in the growth and maintenance of different dendritic compartments. Rop promotes dendrite growth together with the exocyst, an octameric protein complex involved in tethering vesicles to the plasma membrane, with Rop–exocyst complexes and exocytosis predominating in primary dendrites over terminal dendrites. By contrast, membrane-associated proteins readily diffuse from primary dendrites into terminals, but not in the reverse direction, suggesting that diffusion, rather than targeted exocytosis, supplies membranous material for terminal dendritic growth, revealing key differences in the distribution of materials to these expanding dendritic compartments. PMID:26483382

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

Competition between Bending and Internal Pressure Governs the Mechanics of Fluid Nanovesicles.

PubMed

Vorselen, Daan; MacKintosh, Fred C; Roos, Wouter H; Wuite, Gijs J L

2017-03-28

Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14k b T. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae. Conclusion These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae. PMID:17997821

Computational study of small molecule binding for both tethered and free conditions

PubMed Central

2010-01-01

Using a calix[4]arene-benzene complex as a test system we compare the potential of mean force for when the calix[4]arene is tethered versus free. When the complex is in vacuum our results show that the difference between tethered and free is primarily due to the entropic contribution to the potential of mean force resulting in a significant binding free energy difference of 6.6 kJ/mol. By contrast, when the complex is in water our results suggest that there is no appreciable difference between tethered and free. This study elucidates the roles of entropy and enthalpy for this small molecule system and emphasizes the point that tethering the receptor has the potential to dramatically impact the binding properties. These findings should be taken into consideration when using calixarene molecules in nanosensor design. PMID:20369865

α-Synuclein binds to the ER-mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production.

PubMed

Paillusson, Sébastien; Gomez-Suaga, Patricia; Stoica, Radu; Little, Daniel; Gissen, Paul; Devine, Michael J; Noble, Wendy; Hanger, Diane P; Miller, Christopher C J

2017-07-01

α-Synuclein is strongly linked to Parkinson's disease but the molecular targets for its toxicity are not fully clear. However, many neuronal functions damaged in Parkinson's disease are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling involves close physical associations between the two organelles that are mediated by binding of the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) to the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). VAPB and PTPIP51 thus act as a scaffold to tether the two organelles. Here we show that α-synuclein binds to VAPB and that overexpression of wild-type and familial Parkinson's disease mutant α-synuclein disrupt the VAPB-PTPIP51 tethers to loosen ER-mitochondria associations. This disruption to the VAPB-PTPIP51 tethers is also seen in neurons derived from induced pluripotent stem cells from familial Parkinson's disease patients harbouring pathogenic triplication of the α-synuclein gene. We also show that the α-synuclein induced loosening of ER-mitochondria contacts is accompanied by disruption to Ca 2+ exchange between the two organelles and mitochondrial ATP production. Such disruptions are likely to be particularly damaging to neurons that are heavily dependent on correct Ca 2+ signaling and ATP.

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner.

PubMed

Diao, Aipo; Frost, Laura; Morohashi, Yuichi; Lowe, Martin

2008-03-14

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

PubMed

Karim, Mahmoud Abdul; Samyn, Dieter Ronny; Mattie, Sevan; Brett, Christopher Leonard

2018-02-01

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A tethering complex drives the terminal stage of SNARE-dependent membrane fusion

NASA Astrophysics Data System (ADS)

D'Agostino, Massimo; Risselada, Herre Jelger; Lürick, Anna; Ungermann, Christian; Mayer, Andreas

2017-11-01

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore. This last step is highly energy-demanding. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.

The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

PubMed

Ho, Ruoya; Stroupe, Christopher

2016-10-01

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

NASA Astrophysics Data System (ADS)

Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

2016-04-01

Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner. Electronic supplementary information (ESI) available: Two videos of the experiment are shown in Fig. 5, demonstrating the distinctive characteristics of the peptide pair in a mixed population of cells are available in online. Video S1 shows the experiment in the bright field channel including the green channel (calcein-AM stained unfunctionalized cells) and orange channel (rhodamine labeled liposomes). Video S2 shows the exact same frames but combining the fluorescent channels only, including the blue channel for Hoechst nuclear staining. Both videos consist of 31 frames at a frame rate of 5 fps. The labeled liposomes are injected after frame 1. The videos span a total timeframe of 15 minutes. See DOI: 10.1039/c6nr00711b

P-Tether-Mediated, Iterative SN2'-Cuprate Alkylation Strategy to Skipped Polyol Stereotetrads: Utility of an Oxidative "Function Switch" with Phosphite-Borane Tethers.

PubMed

Markley, Jana L; Hanson, Paul R

2017-05-19

The development of a P-tether-mediated, iterative S N 2'-cuprate alkylation protocol for the formation of 1,3-skipped polyol stereotetrads is reported. This two-directional synthetic strategy builds molecular complexity from simple, readily prepared C 2 -symmetric dienediols and unites the chemistry of both temporary phosphite-borane tethers and temporary phosphate tethers-through an oxidative "function switch" of the P-tether itself-to generate intermediates that were previously inaccessible via either method alone.

Dysregulated Arl1, a regulator of post-Golgi vesicle tethering, can inhibit endosomal transport and cell proliferation in yeast

PubMed Central

Benjamin, Jeremy J. R.; Poon, Pak P.; Drysdale, John D.; Wang, Xiangmin; Singer, Richard A.; Johnston, Gerald C.

2011-01-01

Small monomeric G proteins regulated in part by GTPase-activating proteins (GAPs) are molecular switches for several aspects of vesicular transport. The yeast Gcs1 protein is a dual-specificity GAP for ADP-ribosylation factor (Arf) and Arf-like (Arl)1 G proteins, and also has GAP-independent activities. The absence of Gcs1 imposes cold sensitivity for growth and endosomal transport; here we present evidence that dysregulated Arl1 may cause these impairments. We show that gene deletions affecting the Arl1 or Ypt6 vesicle-tethering pathways prevent Arl1 activation and membrane localization, and restore growth and trafficking in the absence of Gcs1. A mutant version of Gcs1 deficient for both ArfGAP and Arl1GAP activity in vitro still allows growth and endosomal transport, suggesting that the function of Gcs1 that is required for these processes is independent of GAP activity. We propose that, in the absence of this GAP-independent regulation by Gcs1, the resulting dysregulated Arl1 prevents growth and impairs endosomal transport at low temperatures. In cells with dysregulated Arl1, an increased abundance of the Arl1 effector Imh1 restores growth and trafficking, and does so through Arl1 binding. Protein sequestration at the trans-Golgi membrane by dysregulated, active Arl1 may therefore be the mechanism of inhibition. PMID:21562219

Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu

2011-07-11

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RCmore » within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.« less

Endocytic pathway rapidly delivers internalized molecules to lysosomes: an analysis of vesicle trafficking, clustering and mass transfer.

PubMed

Pangarkar, Chinmay; Dinh, Anh-Tuan; Mitragotri, Samir

2012-08-20

Lysosomes play a critical role in intracellular drug delivery. For enzyme-based therapies, they represent a potential target site whereas for nucleic acid or many protein drugs, they represent the potential degradation site. Either way, understanding the mechanisms and processes involved in routing of materials to lysosomes after cellular entry is of high interest to the field of drug delivery. Most therapeutic cargoes other than small hydrophobic molecules enter the cells through endocytosis. Endocytosed cargoes are routed to lysosomes via microtubule-based transport and are ultimately shared by various lysosomes via tethering and clustering of endocytic vesicles followed by exchange of their contents. Using a combined experimental and numerical approach, here we studied the rates of mass transfer into and among the endocytic vesicles in a model cell line, 3T3 fibroblasts. In order to understand the relationship of mass transfer with microtubular transport and vesicle clustering, we varied both properties through various pharmacological agents. At the same time, microtubular transport and vesicle clustering were modeled through diffusion-advection equations and the Smoluchowski equations, respectively. Our analysis revealed that the rate of mass transfer is optimally related to microtubular transport and clustering properties of vesicles. Further, the rate of mass transfer is highest in the innate state of the cell. Any perturbation to either microtubular transport or vesicle aggregation led to reduced mass transfer to lysosome. These results suggest that in the absence of an external intervention the endocytic pathway appears to maximize molecular delivery to lysosomes. Strategies are discussed to reduce mass transfer to lysosomes so as to extend the residence time of molecules in endosomes or late endosomes, thus potentially increasing the likelihood of their escape before disposition in the lysosomes. Copyright © 2012 Elsevier B.V. All rights reserved.

In situ formation of magnetopolymersomes via electroporation for MRI

NASA Astrophysics Data System (ADS)

Bain, Jennifer; Ruiz-Pérez, Lorena; Kennerley, Aneurin J.; Muench, Stephen P.; Thompson, Rebecca; Battaglia, Giuseppe; Staniland, Sarah S.

2015-09-01

As the development of diagnostic/therapeutic (and combined: theranostic) nanomedicine grows, smart drug-delivery vehicles become ever more critical. Currently therapies consist of drugs tethered to, or encapsulated within nanoparticles or vesicles. There is growing interest in functionalising them with magnetic nanoparticles (MNPs) to target the therapeutics by localising them using magnetic fields. An alternating magnetic field induces remote heating of the particles (hyperthermia) triggering drug release or cell death. Furthermore, MNPs are diagnostic MRI contrast agents. There is considerable interest in MNP embedded vehicles for nanomedicine, but their development is hindered by difficulties producing consistently monodisperse MNPs and their reliable loading into vesicles. Furthermore, it is highly advantageous to "trigger" MNP production and to tune the MNP's size and magnetic response. Here we present the first example of a tuneable, switchable magnetic delivery vehicle for nanomedical application. These are comprised of robust, tailored polymer vesicles (polymersomes) embedded with superparamagnetic magnetite MNPs (magnetopolymersomes) which show good MRI contrast (R2* = 148.8 s-1) and have a vacant core for loading of therapeutics. Critically, the magnetopolymersomes are produced by a pioneering nanoreactor method whereby electroporation triggers the in situ formation of MNPs within the vesicle membrane, offering a switchable, tuneable magnetic responsive theranostic delivery vehicle.

Janus dendrimersomes coassembled from fluorinated, hydrogenated, and hybrid Janus dendrimers as models for cell fusion and fission.

PubMed

Xiao, Qi; Sherman, Samuel E; Wilner, Samantha E; Zhou, Xuhao; Dazen, Cody; Baumgart, Tobias; Reed, Ellen H; Hammer, Daniel A; Shinoda, Wataru; Klein, Michael L; Percec, Virgil

2017-08-22

A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated-fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

PubMed

Mukai, Chinatsu; Gao, Lizeng; Nelson, Jacquelyn L; Lata, James P; Cohen, Roy; Wu, Lauren; Hinchman, Meleana M; Bergkvist, Magnus; Sherwood, Robert W; Zhang, Sheng; Travis, Alexander J

2017-01-02

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomimicry promotes the efficiency of a 10-step sequential enzymatic reaction on nanoparticles, converting glucose to lactate

PubMed Central

Mukai, Chinatsu; Gao, Lizeng; Nelson, Jacquelyn L.; Lata, James P.; Cohen, Roy; Wu, Lauren; Hinchman, Meleana M.; Bergkvist, Magnus; Sherwood, Robert W.; Zhang, Sheng; Travis, Alexander J.

2016-01-01

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. PMID:27901298

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

PubMed Central

Zheng, Xiangdong; Gooi, Li Ming; Wason, Arpit; Gabriel, Elke; Mehrjardi, Narges Zare; Yang, Qian; Zhang, Xingrun; Debec, Alain; Basiri, Marcus L.; Avidor-Reiss, Tomer; Pozniakovsky, Andrei; Poser, Ina; Šarić, Tomo; Hyman, Anthony A.; Li, Haitao; Gopalakrishnan, Jay

2014-01-01

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication. PMID:24385583

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

PubMed

Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson's Disease

PubMed Central

Bridi, Jessika C.; Hirth, Frank

2018-01-01

Parkinson's disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be the most toxic species, with their accumulation at presynaptic terminals affecting several steps of neurotransmitter release. First, high levels of α-Syn alter the size of synaptic vesicle pools and impair their trafficking. Second, α-Syn overexpression can either misregulate or redistribute proteins of the presynaptic SNARE complex. This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, accompanied by a decrease in AZ protein levels. Furthermore, α-Syn overexpression reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. These presynaptic alterations mediated by accumulation of α-Syn, together impair neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are the most vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes responsible for its synthesis. In addition, α-Syn is able to interact with and reduce the activity of VMAT2 and DAT. The resulting dysregulation of dopamine levels directly contributes to the formation of toxic α-Syn oligomers. Together these data suggest a vicious cycle of accumulating α-Syn and deregulated dopamine that triggers synaptic dysfunction and impaired neuronal communication, ultimately causing synaptopathy and progressive neurodegeneration in Parkinson's disease. PMID:29515354

Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria.

PubMed

Onfelt, Björn; Nedvetzki, Shlomo; Benninger, Richard K P; Purbhoo, Marco A; Sowinski, Stefanie; Hume, Alistair N; Seabra, Miguel C; Neil, Mark A A; French, Paul M W; Davis, Daniel M

2006-12-15

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast*

PubMed Central

Muñoz, Sofía; Manjón, Elvira; Sánchez, Yolanda

2014-01-01

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3+ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation. PMID:24947517

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

PubMed

de Curtis, Ivan; Meldolesi, Jacopo

2012-10-01

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

Atmospheric verification mission for the TSS/STARFAC tethered satellite

NASA Technical Reports Server (NTRS)

Wood, George M., Jr.; Stuart, Thomas D.; Crouch, Donald S.; Deloach, Richard; Brown, Kenneth G.

1991-01-01

Two types of a tethered satellite system (TSS) - a basic 1.8-m-diameter spherical spacecraft and the Shuttle Tethered Aerothermodynamic Research Facility (STARFAC) are considered. Issues related to the deployment and retrieval of a large satellite with exceedingly long tethers are discussed, and the objectives of an Atmospheric Verification Mission (ATM) are outlined. Focus is concentrated on the ATM satellite which will fly after TSS-1 and before the fully instrumented and costlier TSS-2. The differences between the AVM and TSS-2, including the configuration of the aerodynamic stabilizers, instrumentation, and the materials of construction are outlined. The basic Kevlar tether defined for the TSS-2 is being considered for use with the AVM, however, a complex tether is under consideration as well.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

PubMed

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-06-02

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.

Liprin-α3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses.

PubMed

Wong, Man Yan; Liu, Changliang; Wang, Shan Shan H; Roquas, Aram C F; Fowler, Stephen C; Kaeser, Pascal S

2018-02-27

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3-KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3-KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.

Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation.

PubMed

Klip, Amira; Sun, Yi; Chiu, Tim Ting; Foley, Kevin P

2014-05-15

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane. Copyright © 2014 the American Physiological Society.

Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy

NASA Technical Reports Server (NTRS)

Blumberg, Seth; Gajraj, Arivalagan; Pennington, Matthew W.; Meiners, Jens-Christian

2005-01-01

Tethered particle microscopy is a powerful tool to study the dynamics of DNA molecules and DNA-protein complexes in single-molecule experiments. We demonstrate that stroboscopic total internal reflection microscopy can be used to characterize the three-dimensional spatiotemporal motion of DNA-tethered particles. By calculating characteristic measures such as symmetry and time constants of the motion, well-formed tethers can be distinguished from defective ones for which the motion is dominated by aberrant surface effects. This improves the reliability of measurements on tether dynamics. For instance, in observations of protein-mediated DNA looping, loop formation is distinguished from adsorption and other nonspecific events.

Rab from the white shrimp Litopenaeus vannamei: characterization and its regulation upon environmental stress.

PubMed

Wang, Lei; Wang, Xiao-Rong; Liu, Jin; Chen, Chu-Xian; Liu, Yuan; Wang, Wei-Na

2015-10-01

With the destruction of the ecological environment, shrimp cultivation in China has been seriously affected by outbreaks of infectious diseases. Rab, which belong to small GTPase Ras superfamily, can regulate multiple steps in eukaryotic vesicle trafficking including vesicle budding, vesicle tethering, and membrane fusion. Knowledge of Rab in shrimp is essential to understanding regulation and detoxification mechanisms of environmental stress. In this study, we analyzed the functions of Rab from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNA of Rab was obtained, which was 751 bp long, with open reading frame encoding 206 amino acids. In this study, for the first time, the gene expression of Rab of L. vannamei was analyzed by quantitative real-time PCR after exposure to five kinds of environmental stresses (bacteria, pH, Cd, salinity and low temperature). The results demonstrate that Rab is sensitive and involved in bacteria, pH, and Cd stress responses and Rab is more sensitive to bacteria than other stresses. Therefore we infer that Rab may have relationship with the anti-stress mechanism induced by environment stress in shrimp and Rab could be used as critical biomarkers for environmental quality assessment.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER.

PubMed

Costello, Joseph L; Castro, Inês G; Hacker, Christian; Schrader, Tina A; Metz, Jeremy; Zeuschner, Dagmar; Azadi, Afsoon S; Godinho, Luis F; Costina, Victor; Findeisen, Peter; Manner, Andreas; Islinger, Markus; Schrader, Michael

2017-02-01

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering. © 2017 Costello et al.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

PubMed Central

Costello, Joseph L.; Hacker, Christian; Schrader, Tina A.; Zeuschner, Dagmar; Findeisen, Peter

2017-01-01

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering. PMID:28108524

EARP, a multisubunit tethering complex involved in endocytic recycling

PubMed Central

Schindler, Christina; Chen, Yu; Pu, Jing; Guo, Xiaoli; Bonifacino, Juan S.

2015-01-01

Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named Endosome-Associated Recycling Protein (EARP) that is structurally related to the previously described Golgi-Associated Retrograde Protein (GARP) complex. Both complexes share the Ang2, Vps52 and Vps53 subunits, but EARP comprises an uncharacterized protein, Syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target-SNARE Syntaxin 6 and various cognate SNAREs. Depletion of Syndetin or Syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling. PMID:25799061

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

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

PubMed Central

Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

2008-01-01

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila

PubMed Central

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-01-01

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.14226.001 PMID:27253064

Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain.

PubMed

Ghosh, M; Wang, Y; Ebert, C E; Vadlamuri, S; Beattie, D S

2001-01-16

Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity [Obungu et al. (2000) Biochim. Biophys. Acta 1457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.

Forces, energetics, and dynamics of conjugated-carbon ring tethers adhered to CNTs: a computational investigation.

PubMed

Filla, Nicholas; Ramasamy, Ramaraja; Wang, Xianqiao

2018-04-25

The strength and nature of the interactions between carbon nanotubes (CNTs) and molecular tethers plays a vital role in technology such as CNT-enzyme sensors. Tethers that attach noncovalently to CNTs are ideal for retaining the electrical properties of the CNTs since they do not degrade the CNT surface and effect its electrical conductivity. However, leaching due to weak CNT-tether attachment is very common when using noncovalent tethers, and this has limited their use in commercial products including biosensors. Thus, understanding the fundamental mechanics governing the strength of CNT-tether adhesion is crucial for the design of highly sensitive, viable sensors. Here, we computationally investigate the adhesion strength of CNT-tether complexes with 8 different tethering molecules designed to adhere noncovalently to the CNT surface. We study the effects of CNT diameter, CNT chirality, and the size/geometry of the tethering molecule on the adhesion energy and force. Our results show an asymptotic relationship between adhesion strength and CNT diameter. Calculations show that noncovalent tethers tested here can reach adhesion forces and energies that are up to 21% and 54% of the strength of the carbon-carbon single bond force and bond energy respectively. We anticipate our results will help guide CNT-enzyme sensor design to produce sensors with high sensitivity and minimal leaching.

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

PubMed Central

Turnbull, Andrew P; Kümmel, Daniel; Prinz, Bianka; Holz, Caterina; Schultchen, Jeffrey; Lang, Christine; Niesen, Frank H; Hofmann, Klaus-Peter; Delbrück, Heinrich; Behlke, Joachim; Müller, Eva-Christina; Jarosch, Ernst; Sommer, Thomas; Heinemann, Udo

2005-01-01

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-Å resolution. BET3 adopts an α/β-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein–protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions. PMID:15692564

A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis

PubMed Central

Li, Hongmei; Alavian, Kambiz N.; Lazrove, Emma; Mehta, Nabil; Jones, Adrienne; Zhang, Ping; Licznerski, Pawel; Graham, Morven; Uo, Takuma; Guo, Junhua; Rahner, Christoph; Duman, Ronald S.; Morrison, Richard S.; Jonas, Elizabeth A.

2013-01-01

Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, the latter of which is dependent on mitochondrial ATP. The Bcl-2 family protein Bcl-xL, in addition to its role in cell death, regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, however, that, Bcl-xL directly regulates endocytotic vesicle retrieval in hippocampal neurons through protein/protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex of proteins with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytotic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity. PMID:23792689

When intracellular logistics fails--genetic defects in membrane trafficking.

PubMed

Olkkonen, Vesa M; Ikonen, Elina

2006-12-15

The number of human genetic disorders shown to be due to defects in membrane trafficking has greatly increased during the past five years. Defects have been identified in components involved in sorting of cargo into transport carriers, vesicle budding and scission, movement of vesicles along cytoskeletal tracks, as well as in vesicle tethering, docking and fusion at the target membrane. The nervous system is extremely sensitive to such disturbances of the membrane trafficking machinery, and the majority of these disorders display neurological defects--particularly diseases affecting the motility of transport carriers along cytoskeletal tracks. In several disorders, defects in a component that represents a fundamental part of the trafficking machinery fail to cause global transport defects but result in symptoms limited to specific cell types and transport events; this apparently reflects the redundancy of the transport apparatus. In groups of closely related diseases such as Hermansky-Pudlak and Griscelli syndromes, identification of the underlying gene defects has revealed groups of genes in which mutations lead to similar phenotypic consequences. New functionally linked trafficking components and regulatory mechanisms have thus been discovered. Studies of the gene defects in trafficking disorders therefore not only open avenues for new therapeutic approaches but also significantly contribute to our knowledge of the fundamental mechanisms of intracellular membrane transport.

Effects of predation on diel activity and habitat use of the coral-reef shrimp Cinetorhynchus hendersoni (Rhynchocinetidae)

NASA Astrophysics Data System (ADS)

Ory, Nicolas C.; Dudgeon, David; Duprey, Nicolas; Thiel, Martin

2014-09-01

Nonlethal effects of predators on prey behaviour are still poorly understood, although they may have cascading effects through food webs. Underwater observations and experiments were conducted on a shallow fringing coral reef in Malaysia to examine whether predation risks affect diel activity, habitat use, and survival of the rhynchocinetid shrimp Cinetorhynchus hendersoni. The study site was within a protected area where predatory fish were abundant. Visual surveys and tethering experiments were conducted in April-May 2010 to compare the abundance of shrimps and predatory fishes and the relative predation intensity on shrimps during day and night. Shrimps were not seen during the day but came out of refuges at night, when the risk of being eaten was reduced. Shrimp preferences for substrata of different complexities and types were examined at night when they could be seen on the reef; complex substrata were preferred, while simple substrata were avoided. Shrimps were abundant on high-complexity columnar-foliate Porites rus, but tended to make little use of branching Acropora spp. Subsequent tethering experiments, conducted during daytime in June 2013, compared the relative mortality of shrimps on simple (sand-rubble, massive Porites spp.) and complex ( P. rus, branching Acropora spp.) substrata under different predation risk scenarios (i.e., different tether lengths and exposure durations). The mortality of shrimps with short tethers (high risk) was high on all substrata while, under low and intermediate predation risks (long tethers), shrimp mortality was reduced on complex corals relative to that on sand-rubble or massive Porites spp. Overall, mortality was lowest on P. rus. Our study indicates that predation risks constrain shrimp activity and habitat choice, forcing them to hide deep inside complex substrata during the day. Such behavioural responses to predation risks and their consequences for the trophic role of invertebrate mesoconsumers warrant further investigation, especially in areas where predatory fishes have been overexploited.

The effects of tether placement on antibody stability on surfaces

NASA Astrophysics Data System (ADS)

Grawe, Rebecca W.; Knotts, Thomas A.

2017-06-01

Despite their potential benefits, antibody microarrays have fallen short of performing reliably and have not found widespread use outside of the research setting. Experimental techniques have been unable to determine what is occurring on the surface of an atomic level, so molecular simulation has emerged as the primary method of investigating protein/surface interactions. Simulations of small proteins have indicated that the stability of the protein is a function of the residue on the protein where a tether is placed. The purpose of this research is to see whether these findings also apply to antibodies, with their greater size and complexity. To determine this, 24 tethering locations were selected on the antibody Protein Data Bank (PDB) ID: 1IGT. Replica exchange simulations were run on two different surfaces, one hydrophobic and one hydrophilic, to determine the degree to which these tethering sites stabilize or destabilize the antibody. Results showed that antibodies tethered to hydrophobic surfaces were in general less stable than antibodies tethered to hydrophilic surfaces. Moreover, the stability of the antibody was a function of the tether location on hydrophobic surfaces but not hydrophilic surfaces.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity*

PubMed Central

van der Kant, Rik; Jonker, Caspar T. H.; Wijdeven, Ruud H.; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

2015-01-01

Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway. PMID:26463206

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bello, Oscar D.; Zanetti, M. Natalia; Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM

2012-03-10

Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggestedmore » as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black-Right-Pointing-Pointer RIM, Munc13 and Rab3A interplay in human sperm acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Rab3A have critical roles in membrane docking.« less

Sequential Reactions of Surface-Tethered Glycolytic Enzymes

PubMed Central

Mukai, Chinatsu; Bergkvist, Magnus; Nelson, Jacquelyn L.; Travis, Alexander J.

2014-01-01

SUMMARY The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices. PMID:19778729

Vesicle dynamics in a confined Poiseuille flow: from steady state to chaos.

PubMed

Aouane, Othmane; Thiébaud, Marine; Benyoussef, Abdelilah; Wagner, Christian; Misbah, Chaouqi

2014-09-01

Red blood cells (RBCs) are the major component of blood, and the flow of blood is dictated by that of RBCs. We employ vesicles, which consist of closed bilayer membranes enclosing a fluid, as a model system to study the behavior of RBCs under a confined Poiseuille flow. We extensively explore two main parameters: (i) the degree of confinement of vesicles within the channel and (ii) the flow strength. Rich and complex dynamics for vesicles are revealed, ranging from steady-state shapes (in the form of parachute and slipper shapes) to chaotic dynamics of shape. Chaos occurs through a cascade of multiple periodic oscillations of the vesicle shape. We summarize our results in a phase diagram in the parameter plane (degree of confinement and flow strength). This finding highlights the level of complexity of a flowing vesicle in the small Reynolds number where the flow is laminar in the absence of vesicles and can be rendered turbulent due to elasticity of vesicles.

Nuclear pore complex tethers to the cytoskeleton.

PubMed

Goldberg, Martin W

2017-08-01

The nuclear envelope is tethered to the cytoskeleton. The best known attachments of all elements of the cytoskeleton are via the so-called LINC complex. However, the nuclear pore complexes, which mediate the transport of soluble and membrane bound molecules, are also linked to the microtubule network, primarily via motor proteins (dynein and kinesins) which are linked, most importantly, to the cytoplasmic filament protein of the nuclear pore complex, Nup358, by the adaptor BicD2. The evidence for such linkages and possible roles in nuclear migration, cell cycle control, nuclear transport and cell architecture are discussed. Copyright © 2017. Published by Elsevier Ltd.

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

PubMed Central

Li, Cunxi; Hao, Mingming; Cao, Zheng; Ding, Wei; Graves-Deal, Ramona; Hu, Jianyong; Piston, David W.

2007-01-01

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells. PMID:17553928

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

PubMed Central

Helm, Jared R.; Bentley, Marvin; Thorsen, Kevin D.; Wang, Ting; Foltz, Lauren; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C.

2014-01-01

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery. PMID:25006245

Optical absorption, electron spin resonance, and electron spin echo studies of the photoionization of tetramethylbenzidine in cationic and anionic synthetic vesicles: comparison with analogous micellar systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, A.S.W.; Kevan, L.

1983-09-07

The photoionization of N,N,N',N'-tetramethylbenzidine (TMB) in dihexadecylphosphate anionic vesicles and in dioctadecyldimethylammonium chloride cationic vesicles has been studied by optical absorption and electron spin resonance in liquid and frozen solutions. The TMB cation has been observed to be stabilized in both types of vesicles. The photoionization efficiency is about twofold greater in the cationic vesicles compared to the anionic vesicles. Shifts in the optical absorption maximum between micellar and vesicle solutions indicate that TMB is in a less polar environment in the vesicle systems. Electron spin echo modulation spectrometry has been used to detect TMB cation-water interactions that are foundmore » to be weaker than in previously studied micellar solutions. This is consistent with the optical absorption results and with an asymmetric solubilization site for TMB and TMB/sup +/ within the vesicular structure. A new absorption in the photoionized vesicles is assigned to a nonparamagnetic diamine-diimine charge-transfer complex between two TMB cations in the same vesicle. This complex is not formed in micellar systems. 5 figures.« less

Diamidophosphines with six-membered chelates and their coordination chemistry with group 4 metals: development of a trimethylene-methane-tethered [PN2]-type "molecular claw".

PubMed

Batke, S; Kothe, T; Haas, M; Wadepohl, H; Ballmann, J

2016-02-28

The coordination chemistry of the phosphine-tethered diamidophosphine ligands PhP(CH2CH2CH2NHPh)2 (pr[NPN]H2) and PhP(1,2-CH2-C6H4-NHSiMe3)2 (bn[NPN]H2) featuring six-membered N–C3–P chelates was explored with group 4 metals, which allowed for the consecutive development of a new trimethylene-methane-tethered [PN2] scaffold. In the case of the propylene-linked system pr[NPN]H2, access to the sparingly soluble dibenzyl derivative pr[NPN]ZrBn2 (3-Zr) was gained, while thermally sensitive zirconium and hafnium diiodo complexes bn[NPN]MI2 (5-M, M = Zr, Hf) were isolated in the case of the benzylene-linked derivative bn[NPN]H2. Despite the related phosphine-tethered backbone architectures of both of these ligands, their group 4 complexes were found to exhibit either C1-symmetric (bn[NPN]MX2) or averaged CS-symmetric (pr[NPN]MX2) structures in solution. To restrain the overall flexibility of these systems and thereby control the properties of the resulting complexes without disrupting the six-membered chelates, the new trimethylene-methane-tethered N,N′-di-(tert-butyl)-substituted [PN2]H2 protioligand was designed. This tripodal ligand system was prepared on a gram scale and its CS-symmetric dichloro complexes [PN2]MCl2 (6-M, M = Ti, Zr, Hf) were isolated subsequently. The benzene-soluble dibenzyl derivative [PN2]ZrBn2 (7-Zr) was synthesised as well and characterised by X-ray diffraction. These results are discussed not only in conjunction with the known [NPN]-coordinated group 4 complexes incorporating five-membered chelates, but also in the context of “molecular claws” that are related to the new [PN2] tripod.

pH-responsive supramolecular vesicles based on water-soluble pillar[6]arene and ferrocene derivative for drug delivery.

PubMed

Duan, Qunpeng; Cao, Yu; Li, Yan; Hu, Xiaoyu; Xiao, Tangxin; Lin, Chen; Pan, Yi; Wang, Leyong

2013-07-17

The drug delivery system based on supramolecular vesicles that were self-assembled by a novel host-guest inclusion complex between a water-soluble pillar[6]arene (WP6) and hydrophobic ferrocene derivative in water has been developed. The inclusion complexation between WP6 and ferrocene derivative in water was studied by (1)H NMR, UV-vis, and fluorescence spectroscopy, which showed a high binding constant of (1.27 ± 0.42) × 10(5) M(-1) with 1:1 binding stoichiometry. This resulting inclusion complex could self-assemble into supramolecular vesicles that displayed a significant pH-responsive behavior in aqueous solution, which were investigated by fluorescent probe technique, dynamic laser scattering, and transmission electron microscopy. Furthermore, the drug loading and in vitro drug release studies demonstrated that these supramolecular vesicles were able to encapsulate mitoxantrone (MTZ) to achieve MTZ-loaded vesicles, which particularly showed rapid MTZ release at low-pH environment. More importantly, the cellular uptake of these pH-responsive MTZ-loaded vesicles by cancer cells was observed by living cell imaging techniques, and their cytotoxicity assay indicated that unloaded vesicles had low toxicity to normal cells, which could dramatically reduce the toxicity of MTZ upon loading of MTZ. Meanwhile, MTZ-loaded vesicles exhibited comparable anticancer activity in vitro as free MTZ to cancer cells under examined conditions. This study suggests that such supramolecular vesicles have great potential as controlled drug delivery systems.

The motion and control of a complex three-body space tethered system

NASA Astrophysics Data System (ADS)

Shi, Gefei; Zhu, Zhanxia; Chen, Shiyu; Yuan, Jianping; Tang, Biwei

2017-11-01

This paper is mainly devoted to investigating the dynamics and stability control of a three body-tethered satellite system which contains a main satellite and two subsatellites connected by two straight, massless and inextensible tethers. Firstly, a detailed mathematical model is established in the central gravitational field. Then, the dynamic characteristics of the established system are investigated and analyzed. Based on the dynamic analysis, a novel sliding mode prediction model (SMPM) control strategy is proposed to suppress the motion of the built tethered system. The numerical results show that the proposed underactuated control law is highly effective in suppressing the attitude/libration motion of the underactuated three-body tethered system. Furthermore, cases of different target angles are also examined and analyzed. The simulation results reveal that even if the final equilibrium states differ from different selections of the target angles, the whole system can still be maintained in acceptable areas.

Axel Robotic Platform for Crater and Extreme Terrain Exploration

NASA Technical Reports Server (NTRS)

Nesnas, Issa A.; Matthews, Jaret B.; Edlund, Jeffrey A.; Burdick, Joel W.; Abad-Manterola, Pablo

2012-01-01

To be able to conduct science investigations on highly sloped and challenging terrains, it is necessary to deploy science payloads to such locations and collect and process in situ samples. A tethered robotic platform has been developed that is capable of exploring very challenging terrain. The Axel rover is a symmetrical rover that is minimally actuated, can traverse arbitrary paths, and operate upside-down or right-side up. It can be deployed from a larger platform (rover, lander, or aerobot) or from a dual Axel configuration. Axel carries and manages its own tether, reducing damage to the tether during operations. Fundamentally, Axel is a two-wheeled rover with a symmetric body and a trailing link. Because the primary goal is minimal complexity, this version of the Axel rover uses only four primary actuators to control its wheels, tether, and a trailing link. A fifth actuator is used for level winding of tether onto Axel s spool.

Elastic issues and vibration reduction in a tethered deorbiting mission

NASA Astrophysics Data System (ADS)

Sabatini, Marco; Gasbarri, Paolo; Palmerini, Giovanni B.

2016-05-01

Recently proposed mission concepts involving harpoons or nets to capture and de-orbit debris represent an interesting application of the tethered systems, where the orbiting bodies are connected by a flexible link. These systems present a complex behavior, as flexible characteristics combine with orbital dynamics. The focus of the paper is on the dynamic behavior of the tethered system in the final phase of the de-orbiting mission, when a powerful apogee motor is used to change the debris orbit. The thrust action introduces significant issues, as elastic waves propagate along the tether, and the relevant oscillations couple with the orbital dynamics. Input shaping techniques are proposed to limit or cancel these oscillations. However, the performance of these techniques drops when non-ideal scenarios are considered. In particular, an initially slack tether is a serious issue that must be solved if acceptably low oscillations of the tether are to be obtained. Three strategies are proposed and discussed in this paper to remove the slack condition: a natural drift of the chaser by means of a single impulse, a controlled maneuver for precisely adjusting the relative distance between chaser spacecraft and debris, and a retrieval mechanism for changing the tether length.

Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

PubMed

Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

2010-02-16

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

BIOCHEMICAL CHARACTERIZATION OF NATIVE USHER PROTEIN COMPLEXES FROM A VESICULAR SUBFRACTION OF TRACHEAL EPITHELIAL CELLS†

PubMed Central

Zallocchi, Marisa; Sisson, Joseph H.; Cosgrove, Dominic

2010-01-01

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells, and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in non-detergent buffer, and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15 and VLGR-1, and a different one at the top of the gradient that included all the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100–200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins co-sediment into the gradient at a sedimentation coefficient of approximately 50S, correlating with a predicted molecular mass of 2 × 106 Daltons. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher proteins complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors. PMID:20058854

Tethered orbital propellant depot

NASA Technical Reports Server (NTRS)

Fester, D. A.; Rudolph, L. K.; Kiefel, E. R.

1985-01-01

A planned function of the Space Station is to refurbish and refuel an advanced space-based LO2/LH2 orbit transfer vehicle. An alternative to propellant storage at the station is to use a remote facility tied to the station with a log tether. Preliminary design of such a facility is described with emphasis on fluid transfer and storage requirements. Using tether lengths of at least 300 ft, gravity gradient forces will dominate surface tension in such a system. Although gravity given transfer is difficult because of line pressure drops, fluid settling over the tank outlet greatly alleviates acquisition concerns and will facilitate vented tank fills. The major concern with a tethered orbital refueling facility is its considerable operational complexity including transport of the OTV to and from the facility.

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

PubMed Central

2012-01-01

Background Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. Results Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. Conclusions Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types. PMID:22873208

Command Generation and Control of Momentum Exchange Electrodynamic Reboost Tethered Satellite

NASA Technical Reports Server (NTRS)

Robertson, Michael J.

2005-01-01

The research completed for this NASA Graduate Student Research Program Fellowship sought to enhance the current state-of-the-art dynamic models and control laws for Momentum Exchange Electrodynamic Reboost satellite systems by utilizing command generation, specifically Input Shaping. The precise control of tethered spacecraft with flexible appendages is extremely difficult. The complexity is magnified many times when the satellite must interact with other satellites as in a momentum exchange via a tether. The Momentum Exchange Electronic Reboost Tether (MXER) concept encapsulates all of these challenging tasks [l]. Input Shaping is a command generation technique that allows flexible spacecraft to move without inducing residual vibration [2], limit transient deflection [3] and utilize fuel-efficient actuation [4]. Input shaping is implemented by convolving a sequence of impulses, known as the input shaper, with a desired system command to produce a shaped input that is then used to drive the system. This process is demonstrated in Figure 1. The shaped command is then use to drive the system without residual vibration while meeting many other performance specifications. The completed work developed tether control algorithms for retrieval. A simple model of the tether response has been developed and command shaping was implemented to minimize unwanted dynamics. A model of a flexible electrodynamic tether has been developed to investigate the tether s response during reboost. Command shaping techniques have been developed to eliminate the tether oscillations and reduce the tether s deflection to pre-specified levels during reboost. Additionally, a model for the spin-up of a tethered system was developed. This model was used in determining the parameters for optimization the resulting angular velocity.

Replication of Simulated Prebiotic Amphiphilic Vesicles in a Finite Environment Exhibits Complex Behavior That Includes High Progeny Variability and Competition

PubMed Central

Armstrong, Don L.; Lancet, Doron

2018-01-01

Abstract We studied the simulated replication and growth of prebiotic vesicles composed of 140 phospholipids and cholesterol using our R-GARD (Real Graded Autocatalysis Replication Domain) formalism that utilizes currently extant lipids that have known rate constants of lipid-vesicle interactions from published experimental data. R-GARD normally modifies kinetic parameters of lipid-vesicle interactions based on vesicle composition and properties. Our original R-GARD model tracked the growth and division of one vesicle at a time in an environment with unlimited lipids at a constant concentration. We explore here a modified model where vesicles compete for a finite supply of lipids. We observed that vesicles exhibit complex behavior including initial fast unrestricted growth, followed by intervesicle competition for diminishing resources, then a second growth burst driven by better-adapted vesicles, and ending with a final steady state. Furthermore, in simulations without kinetic parameter modifications (“invariant kinetics”), the initial replication was an order of magnitude slower, and vesicles' composition variability at the final steady state was much lower. The complex kinetic behavior was not observed either in the previously published R-GARD simulations or in additional simulations presented here with only one lipid component. This demonstrates that both a finite environment (inducing selection) and multiple components (providing variation for selection to act upon) are crucial for portraying evolution-like behavior. Such properties can improve survival in a changing environment by increasing the ability of early protocellular entities to respond to rapid environmental fluctuations likely present during abiogenesis both on Earth and possibly on other planets. This in silico simulation predicts that a relatively simple in vitro chemical system containing only lipid molecules might exhibit properties that are relevant to prebiotic processes. Key Words: Phospholipid vesicles—Prebiotic compartments—Prebiotic vesicle competition—Prebiotic vesicle variability. Astrobiology 18, 419–430. PMID:29634319

Self-Assembly of Semiconducting-Plasmonic Gold Nanoparticles with Enhanced Optical Property for Photoacoustic Imaging and Photothermal Therapy

PubMed Central

Yang, Zhen; Song, Jibin; Dai, Yunlu; Chen, Jingyi; Wang, Feng; Lin, Lisen; Liu, Yijing; Zhang, Fuwu; Yu, Guocan; Zhou, Zijian; Fan, Wenpei; Huang, Wei; Fan, Quli; Chen, Xiaoyuan

2017-01-01

Although various noble metal and semiconducting molecules have been developed as photoacoustic (PA) agents, the use of semiconducting polymer-metal nanoparticle hybrid materials to enhance PA signal has not been explored. A novel semiconducting-plasmonic nanovesicle was fabricated by self-assembly of semiconducting poly(perylene diimide) (PPDI) and poly(ethylene glycol (PEG) tethered gold nanoparticles (Au@PPDI/PEG). A highly localized and strongly enhanced electromagnetic (EM) field is distributed between adjacent gold nanoparticles in the vesicular shell, where the absorbing collapsed PPDI is present. Significantly, the EM field in turn enhances the light absorption efficiency of PPDI, leading to a much greater photothermal effect and a stronger photoacoustic signal compared to PDI nanoparticle or gold nanovesicle alone. The optical property of the hybrid vesicle can be further tailored by controlling the ratio of PPDI and gold nanoparticle as well as the adjustable interparticle distance of gold nanoparticles localized in the vesicular shell. In vivo imaging and therapeutic evaluation demonstrated that the hybrid vesicle is an excellent probe for cancer theranostics. PMID:28740543

Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells

PubMed Central

Gálvez-Santisteban, Manuel; Rodriguez-Fraticelli, Alejo E.; Bryant, David M.; Vergarajauregui, Silvia; Yasuda, Takao; Bañón-Rodríguez, Inmaculada; Bernascone, Ilenia; Datta, Anirban; Spivak, Natalie; Young, Kitty; Slim, Christiaan L.; Brakeman, Paul R.; Fukuda, Mitsunori; Mostov, Keith E.; Martín-Belmonte, Fernando

2012-01-01

SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell. PMID:22820376

Self-Assembly of Semiconducting-Plasmonic Gold Nanoparticles with Enhanced Optical Property for Photoacoustic Imaging and Photothermal Therapy.

PubMed

Yang, Zhen; Song, Jibin; Dai, Yunlu; Chen, Jingyi; Wang, Feng; Lin, Lisen; Liu, Yijing; Zhang, Fuwu; Yu, Guocan; Zhou, Zijian; Fan, Wenpei; Huang, Wei; Fan, Quli; Chen, Xiaoyuan

2017-01-01

Although various noble metal and semiconducting molecules have been developed as photoacoustic (PA) agents, the use of semiconducting polymer-metal nanoparticle hybrid materials to enhance PA signal has not been explored. A novel semiconducting-plasmonic nanovesicle was fabricated by self-assembly of semiconducting poly(perylene diimide) (PPDI) and poly(ethylene glycol (PEG) tethered gold nanoparticles (Au@PPDI/PEG). A highly localized and strongly enhanced electromagnetic (EM) field is distributed between adjacent gold nanoparticles in the vesicular shell, where the absorbing collapsed PPDI is present. Significantly, the EM field in turn enhances the light absorption efficiency of PPDI, leading to a much greater photothermal effect and a stronger photoacoustic signal compared to PDI nanoparticle or gold nanovesicle alone. The optical property of the hybrid vesicle can be further tailored by controlling the ratio of PPDI and gold nanoparticle as well as the adjustable interparticle distance of gold nanoparticles localized in the vesicular shell. In vivo imaging and therapeutic evaluation demonstrated that the hybrid vesicle is an excellent probe for cancer theranostics.

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

PubMed Central

Bendezú, Felipe O.; Martin, Sophie G.

2011-01-01

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types. PMID:21148300

A Space Station tethered orbital refueling facility

NASA Technical Reports Server (NTRS)

Fester, D. A.; Rudolph, L. K.; Kiefel, E. R.

1985-01-01

A planned function of the Space Station is to refurbish and refuel an advanced space-based LO2/LH2 orbit transfer vehicle. An alternative to propellant storage at the station is to use a remote facility tied to the station with a long tether. Preliminary design of such a facility is described with emphasis on fluid transfer and storage requirements. Using tether lengths of at least 300 ft, gravity gradient forces will dominate surface tension in such a system. Although gravity driven transfer is difficult because of line pressure drops, fluid settling over the tank outlet greatly alleviates acquisition concerns and will facilitate vented tank fills. The major concern with a tethered orbital refueling facility is its considerable operational complexity including transport of the OTV to and from the facility.

On the dynamical stability of the space 'monorail'

NASA Astrophysics Data System (ADS)

Bergamaschi, S.; Manni, D.

The dynamical stability of 'monorail' tethered-satellite/elevator configurations being studied for the Space Station is investigated analytically, treating the end platforms and elevator as point masses, neglecting tether elasticity, and taking the Coriolis force and the complex gravitational field into account in analyzing the orbital-plane motion of the system. A mathematical model is constructed; the equations of motion are derived; and results obtained by numerical integration for platform masses 100,000 and 10,000 kg, elevator mass 5000 kg, and a 10-km-long 6-mm-diameter 4070-kg-mass tether are presented in graphs and briefly characterized.

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

PubMed Central

Cirnaru, Maria D.; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

2014-01-01

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

Promyelocytic leukemia bodies tether to early endosomes during mitosis.

PubMed

Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

2014-01-01

During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis.

PubMed

Khodthong, Chuenchanok; Kabachinski, Greg; James, Declan J; Martin, Thomas F J

2011-08-03

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

Dissecting the telomere-inner nuclear membrane interface formed in meiosis.

PubMed

Pendlebury, Devon F; Fujiwara, Yasuhiro; Tesmer, Valerie M; Smith, Eric M; Shibuya, Hiroki; Watanabe, Yoshinori; Nandakumar, Jayakrishnan

2017-12-01

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

PubMed Central

Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

2013-01-01

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru; Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru; St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time ofmore » slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.« less

The mechanism of a nuclear pore assembly: a molecular biophysics view.

PubMed

Kuvichkin, Vasily V

2011-06-01

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA-PC liposomes-Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) < 100 nm in diameter, a "big" liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The "big" membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.

Lst1p and Sec24p Cooperate in Sorting of the Plasma Membrane Atpase into Copii Vesicles in Saccharomyces cerevisiae

PubMed Central

Shimoni, Yuval; Kurihara, Tatsuo; Ravazzola, Mariella; Amherdt, Mylène; Orci, Lelio; Schekman, Randy

2000-01-01

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of α-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles. PMID:11086000

Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

PubMed

Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

2016-02-23

Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering*

PubMed Central

Heinrich, Frank; Nanda, Hirsh; Goh, Haw Zan; Bachert, Collin; Lösche, Mathias; Linstedt, Adam D.

2014-01-01

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. PMID:24505136

Selective Metal-Ion-Mediated Vesicle Adhesion Based on Dynamic Self-Organization of a Pyrene-Appended Glutamic Acid.

PubMed

Xing, Pengyao; Wang, Yajie; Yang, Minmin; Zhang, Yimeng; Wang, Bo; Hao, Aiyou

2016-07-13

Vesicles with dynamic membranes provide an ideal model system for investigating biological membrane activities, whereby vesicle aggregation behaviors including adhesion, fusion, fission, and membrane contraction/extension have attracted much attention. In this work we utilize an aromatic amino acid (pyrene-appended glutamic acid, PGlu) to prepare nanovesicles that aggregate to form vesicle clusters selectively induced by Fe(3+) or Cu(2+), and the vesicles transform into irregular nano-objects when interacting with Al(3+). Vesicle clusters have better stability than pristine vesicles, which hinders the spontaneous morphological transformation from vesicles into lamellar nanosheets with long incubation period. The difference between complexation of Fe(3+) and Al(3+) with vesicles was studied by various techniques. On the basis of metal ion-vesicle interactions, this self-assembled nanovesicle system also behaves as an effective fluorescent sensor for Fe(3+) and Al(3+), which cause fluorescence quenching and enhanced excimer emission, respectively.

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Yeongseon; Choi, Won Tae; Heller, William T.

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE PAGES

Jang, Yeongseon; Choi, Won Tae; Heller, William T.; ...

2017-07-27

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

COPII-coated membranes function as transport carriers of intracellular procollagen I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorur, Amita; Yuan, Lin; Kenny, Samuel J.

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completelymore » encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.« less

The effect of spontaneous curvature on a two-phase vesicle

PubMed Central

Cox, Geoffrey; Lowengrub, John

2015-01-01

Vesicles are membrane-bound structures commonly known for their roles in cellular transport and the shape of a vesicle is determined by its surrounding membrane (lipid bilayer). When the membrane is composed of different lipids, it is natural for the lipids of similar molecular structure to migrate towards one another (via spinodal decomposition), creating a multi-phase vesicle. In this article, we consider a two-phase vesicle model which is driven by nature’s propensity to maintain a minimal state of elastic energy. The model assumes a continuum limit, thereby treating the membrane as a closed three-dimensional surface. The main purpose of this study is to reveal the complexity of the Helfrich two-phase vesicle model with non-zero spontaneous curvature and provide further evidence to support the relevance of spontaneous curvature as a modelling parameter. In this paper, we illustrate the complexity of the Helfrich two-phase model by providing multiple examples of undocumented solutions and energy hysteresis. We also investigate the influence of spontaneous curvature on morphological effects and membrane phenomena such as budding and fusion. PMID:26097287

COPII-coated membranes function as transport carriers of intracellular procollagen I

DOE PAGES

Gorur, Amita; Yuan, Lin; Kenny, Samuel J.; ...

2017-04-20

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completelymore » encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.« less

Low-resolution simulations of vesicle suspensions in 2D

NASA Astrophysics Data System (ADS)

Kabacaoğlu, Gökberk; Quaife, Bryan; Biros, George

2018-03-01

Vesicle suspensions appear in many biological and industrial applications. These suspensions are characterized by rich and complex dynamics of vesicles due to their interaction with the bulk fluid, and their large deformations and nonlinear elastic properties. Many existing state-of-the-art numerical schemes can resolve such complex vesicle flows. However, even when using provably optimal algorithms, these simulations can be computationally expensive, especially for suspensions with a large number of vesicles. These high computational costs can limit the use of simulations for parameter exploration, optimization, or uncertainty quantification. One way to reduce the cost is to use low-resolution discretizations in space and time. However, it is well-known that simply reducing the resolution results in vesicle collisions, numerical instabilities, and often in erroneous results. In this paper, we investigate the effect of a number of algorithmic empirical fixes (which are commonly used by many groups) in an attempt to make low-resolution simulations more stable and more predictive. Based on our empirical studies for a number of flow configurations, we propose a scheme that attempts to integrate these fixes in a systematic way. This low-resolution scheme is an extension of our previous work [51,53]. Our low-resolution correction algorithms (LRCA) include anti-aliasing and membrane reparametrization for avoiding spurious oscillations in vesicles' membranes, adaptive time stepping and a repulsion force for handling vesicle collisions and, correction of vesicles' area and arc-length for maintaining physical vesicle shapes. We perform a systematic error analysis by comparing the low-resolution simulations of dilute and dense suspensions with their high-fidelity, fully resolved, counterparts. We observe that the LRCA enables both efficient and statistically accurate low-resolution simulations of vesicle suspensions, while it can be 10× to 100× faster.

Assessing the Potential of Societal Verification by Means of New Media

DTIC Science & Technology

2014-01-01

the Defense Advanced Research Projects Agency (DARPA) “Red Balloon Challenge” in 2009 by finding 10 tethered weather balloons scattered across the...Institute of Technology (MIT) managed to locate 10 weather balloons tethered at undisclosed locations across the continental United States in less than...suited for complex problem solving, and the 2009 Defense Advanced Research Projects Agency’s (DARPA) “Red Balloon Challenge” has already demonstrated

Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

PubMed

Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

2014-10-01

To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

Dissecting the telomere–inner nuclear membrane interface formed in meiosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pendlebury, Devon F.; Fujiwara, Yasuhiro; Tesmer, Valerie M.

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere–INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1–TERB1 interface to reveal the structural basis for telomere–INM linkage. Disruption of this interface abrogates binding and compromises telomere–INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1–TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, ourmore » biochemical analysis implicates distinct complexes for telomere–INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere–INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.« less

Tiam–Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion

PubMed Central

2016-01-01

Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB+ cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB+ cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion. PMID:27597758

Formation Flying of Tethered and Nontethered Spacecraft

NASA Technical Reports Server (NTRS)

Quadrelli, Marco B.

2005-01-01

A paper discusses the effect of the dynamic interaction taking place within a formation composed of a rigid and a deformable vehicle, and presents the concept of two or more tethered spacecraft flying in formation with one or more separated free-flying spacecraft. Although progress toward formation flight of nontethered spacecraft has already been achieved, the document cites potential advantages of tethering, including less consumption of fuel to maintain formation, very high dynamic stability of a rotating tethered formation, and intrinsically passive gravity-gradient stabilization. The document presents a theoretical analysis of the dynamics of a system comprising one free-flying spacecraft and two tethered spacecraft in orbit, as a prototype of more complex systems. The spacecraft are modeled as rigid bodies and the tether as a mass-less spring with structural viscous damping. Included in the analysis is a study of the feasibility of a centralized control system for maintaining a required formation in low Earth orbit. A numerical simulation of a retargeting maneuver is reported to show that even if the additional internal dynamics of the system caused by flexibility is considered, high pointing precision can be achieved if a fictitious rigid frame is used to track the tethered system, and it should be possible to position the spacecraft with centimeter accuracy and to orient the formation within arc seconds of the desired direction also in the presence of low Earth orbit environmental perturbations. The results of the study demonstrate that the concept is feasible in Earth orbit and point the way to further study of these hybrid tethered and free-flying systems for related applications in orbit around other Solar System bodies.

Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation.

PubMed

Smith, Benjamin A; Padrick, Shae B; Doolittle, Lynda K; Daugherty-Clarke, Karen; Corrêa, Ivan R; Xu, Ming-Qun; Goode, Bruce L; Rosen, Michael K; Gelles, Jeff

2013-09-03

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it. DOI:http://dx.doi.org/10.7554/eLife.01008.001.

Rigid biimidazole ancillary ligands as an avenue to bright deep blue cationic iridium(iii) complexes.

PubMed

Henwood, Adam F; Evariste, Sloane; Slawin, Alexandra M Z; Zysman-Colman, Eli

2014-01-01

Herein we report the synthesis and optoelectronic characterisation of three deep blue-emitting cationic iridium complexes, of the form [Ir(dFppy)(2)(N^N)]PF(6), bearing biimidazole-type N^N ancillary ligands (dFppyH = 2-(2,4-difluorophenyl)pyridine). Complex 1 contains the parent biimidazole, biim, while 2 contains a dimethylated analog, dMebiim, and 3 contains an ortho-xylyl-tethered biimidzole, o-xylbiim. We explore a strategy of tethering the biimidazole in order to rigidify the complex and increase the photoluminescent quantum yield, culminating in deep blue (λ(max): 457 nm in MeOH at 298 K) ligand-centered emission with a very high photoluminescent quantum yield of 68% and microsecond emission lifetime. Density functional theory calculations elucidate the origin of such disparate excited state kinetics across this series, especially in light of virtually identical optoelectronic properties observed for these compounds.

Overall energy conversion efficiency of a photosynthetic vesicle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

Overall energy conversion efficiency of a photosynthetic vesicle

PubMed Central

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; Hunter, C Neil; Schulten, Klaus

2016-01-01

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12–0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination. DOI: http://dx.doi.org/10.7554/eLife.09541.001 PMID:27564854

Overall energy conversion efficiency of a photosynthetic vesicle

DOE PAGES

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; ...

2016-08-26

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

PubMed Central

Groves, J T; Wülfing, C; Boxer, S G

1996-01-01

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8913608

Ultrastructure of spermatogenesis in the sea star, Asterina minor.

PubMed

Yamagata, A

1988-02-01

The ultrastructural features of spermatogenesis were investigated in the hermaphroditic sea star Asterina minor. The primordial germ cells in the genital rachis contain small clusters of electron-dense material (nuage material) and a stack of annulate lamellae. They also have a flagellum and basal body complex situated close to the Golgi complex. After the development of the genital rachis into the ovotestis, spermatogenic cells increase in number and differentiation begins. Nuage material is observed in spermatogonia, but it gradually disappears in spermatocytes. The annulate lamellae do not exist beyond the early spermatogonial stage. By contrast, a flagellum and basal body complex are found throughout spermatogenesis. The Golgi-derived proacrosomal vesicles appear in the spermatocyte and coalesce to form an acrosomal vesicle in the early spermatid. The process of acrosome formation is as follows: (1) a lamella of endoplasmic reticulum (ER) continuous with the outer nuclear membrane encloses the posterior portion of the acrosomal vesicle; (2) the vesicle attaches to the cell membrane with its anterior portion; (3) periacrosomal material accumulates in the space between the acrosomal vesicle and the ER; (4) the nucleus proper changes its features to surround the acrosome; (5) amorphous, electron-dense material is deposited under the electron-dense disk; and (6) the nucleus forms a hollow opposite the electron-dense material.

SNARE interactions in membrane trafficking: a perspective from mammalian central synapses.

PubMed

Kavalali, Ege T

2002-10-01

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are a large family of proteins that are present on all organelles involved in intracellular vesicle trafficking and secretion. The interaction of complementary SNAREs found on opposing membranes presents an attractive lock-and-key mechanism, which may underlie the specificity of vesicle trafficking. Moreover, formation of the tight complex between a vesicle membrane SNARE and corresponding target membrane SNAREs could drive membrane fusion. In synapses, this tight complex, also referred to as the synaptic core complex, is essential for neurotransmitter release. However, recent observations in knockout mice lacking major synaptic SNAREs challenge the prevailing notion on the executive role of these proteins in fusion and open up several questions about their exact role(s) in neurotransmitter release. Persistence of a form of regulated neurotransmitter release in these mutant mice also raises the possibility that other cognate or non-cognate SNAREs may partially compensate for the loss of a particular SNARE. Future analysis of SNARE function in central synapses will also have implications for the role of these molecules in other vesicle trafficking events such as endocytosis and vesicle replenishment. Such analysis can provide a molecular basis for synaptic processes including certain forms of short-term synaptic plasticity. Copyright 2002 Wiley Periodicals, Inc.

Multiple Hydrogen Bond Tethers for Grazing Formic Acid in Its Complexes with Phenylacetylene.

PubMed

Karir, Ginny; Kumar, Gaurav; Kar, Bishnu Prasad; Viswanathan, K S

2018-03-01

Complexes of phenylacetylene (PhAc) and formic acid (FA) present an interesting picture, where the two submolecules are tethered, sometimes multiply, by hydrogen bonds. The multiple tentacles adopted by PhAc-FA complexes stem from the fact that both submolecules can, in the same complex, serve as proton acceptors and/or proton donors. The acetylenic and phenyl π systems of PhAc can serve as proton acceptors, while the ≡C-H or -C-H of the phenyl ring can act as a proton donor. Likewise, FA also is amphiprotic. Hence, more than 10 hydrogen-bonded structures, involving O-H···π, C-H···π, and C-H···O contacts, were indicated by our computations, some with multiple tentacles. Interestingly, despite the multiple contacts in the complexes, the barrier between some of the structures is small, and hence, FA grazes around PhAc, even while being tethered to it, with hydrogen bonds. We used matrix isolation infrared spectroscopy to experimentally study the PhAc-FA complexes, with which we located global and a few local minima, involving primarily an O-H···π interaction. Experiments were corroborated by ab initio computations, which were performed using MP2 and M06-2X methods, with 6-311++G (d,p) and aug-cc-pVDZ basis sets. Single-point energy calculations were also done at MP2/CBS and CCSD(T)/CBS levels. The nature, strength, and origin of these noncovalent interactions were studied using AIM, NBO, and LMO-EDA analysis.

Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses

PubMed Central

Sinha, Raunak; Ahmed, Saheeb; Jahn, Reinhard; Klingauf, Jurgen

2011-01-01

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission. PMID:21844343

TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes

PubMed Central

Santos, António JM; Foresti, Ombretta; Zhang, Chong; Garcia-Parajo, Maria F; Campelo, Felix

2018-01-01

Collagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats, and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER. PMID:29513218

AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

PubMed Central

Glyvuk, Nataliya; Tsytsyura, Yaroslav; Geumann, Constanze; D'Hooge, Rudi; Hüve, Jana; Kratzke, Manuel; Baltes, Jennifer; Böning, Daniel; Klingauf, Jürgen; Schu, Peter

2010-01-01

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation. PMID:20203623

Fabrication of Propeller-Shaped Supra-amphiphile for Construction of Enzyme-Responsive Fluorescent Vesicles.

PubMed

Li, Jie; Liu, Kaerdun; Han, Yuchun; Tang, Ben Zhong; Huang, Jianbin; Yan, Yun

2016-10-04

Propeller-shaped molecules have been recognized to display fantastic AIE (aggregation induced emission), but they can hardly self-assemble into nanostructures. Herein, we for the first time report that ionic complexation between a water-soluble tetrapheneyl derivative and an enzyme substrate in aqueous media produces a propeller-shaped supra-amphiphile that self-assembles into enzyme responsive fluorescent vesicles. The supra-amphiphile was fabricated upon complexation between a water-soluble propeller-shaped AIE luminogen TPE-BPA and myristoylcholine chloride (MChCl) in aqueous media. MChCl filled in the intramolecular voids of propeller-shaped TPE-BPA upon supra-amphiphile formation, which endows the supra-amphiphile superior self-assembling ability to the component molecules thus leading to the formation of fluorescent vesicles. Because MChCl is the substrate of cholinesterases, the vesicles dissemble in the presence of cholinesterases, and the fluorescent intensity can be correlated to the level of enzymes. The resulting fluorescent vesicles may be used to recognize the site of Alzheimer's disease, to encapsulate the enzyme inhibitor, and to release the inhibitor at the disease site.

Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

PubMed Central

Zhang, Xinming; Rebane, Aleksander A.; Ma, Lu; Li, Feng; Jiao, Junyi; Qu, Hong; Pincet, Frederic; Rothman, James E.

2016-01-01

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission. PMID:27911771

From artificial red blood cells, oxygen carriers, and oxygen therapeutics to artificial cells, nanomedicine, and beyond

PubMed Central

Chang, Thomas M. S.

2013-01-01

The first experimental artificial red blood cells have all three major functions of red blood cells (rbc). However, the first practical one is a simple polyhemoglobin (PolyHb) that only has an oxygen-carrying function. This is now in routine clinical use in South Africa and Russia. An oxygen carrier with antioxidant functions, PolyHb-catalase-superoxide dismutase, can fulfill two of the three functions of rbc. Even more complete is one with all three functions of rbc in the form of PolyHb-catalase-superoxide dismutase-carbonic anhydrase. The most advanced ones are nanodimension artificial rbc with either PEG-lipid membrane or PEG-PLA polymermembrane. Extensions in to oxygen therapeutics include a PolyHb-tyrosinase that suppresses the growth of melanoma in a mice model. Another is a PolyHb-fibrinogen that is an oxygen carrier with platelet-like function. Research has now extended well beyond the original research on artificial rbc into many areas of artificial cells. These include nanoparticles, nanotubules, lipid vesicles, liposomes, polymer-tethered lipid vesicles, polymersomes, microcapsules, bioencapsulation, nanocapules, macroencapsulation, synthetic cells, and others. These are being used in nanotechnology, nanomedicine, regenerative medicine, enzyme/gene therapy, cell/stem cell therapy, biotechnology, drug delivery, hemoperfusion, nanosensers, and even by some groups in agriculture, industry, aquatic culture, nanocomputers, and nanorobotics. PMID:22409281

Loss of the homotypic fusion and vacuole protein sorting or golgi-associated retrograde protein vesicle tethering complexes results in gentamicin sensitivity in the yeast Saccharomyces cerevisiae.

PubMed

Wagner, Mark C; Molnar, Elizabeth E; Molitoris, Bruce A; Goebl, Mark G

2006-02-01

Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity.

Loss of the Homotypic Fusion and Vacuole Protein Sorting or Golgi-Associated Retrograde Protein Vesicle Tethering Complexes Results in Gentamicin Sensitivity in the Yeast Saccharomyces cerevisiae†

PubMed Central

Wagner, Mark C.; Molnar, Elizabeth E.; Molitoris, Bruce A.; Goebl, Mark G.

2006-01-01

Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity. PMID:16436714

Synaptic Vesicle-Recycling Machinery Components as Potential Therapeutic Targets

PubMed Central

Li, Ying C.

2017-01-01

Presynaptic nerve terminals are highly specialized vesicle-trafficking machines. Neurotransmitter release from these terminals is sustained by constant local recycling of synaptic vesicles independent from the neuronal cell body. This independence places significant constraints on maintenance of synaptic protein complexes and scaffolds. Key events during the synaptic vesicle cycle—such as exocytosis and endocytosis—require formation and disassembly of protein complexes. This extremely dynamic environment poses unique challenges for proteostasis at synaptic terminals. Therefore, it is not surprising that subtle alterations in synaptic vesicle cycle-associated proteins directly or indirectly contribute to pathophysiology seen in several neurologic and psychiatric diseases. In contrast to the increasing number of examples in which presynaptic dysfunction causes neurologic symptoms or cognitive deficits associated with multiple brain disorders, synaptic vesicle-recycling machinery remains an underexplored drug target. In addition, irrespective of the involvement of presynaptic function in the disease process, presynaptic machinery may also prove to be a viable therapeutic target because subtle alterations in the neurotransmitter release may counter disease mechanisms, correct, or compensate for synaptic communication deficits without the need to interfere with postsynaptic receptor signaling. In this article, we will overview critical properties of presynaptic release machinery to help elucidate novel presynaptic avenues for the development of therapeutic strategies against neurologic and neuropsychiatric disorders. PMID:28265000

Jupiter, Tether, and Lenz's Law

NASA Technical Reports Server (NTRS)

Lee, Russell

1999-01-01

Jupiter has a large, complex, and intense magnetic field that is thought to arise from electrical currents in the rapidly spinning metallic hydrogen interior. The strong magnetic field can induce currents when the conductive tether is directed toward or away from Jupiter. The currents can be stored and used for both propulsion and power generation. Therefore, our spacecraft might be able to visit several Jovian moons or maintain in the orbit around Jupiter. In our future space traveling, we also can use this technical skill to travel to other planets without any fuel. First-year physics textbooks describe Lenz's Law in which current is induced in a conductor moving through a stationary magnetic field. A demonstration of induced current in a stationary conductor and moving magnetic field is described, which may have space-tether application.

Self-assembled tethered bimolecular lipid membranes.

PubMed

Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

2009-01-01

This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

Dynamics of coarsening in multicomponent lipid vesicles with non-uniform mechanical properties

NASA Astrophysics Data System (ADS)

Funkhouser, Chloe M.; Solis, Francisco J.; Thornton, K.

2014-04-01

Multicomponent lipid vesicles are commonly used as a model system for the complex plasma membrane. One phenomenon that is studied using such model systems is phase separation. Vesicles composed of simple lipid mixtures can phase-separate into liquid-ordered and liquid-disordered phases, and since these phases can have different mechanical properties, this separation can lead to changes in the shape of the vesicle. In this work, we investigate the dynamics of phase separation in multicomponent lipid vesicles, using a model that couples composition to mechanical properties such as bending rigidity and spontaneous curvature. The model allows the vesicle surface to deform while conserving surface area and composition. For vesicles initialized as spheres, we study the effects of phase fraction and spontaneous curvature. We additionally initialize two systems with elongated, spheroidal shapes. Dynamic behavior is contrasted in systems where only one phase has a spontaneous curvature similar to the overall vesicle surface curvature and systems where the spontaneous curvatures of both phases are similar to the overall curvature. The bending energy contribution is typically found to slow the dynamics by stabilizing configurations with multiple domains. Such multiple-domain configurations are found more often in vesicles with spheroidal shapes than in nearly spherical vesicles.

Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

PubMed Central

Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

2014-01-01

Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

PubMed

Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

2014-09-01

Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. Published by Elsevier Inc.

Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation

PubMed Central

Smith, Benjamin A; Padrick, Shae B; Doolittle, Lynda K; Daugherty-Clarke, Karen; Corrêa, Ivan R; Xu, Ming-Qun; Goode, Bruce L; Rosen, Michael K; Gelles, Jeff

2013-01-01

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it. DOI: http://dx.doi.org/10.7554/eLife.01008.001 PMID:24015360

Technology Advances Enabling a New Class of Hybrid Underwater Vehicles

NASA Astrophysics Data System (ADS)

Bowen, A.

2016-02-01

Both tethered (ROV) and untethered (AUV) systems have proven to be highly valuable tools for a range of application undersea. Certain enabling technologies coupled with recent advances in robotic systems make it possible to consider supplementing many of the functions performed by these platforms with appropriately designed semi-autonomous vehicles that may be less expensive operate than traditional deep-water ROVs. Such vehicles can be deployed from smaller ships and may lead to sea-floor resident systems able to perform a range of interventions under direct human control when required. These systems are effectively a hybrid cross between ROV and AUV vehicles and poised to enable an important new class of undersea vehicle. It is now possible to radically redefine the meaning of the words "tethered vehicle" to include virtual tethering via acoustic and optical means or through the use of small diameter re-useable tethers, providing not power but only high bandwidth communications. The recent developments at Woods Hole Oceanographic Institution (WHOI), paves the way for a derivative vehicle type able to perform a range of interventions in deep water. Such battery-powered, hybrid-tethered vehicles will be able to perform tasks that might otherwise require a conventional ROV. These functions will be possible from less complex ships because of a greatly reduced dependence on large, heavy tethers and associated vehicle handling equipment. In certain applications, such vehicles can be resident within subsea facilities, able to provide operators with near instant access when required. Several key emerging technologies and capabilities make such a vehicle possible. Advances in both acoustic and optical "wireless" underwater communications and mico-tethers as pioneered by the HROV Nereus offer the potential to transform ROV type operations and thus offer planners and designers an important new dimension to subsea robotic intervention

Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation.

PubMed

Tarpey, Patrick S; Stevens, Claire; Teague, Jon; Edkins, Sarah; O'Meara, Sarah; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Butler, Adam; Cole, Jennifer; Dicks, Ed; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathon; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Tofts, Calli; Varian, Jennifer; West, Sofie; Widaa, Sara; Yates, Andy; Catford, Rachael; Butler, Julia; Mallya, Uma; Moon, Jenny; Luo, Ying; Dorkins, Huw; Thompson, Deborah; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Carpenter, Nancy; Simensen, Richard J; Schwartz, Charles E; Stevenson, Roger E; Turner, Gillian; Partington, Michael; Gecz, Jozef; Stratton, Michael R; Futreal, P Andrew; Raymond, F Lucy

2006-12-01

In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.

ABC triblock copolymer vesicles with mesh-like morphology.

PubMed

Zhao, Wei; Chen, Dian; Hu, Yunxia; Grason, Gregory M; Russell, Thomas P

2011-01-25

Polymer vesicles made from poly(isoprene-b-styrene-b-2-vinyl pyridine) (PI-b-PS-b-P2VP) triblock copolymer confined within the nanopores of an anodic aluminum oxide (AAO) membrane are studied. It was found that these vesicles have well-defined, nanoscopic size, and complex microphase-separated hydrophobic membranes, comprised of the PS and PI blocks, while the coronas are formed by the P2VP block. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the membrane at a well-defined composition of the three blocks that can be tuned by changing the copolymer composition. The nanoscale confinement, copolymer composition, and subtle molecular interactions contribute to the generation of these vesicles with such unusual morphologies.

Tethered Forth system for FPGA applications

NASA Astrophysics Data System (ADS)

Goździkowski, Paweł; Zabołotny, Wojciech M.

2013-10-01

This paper presents the tethered Forth system dedicated for testing and debugging of FPGA based electronic systems. Use of the Forth language allows to interactively develop and run complex testing or debugging routines. The solution is based on a small, 16-bit soft core CPU, used to implement the Forth Virtual Machine. Thanks to the use of the tethered Forth model it is possible to minimize usage of the internal RAM memory in the FPGA. The function of the intelligent terminal, which is an essential part of the tethered Forth system, may be fulfilled by the standard PC computer or by the smartphone. System is implemented in Python (the software for intelligent terminal), and in VHDL (the IP core for FPGA), so it can be easily ported to different hardware platforms. The connection between the terminal and FPGA may be established and disconnected many times without disturbing the state of the FPGA based system. The presented system has been verified in the hardware, and may be used as a tool for debugging, testing and even implementing of control algorithms for FPGA based systems.

Annexin A1 Complex Mediates Oxytocin Vesicle Transport

PubMed Central

Makani, Vishruti; Sultana, Rukhsana; Sie, Khin Sander; Orjiako, Doris; Tatangelo, Marco; Dowling, Abigail; Cai, Jian; Pierce, William; Butterfield, D. Allan; Hill, Jennifer; Park, Joshua

2013-01-01

Oxytocin is a major neuropeptide that modulates the brain functions involved in social behavior and interaction. Despite of the importance of oxytocin for neural control of social behavior, little is known about the molecular mechanism(s) by which oxytocin secretion in the brain is regulated. Pro-oxytocin is synthesized in the cell bodies of hypothalamic neurons in the supraoptic and paraventricular nuclei and processed to a 9-amino-acid mature form during post-Golgi transport to the secretion sites at the axon terminals and somatodendritic regions. Oxytocin secreted from the somatodendritic regions diffuses throughout the hypothalamus and its neighboring brain regions. Some oxytocin-positive axons innervate and secrete oxytocin to the brain regions distal to the hypothalamus. Brain oxytocin binds to its receptors in the brain regions involved in social behavior. Oxytocin is also secreted from the axon terminal at the posterior pituitary gland into the blood circulation. We have discovered a new molecular complex consisting of annexin A1 (ANXA1), A-kinase anchor protein 150 (AKAP150), and microtubule motor, that controls the distribution of oxytocin vesicles between the axon and the cell body in a protein kinase A (PKA)- and protein kinase C (PKC)-sensitive manner. ANXA1 showed significant co-localization with oxytocin vesicles. Activation of PKA enhanced the association of kinesin-2 with ANXA1, thus increasing the axon-localization of oxytocin vesicles. Conversely, activation of PKC decreased the binding of kinesin-2 to ANXA1, thus attenuating the axon-localization of oxytocin vesicles. Our study suggests that ANXA1 complex coordinates the actions of PKA and PKC to control the distribution of oxytocin vesicles between the axon and the cell body. PMID:24118254

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

PubMed

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

Study of Dynamic Membrane Behavior in Applied DC Electric Field

NASA Astrophysics Data System (ADS)

Dutta, Prashanta; Morshed, Adnan; Hossan, Mohammad

2017-11-01

Electrodeformation of vesicles can be used as a useful tool to understand the characteristics of biological soft matter, where vesicles immersed in a fluid medium are subjected to an applied electric field. The complex response of the vesicle membrane strongly depends on the conductivity of surrounding fluid, vesicle size and shape, and applied electric field We studied the electrodeformation of vesicles immersed in a fluid media under a short DC electric pulse. An immersed interface method is used to solve the electric field over the domain with conductive or non-conductive vesicles while an immersed boundary scheme is employed to solve fluid flow, fluid-solid interaction, membrane mechanics and vesicle movement. Force analysis on the membrane surface reveals almost linear relation with vesicle size, but highly nonlinear influence of applied field as well as the conductivity ratios inside and outside of the vesicle. Results also point towards an early linear deformation regime followed by an equilibrium stage for the membranes. Moreover, significant influence of the initial aspect ratio of the vesicle on the force distribution is observed across a range of conductivity ratios. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM122081.

Oxoiron(IV) Tetramethylcyclam Complexes with Axial Carboxylate Ligands: Effect of Tethering the Carboxylate on Reactivity.

PubMed

Bigelow, Jennifer O; England, Jason; Klein, Johannes E M N; Farquhar, Erik R; Frisch, Jonathan R; Martinho, Marlène; Mandal, Debasish; Münck, Eckard; Shaik, Sason; Que, Lawrence

2017-03-20

Oxoiron(IV) species are implicated as reactive intermediates in nonheme monoiron oxygenases, often acting as the agent for hydrogen-atom transfer from substrate. A histidine is the most likely ligand trans to the oxo unit in most enzymes characterized thus far but is replaced by a carboxylate in the case of isopenicillin N synthase. As the effect of a trans carboxylate ligand on the properties of the oxoiron(IV) unit has not been systematically studied, we have synthesized and characterized four oxoiron(IV) complexes supported by the tetramethylcyclam (TMC) macrocycle and having a carboxylate ligand trans to the oxo unit. Two complexes have acetate or propionate axial ligands, while the other two have the carboxylate functionality tethered to the macrocyclic ligand framework by one or two methylene units. Interestingly, these four complexes exhibit substrate oxidation rates that differ by more than 100-fold, despite having E p,c values for the reduction of the Fe═O unit that span a range of only 130 mV. Eyring parameters for 1,4-cyclohexadiene oxidation show that reactivity differences originate from differences in activation enthalpy between complexes with tethered carboxylates and those with untethered carboxylates, in agreement with computational results. As noted previously for the initial subset of four complexes, the logarithms of the oxygen atom transfer rates of 11 complexes of the Fe IV (O)TMC(X) series increase linearly with the observed E p,c values, reflecting the electrophilicity of the Fe═O unit. In contrast, no correlation with E p,c values is observed for the corresponding hydrogen atom transfer (HAT) reaction rates; instead, the HAT rates increase as the computed triplet-quintet spin state gap narrows, consistent with Shaik's two-state-reactivity model. In fact, the two complexes with untethered carboxylates are among the most reactive HAT agents in this series, demonstrating that the axial ligand can play a key role in tuning the HAT reactivity in a nonheme iron enzyme active site.

Activity-Dependence of Synaptic Vesicle Dynamics

PubMed Central

Forte, Luca A.

2017-01-01

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release. SIGNIFICANCE STATEMENT Mechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function. PMID:28954868

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

PubMed

James, Declan J; Kowalchyk, Judith; Daily, Neil; Petrie, Matt; Martin, Thomas F J

2009-10-13

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

Synaptic Vesicle Mobility and Presynaptic F-Actin Are Disrupted in a N-ethylmaleimide–sensitive Factor Allele of Drosophila

PubMed Central

Nunes, Paula; Haines, Nicola; Kuppuswamy, Venkat; Fleet, David J.

2006-01-01

N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin. PMID:16914524

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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Tiny individuals attached to a new Silurian arthropod suggest a unique mode of brood care

PubMed Central

Briggs, Derek E. G.; Siveter, Derek J.; Siveter, David J.; Sutton, Mark D.; Legg, David

2016-01-01

The ∼430-My-old Herefordshire, United Kingdom, Lagerstätte has yielded a diversity of remarkably preserved invertebrates, many of which provide fundamental insights into the evolutionary history and ecology of particular taxa. Here we report a new arthropod with 10 tiny arthropods tethered to its tergites by long individual threads. The head of the host, which is covered by a shield that projects anteriorly, bears a long stout uniramous antenna and a chelate limb followed by two biramous appendages. The trunk comprises 11 segments, all bearing limbs and covered by tergites with long slender lateral spines. A short telson bears long parallel cerci. Our phylogenetic analysis resolves the new arthropod as a stem-group mandibulate. The evidence suggests that the tethered individuals are juveniles and the association represents a complex brooding behavior. Alternative possibilities—that the tethered individuals represent a different epizoic or parasitic arthropod—appear less likely. PMID:27044103

Glucocorticoid-induced tethered transrepression requires SUMOylation of GR and formation of a SUMO-SMRT/NCoR1-HDAC3 repressing complex

PubMed Central

Hua, Guoqiang; Ganti, Krishna Priya; Chambon, Pierre

2016-01-01

Upon binding of a glucocorticoid (GC), the GC receptor (GR) can exert one of three transcriptional regulatory functions. We recently reported that SUMOylation of the GR at position K293 in humans (K310 in mice) within the N-terminal domain is indispensable for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression. We now demonstrate that the integrity of this GR SUMOylation site is mandatory for the formation of a GR-small ubiquitin-related modifiers (SUMOs)-SMRT/NCoR1-HDAC3 repressing complex, which is indispensable for NF-κB/AP1-mediated GC-induced tethered indirect transrepression in vitro. Using GR K310R mutant mice or mice containing the N-terminal truncated GR isoform GRα-D3 lacking the K310 SUMOylation site, revealed a more severe skin inflammation than in WT mice. Importantly, cotreatment with dexamethasone (Dex) could not efficiently suppress a 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced skin inflammation in these mutant mice, whereas it was clearly decreased in WT mice. In addition, in mice selectively ablated in skin keratinocytes for either nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors or histone deacetylase 3 (HDAC3), Dex-induced tethered transrepression and the formation of a repressing complex on DNA-bound NF-κB/AP1 were impaired. We previously suggested that GR ligands that would lack both (+)GRE-mediated transactivation and IR nGRE-mediated direct transrepression activities of GCs may preferentially exert the therapeutically beneficial GC antiinflammatory properties. Interestingly, we now identified a nonsteroidal antiinflammatory selective GR agonist (SEGRA) that selectively lacks both Dex-induced (+)GRE-mediated transactivation and IR nGRE-mediated direct transrepression functions, while still exerting a tethered indirect transrepression activity and could therefore be clinically lesser debilitating on long-term GC therapy. PMID:26712006

Tethering of SCFDia2 to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase

PubMed Central

Maculins, Timurs; Nkosi, Pedro Junior; Nishikawa, Hiroko; Labib, Karim

2015-01-01

Summary Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood [1, 2]. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase [3]. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex [4, 5], which assembles around the CMG helicase at replication forks [6]. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 [4] or else tethering SCFDia2 (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks [5]. Here, we show that SCFDia2 does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCFDia2 to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned. PMID:26255844

EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research.

PubMed

Kim, Dae-Kyum; Lee, Jaewook; Simpson, Richard J; Lötvall, Jan; Gho, Yong Song

2015-04-01

For cell-to-cell communication, all living cells including archaea, bacteria, and eukaryotes secrete nano-sized membrane vesicles into the extracellular space. These extracellular vesicles harbor specific subsets of proteins, mRNAs, miRNAs, lipids, and metabolites that represent their cellular status. These vesicle-specific cargos are considered as novel diagnostic biomarkers as well as therapeutic targets. With the advancement in high-throughput technologies on multiomics studies and improvements in bioinformatics approaches, a huge number of vesicular proteins, mRNAs, miRNAs, lipids, and metabolites have been identified, and our understanding of these complex extracellular organelles has considerably increased during these past years. In this review, we highlight EVpedia (http://evpedia.info), a community web portal for systematic analyses of prokaryotic and eukaryotic extracellular vesicles research. Copyright © 2015 Elsevier Ltd. All rights reserved.

Photosensitized electron transport across lipid vesicle walls: quantum yield dependence on sensitizer concentration.

PubMed Central

Ford, W E; Otvos, J W; Calvin, M

1979-01-01

An amphiphilic tris(2,2'-bipyridine)ruthenium(2+) derivative that is incorporated into the walls of phosphatidylcholine vesicles photosensitizes the irreversible oxidation of ethylenediaminetetraacetate(3-) dissolved in the inner aqueous compartments of the vesicle suspension and the one-electron reduction of heptylviologen(2+) dissolved in the continuous aqueous phase. The quantum yield of viologen radical production depends on the phospholipid-to-ruthenium complex mole ratios. A kinetic model is used to derive an order-of-magnitude estimate for the rate constant of electron transport across the vesicle walls. The results are inconsistent with a diffusional mechanism for electron transport and are interpreted in terms of electron exchange. PMID:291027

Magneto-Plasmonic Janus Vesicles for Magnetic Field-Enhanced Photoacoustic and Magnetic Resonance Imaging of Tumors.

PubMed

Liu, Yijing; Yang, Xiangyu; Huang, Zhiqi; Huang, Peng; Zhang, Yang; Deng, Lin; Wang, Zhantong; Zhou, Zijian; Liu, Yi; Kalish, Heather; Khachab, Niveen M; Chen, Xiaoyuan; Nie, Zhihong

2016-12-05

Magneto-plasmonic Janus vesicles (JVs) integrated with gold nanoparticles (AuNPs) and magnetic NPs (MNPs) were prepared asymmetrically in the membrane for in vivo cancer imaging. The hybrid JVs were produced by coassembling a mixture of hydrophobic MNPs, free amphiphilic block copolymers (BCPs), and AuNPs tethered with amphiphilic BCPs. Depending on the size and content of NPs, the JVs acquired spherical or hemispherical shapes. Among them, hemispherical JVs containing 50 nm AuNPs and 15 nm MNPs showed a strong absorption in the near-infrared (NIR) window and enhanced the transverse relaxation (T 2 ) contrast effect, as a result of the ordering and dense packing of AuNPs and MNPs in the membrane. The magneto-plasmonic JVs were used as drug delivery vehicles, from which the release of a payload can be triggered by NIR light and the release rate can be modulated by a magnetic field. Moreover, the JVs were applied as imaging agents for in vivo bimodal photoacoustic (PA) and magnetic resonance (MR) imaging of tumors by intravenous injection. With an external magnetic field, the accumulation of the JVs in tumors was significantly increased, leading to a signal enhancement of approximately 2-3 times in the PA and MR imaging, compared with control groups without a magnetic field. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Somanath, Sangeeta; Partridge, Christopher J.; Marshall, Catriona

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

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes

PubMed Central

Ptak, Christopher; Roesner, Ulyss K.

2017-01-01

Interactions occurring at the nuclear envelope (NE)–chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE–chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins—the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170—physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. PMID:28883038

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes.

PubMed

Lapetina, Diego L; Ptak, Christopher; Roesner, Ulyss K; Wozniak, Richard W

2017-10-02

Interactions occurring at the nuclear envelope (NE)-chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE-chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins-the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170-physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. © 2017 Lapetina et al.

An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

PubMed Central

Choudhary, Vineet

2017-01-01

Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides. PMID:28011845

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication

PubMed Central

Sindhwani, Aastha; Kaur, Harmeet; Tuli, Amit

2017-01-01

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication. PMID:29084291

Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

PubMed

Bieberich, Erhard

2011-04-26

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B.

PubMed

Hunter, Morag R; Hesketh, Geoffrey G; Benedyk, Tomasz H; Gingras, Anne-Claude; Graham, Stephen C

2018-05-17

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. Class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) are two such complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named "CHEVI"), perhaps analogous in configuration to CORVET and HOPS. We utilized the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting that these proteins have distinct sub-cellular localizations. Consistent with previous reports, we observed that VPS33A was co-localized with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localized with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localize with VPS33B. Instead, we identified that VPS33B interacts directly with CCDC22, a member of the CCC complex. CCDC22 does not co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates, suggesting that CCDC22 interacts transiently with VPS33B/VIPAR rather than forming a stable complex with these proteins in cells. We also observed that the protein complex containing VPS33B and VIPAR is considerably smaller than CORVET/HOPS, suggesting that the CHEVI complex comprises just VPS33B and VIPAR. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity

PubMed Central

Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A. Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M.; Helms, Jill A.; Nusse, Roel

2008-01-01

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373

Liposomal packaging generates Wnt protein with in vivo biological activity.

PubMed

Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

2008-08-13

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation

PubMed Central

van den Bogaart, Geert; Thutupalli, Shashi; Risselada, Jelger H.; Meyenberg, Karsten; Holt, Matthew; Riedel, Dietmar; Diederichsen, Ulf; Herminghaus, Stephan; Grubmüller, Helmut; Jahn, Reinhard

2011-01-01

Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting the interaction of SNARE proteins between the synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is controversial, and the massive increase in membrane fusion efficiency of Ca2+-synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator: tethering the liposomes too far for SNARE nucleation in the absence of Ca2+, but brings the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions of synaptotagmin-1 with membranes substantially accelerate fusion. PMID:21642968

DNA condensation by partially acetylated poly(amido amine) dendrimers: effects of dendrimer charge density on complex formation.

PubMed

Yu, Shi; Li, Ming-Hsin; Choi, Seok Ki; Baker, James R; Larson, Ronald G

2013-09-03

The ability of poly(amido amine) (or PAMAM) dendrimers to condense semiflexible dsDNA and penetrate cell membranes gives them great potential in gene therapy and drug delivery but their high positive surface charge makes them cytotoxic. Here, we describe the effects of partial neutralization by acetylation on DNA condensation using light scattering, circular dichroism, and single molecule imaging of dendrimer-DNA complexes combed onto surfaces and tethered to those surfaces under flow. We find that DNA can be condensed by generation-five (G5) dendrimers even when the surface charges are more than 65% neutralized, but that such dendrimers bind negligibly when an end-tethered DNA is stretched in flow. We also find that when fully charged dendrimers are introduced by flow to end-tethered DNA, all DNA molecules become equally highly coated with dendrimers at a rate that becomes very fast at high dendrimer concentration, and that dendrimers remain bound during subsequent flow of dendrimer-free buffer. These results suggest that the presence of dendrimer-free DNA coexisting with dendrimer-bound DNA after bulk mixing of the two in solution may result from diffusion-limited irreversible dendrimer-DNA binding, rather than, or in addition to, the previously proposed cooperative binding mechanism of dendrimers to DNA.

Protein Assembly and Building Blocks: Beyond the Limits of the LEGO Brick Metaphor.

PubMed

Levy, Yaakov

2017-09-26

Proteins, like other biomolecules, have a modular and hierarchical structure. Various building blocks are used to construct proteins of high structural complexity and diverse functionality. In multidomain proteins, for example, domains are fused to each other in different combinations to achieve different functions. Although the LEGO brick metaphor is justified as a means of simplifying the complexity of three-dimensional protein structures, several fundamental properties (such as allostery or the induced-fit mechanism) make deviation from it necessary to respect the plasticity, softness, and cross-talk that are essential to protein function. In this work, we illustrate recently reported protein behavior in multidomain proteins that deviates from the LEGO brick analogy. While earlier studies showed that a protein domain is often unaffected by being fused to another domain or becomes more stable following the formation of a new interface between the tethered domains, destabilization due to tethering has been reported for several systems. We illustrate that tethering may sometimes result in a multidomain protein behaving as "less than the sum of its parts". We survey these cases for which structure additivity does not guarantee thermodynamic additivity. Protein destabilization due to fusion to other domains may be linked in some cases to biological function and should be taken into account when designing large assemblies.

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. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Dynamics of vesicles in electric fields

NASA Astrophysics Data System (ADS)

Vlahovska, Petia; Gracia, Ruben

2007-11-01

Electromechanical forces are widely used for cell manipulation. Knowledge of the physical mechanisms underlying the interaction of cells and external fields is essential for practical applications. Vesicles are model cells made of a lipid bilayer membrane. They are examples of ``soft'' particles, i.e., their shape when subjected to flow or electric field is not given a priori but it is governed by the balance of membrane, fluid and electrical stresses. This generic ``softness'' gives rise to a very complex vesicle dynamics in external fields. In an AC electric field, as the frequency is increased, vesicles filled with a fluid less conducting than the surrounding fluid undergo shape transition from prolate to oblate ellipsoids. The opposite effect is observed with drops. We present an electro- hydrodynamic theory based on the leaky dielectric model that quantitatively describes experimental observations. We compare drops and vesicles, and show how their distinct behavior stems from different interfacial properties.

Self-assembly of star micelle into vesicle in solvents of variable quality: the star micelle retains its core-shell nanostructure in the vesicle.

PubMed

Liu, Nijuan; He, Qun; Bu, Weifeng

2015-03-03

Intra- and intermolecular interactions of star polymers in dilute solutions are of fundamental importance for both theoretical interest and hierarchical self-assembly into functional nanostructures. Here, star micelles with a polystyrene corona and a small ionic core bearing platinum(II) complexes have been regarded as a model of star polymers to mimic their intra- and interstar interactions and self-assembled behaviors in solvents of weakening quality. In the chloroform/methanol mixture solvents, the star micelles can self-assemble to form vesicles, in which the star micelles shrink significantly and are homogeneously distributed on the vesicle surface. Unlike the morphological evolution of conventional amphiphiles from micellar to vesicular, during which the amphiphilic molecules are commonly reorganized, the star micelles still retain their core-shell nanostructures in the vesicles and the coronal chains of the star micelle between the ionic cores are fully interpenetrated.

Bioengineering anembryonic human trophoblast vesicles.

PubMed

Robins, Jared C; Morgan, Jeffrey R; Krueger, Paula; Carson, Sandra A

2011-02-01

Trophoblast cells in vivo form a 3-dimensional structure that promotes complex cell-to-cell interactions that cannot be studied with traditional monolayer culture. We describe a 3-dimensional trophoblast bioreactor to study cellular interactions. Nonadhesive agarose hydrogels were cast from molds using computer-assisted prototyping. Trophoblast cells were seeded into the gels for 10 days. Morphology, viability, and vesicle behavior were assessed. Trophoblast cells formed uniform spheroids. Serial sectioning on days 3, 7, and 10 revealed central vacuolization with a consistent outer rim 12.3-μ thick. The vesicle configuration has been confirmed with confocal imaging. Electron Microscopic (EM) imaging revealed its ultrastructure. The vesicles migrate across a fibronectin-coated surface and invaded basement membrane. Trophoblast cells cultured in a novel substrate-free 3-dimensional system form trophoblast vesicles. This new cell culture technique allows us to better study placental cell-to-cell interactions with the potential of forming microtissues.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro.

PubMed

Matsuno, Yuta; Onuma, Asuka; Fujioka, Yoshie A; Yasuhara, Kazuma; Fujii, Wataru; Naito, Kunihiko; Sugiura, Koji

2017-02-16

Cell-secreted vesicles, such as exosomes, have recently been recognized as mediators of cell communication. A recent study in cattle showed the involvement of exosome-like vesicles in the control of cumulus expansion, a prerequisite process for normal ovulation; however, whether this is the case in other mammalian species is not known. Therefore, this study aimed to examine the presence of exosome-like vesicles in ovarian follicles and their effects on cumulus expansion in vitro in pigs. The presence of exosome-like vesicles in porcine follicular fluid (pFF) was confirmed by transmission electron microscopic observation, the detection of marker proteins, and RNA profiles specific to exosomes. Fluorescently labeled exosome-like vesicles isolated from pFF were incorporated into both cumulus and mural granulosa cells in vitro. Exosome-like vesicles were not capable of inducing cumulus expansion to a degree comparable to that induced by follicle-stimulating hormone (FSH). Moreover, exosome-like vesicles had no significant effects on the expression levels of transcripts required for the normal expansion process (HAS2, TNFAIP6, and PTGS2). Interestingly, FSH-induced expression of HAS2 and TNFAIP6 mRNA, but not of PTGS2 mRNA, was significantly increased by the presence of exosome-like vesicles; however, the degree of FSH-induced expansion was not affected. In addition, porcine exosome-like vesicles had no significant effects on the expansion of mouse cumulus-oocyte complexes. Collectively, the present results suggest that exosome-like vesicles are present in pFF, but they are not efficient in inducing cumulus expansion in pigs.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro

PubMed Central

MATSUNO, Yuta; ONUMA, Asuka; FUJIOKA, Yoshie A; YASUHARA, Kazuma; FUJII, Wataru; NAITO, Kunihiko; SUGIURA, Koji

2017-01-01

Cell-secreted vesicles, such as exosomes, have recently been recognized as mediators of cell communication. A recent study in cattle showed the involvement of exosome-like vesicles in the control of cumulus expansion, a prerequisite process for normal ovulation; however, whether this is the case in other mammalian species is not known. Therefore, this study aimed to examine the presence of exosome-like vesicles in ovarian follicles and their effects on cumulus expansion in vitro in pigs. The presence of exosome-like vesicles in porcine follicular fluid (pFF) was confirmed by transmission electron microscopic observation, the detection of marker proteins, and RNA profiles specific to exosomes. Fluorescently labeled exosome-like vesicles isolated from pFF were incorporated into both cumulus and mural granulosa cells in vitro. Exosome-like vesicles were not capable of inducing cumulus expansion to a degree comparable to that induced by follicle-stimulating hormone (FSH). Moreover, exosome-like vesicles had no significant effects on the expression levels of transcripts required for the normal expansion process (HAS2, TNFAIP6, and PTGS2). Interestingly, FSH-induced expression of HAS2 and TNFAIP6 mRNA, but not of PTGS2 mRNA, was significantly increased by the presence of exosome-like vesicles; however, the degree of FSH-induced expansion was not affected. In addition, porcine exosome-like vesicles had no significant effects on the expansion of mouse cumulus-oocyte complexes. Collectively, the present results suggest that exosome-like vesicles are present in pFF, but they are not efficient in inducing cumulus expansion in pigs. PMID:28163264

In-Space Transportation with Tethers

NASA Technical Reports Server (NTRS)

Lorenzini, Enrico; Estes, Robert D.; Cosmo, Mario L.

1998-01-01

The annual report covers the research conducted on the following topics related to the use of spaceborne tethers for in-space transportation: ProSEDS tether modeling (current collection analyses, influence of a varying tether temperature); proSEDS mission analysis and system dynamics (tether thermal model, thermo-electro-dynamics integrated simulations); proSEDS-tether development and testing (tether requirements, deployment test plan, tether properties testing, deployment tests); and tethers for reboosting the space-based laser (mission analysis, tether system preliminary design, evaluation of attitude constraints).

Kinetics and mechanism of electron transfer reaction of single and double chain surfactant cobalt(III) complex by Fe2+ ions in liposome (dipalmitoylphosphotidylcholine) vesicles: effects of phase transition

NASA Astrophysics Data System (ADS)

Nagaraj, Karuppiah; Senthil Murugan, Krishnan; Thangamuniyandi, Pilavadi

2015-05-01

In this study, we report the kinetics of reduction reactions of single and double chain surfactant cobalt(III) complexes of octahedral geometry, cis-[Co(en)2(4AMP)(DA)](ClO4)3 and cis-[Co(dmp)2(C12H25NH2)2](ClO4)3 (en = ethylenediamine, dmp = 1,3-diaminopropane, 4AMP = 4-aminopropane, C12H25NH2 = dodecylamine) by Fe2+ ion in dipalmitoylphosphotidylcholine (DPPC) vesicles at different temperatures under pseudo first-order conditions. The kinetics of these reactions is followed by spectrophotometry method. The reactions are found to be second order and the electron transfer is postulated as outer sphere. The remarkable findings in the present investigation are that, below the phase transition temperature of DPPC, the rate decreases with an increase in the concentration of DPPC, while above the phase transition temperature the rate increases with an increase in the concentration of DPPC. The main driving force for this phenomenon is considered to be the intervesicular hydrophobic interaction between vesicles surface and hydrophobic part of the surfactant complexes. Besides, comparing the values of rate constants of these outer-sphere electron transfer reactions in the absence and in the presence of DPPC, the rate constant values in the presence of DPPC are always found to be greater than in the absence of DPPC. This is ascribed to the double hydrophobic fatty acid chain in the DPPC that gives the molecule an overall tubular shape due to the intervesicular hydrophobic interaction between vesicles surface and hydrophobic part of the surfactant complexes more suitable for vesicle aggregation which facilitates lower activation energy, and consequently higher rate is observed in the presence of DPPC. The activation parameters (ΔS# and ΔH#) of the reactions at different temperatures have been calculated which corroborate the kinetics of the reaction.

Space station operations enhancement using tethers

NASA Astrophysics Data System (ADS)

Bekey, I.

1984-10-01

Space tethers represent a tool of unusual versatility for applications to operations involving space stations. The present investigation is concerned with a number of applications which exploit the dynamic, static, and electrodynamic properties of tethers. One of the simplest applications of a tethered system on the Space Station might be that of a remote docking port, allowing the Shuttle to dock with no contamination or disturbance effects. Attention is also given to tethered platforms, a tethered microgravity facility, a tethered space station propellant facility, electrodynamic tether principles, a tether power generator, a tether thrust generator (motor), and an electrodynamic tether for drag makeup and energy storage.

ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations.

PubMed

Cheng, Aifang; Zhao, Teng; Tse, Kai-Hei; Chow, Hei-Man; Cui, Yong; Jiang, Liwen; Du, Shengwang; Loy, Michael M T; Herrup, Karl

2018-01-09

ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) are large PI3 kinases whose human mutations result in complex syndromes that include a compromised DNA damage response (DDR) and prominent nervous system phenotypes. Both proteins are nuclear-localized in keeping with their DDR functions, yet both are also found in cytoplasm, including on neuronal synaptic vesicles. In ATM- or ATR-deficient neurons, spontaneous vesicle release is reduced, but a drop in ATM or ATR level also slows FM4-64 dye uptake. In keeping with this, both proteins bind to AP-2 complex components as well as to clathrin, suggesting roles in endocytosis and vesicle recycling. The two proteins play complementary roles in the DDR; ATM is engaged in the repair of double-strand breaks, while ATR deals mainly with single-strand damage. Unexpectedly, this complementarity extends to these proteins' synaptic function as well. Superresolution microscopy and coimmunoprecipitation reveal that ATM associates exclusively with excitatory (VGLUT1 + ) vesicles, while ATR associates only with inhibitory (VGAT + ) vesicles. The levels of ATM and ATR respond to each other; when ATM is deficient, ATR levels rise, and vice versa. Finally, blocking NMDA, but not GABA, receptors causes ATM levels to rise while ATR levels respond to GABA, but not NMDA, receptor blockade. Taken together, our data suggest that ATM and ATR are part of the cellular "infrastructure" that maintains the excitatory/inhibitory balance of the nervous system. This idea has important implications for the human diseases resulting from their genetic deficiency.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

Extracellular wire tetrode recording in brain of freely walking insects.

PubMed

Guo, Peiyuan; Pollack, Alan J; Varga, Adrienn G; Martin, Joshua P; Ritzmann, Roy E

2014-04-01

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

beta'-COP, a novel subunit of coatomer.

PubMed Central

Stenbeck, G; Harter, C; Brecht, A; Herrmann, D; Lottspeich, F; Orci, L; Wieland, F T

1993-01-01

Several lines of evidence favour the hypothesis that intracellular biosynthetic protein transport in eukaryotes is mediated by non-clathrin-coated vesicles (for a review see Rothman and Orci, 1992). The vesicles have been isolated and a set of their surface proteins has been characterized as coat proteins (COPs). These COPs exist in the cytosol as a preformed complex, the coatomer, which was prior to this study known to contain six subunits: four (alpha-, beta-, gamma- and delta-COP) with molecular weights between 160 and 58 kDa, and two additional proteins of approximately 36 and 20 kDa, epsilon- and xi-COP. Here we describe a novel subunit of the coatomer complex, beta'-COP. This subunit occurs in amounts stoichiometric to the established COPs both in the coatomer and in nonclathrin-coated vesicles and shows homology to the beta-subunits of trimeric G proteins. Images PMID:8334999

Layer-by-layer cell membrane assembly

NASA Astrophysics Data System (ADS)

Matosevic, Sandro; Paegel, Brian M.

2013-11-01

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Model Systems of Precursor Cellular Membranes: Long-Chain Alcohols Stabilize Spontaneously Formed Oleic Acid Vesicles

PubMed Central

Rendón, Adela; Carton, David Gil; Sot, Jesús; García-Pacios, Marcos; Montes, Ruth; Valle, Mikel; Arrondo, José-Luis R.; Goñi, Felix M.; Ruiz-Mirazo, Kepa

2012-01-01

Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid. PMID:22339864

Complementary and partially complementary DNA duplexes tethered to a functionalized substrate: a molecular dynamics approach to biosensing.

PubMed

Monti, Susanna; Cacelli, Ivo; Ferretti, Alessandro; Prampolini, Giacomo; Barone, Vincenzo

2011-07-21

Molecular dynamics simulations (90 ns) of different DNA complexes attached to a functionalized substrate in solution were performed in order to clarify the behavior of mismatched DNA sequences captured by a tethered DNA probe (biochip). Examination of the trajectories revealed that the substrate influence and a series of cooperative events, including recognition, reorientation and reorganization of the bases, could induce the formation of stable duplexes having non-canonical arrangements. Major adjustment of the structures was observed when the mutated base was located in the end region of the chain close to the surface. This journal is © the Owner Societies 2011

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.

Development of the Flight Tether for ProSEDS

NASA Technical Reports Server (NTRS)

Curtis, Leslie; Vaughn, Jason; Welzyn, Ken; Carroll, Joe; Brown, Norman S. (Technical Monitor)

2002-01-01

The Propulsive Small Expendable Deployer System (ProSEDS) space experiment will demonstrate the use of an electrodynamic tether propulsion system to generate thrust in space by decreasing the orbital altitude of a Delta 11 Expendable Launch Vehicle second stage. ProSEDS will use the flight-proven Small Expendable Deployer System to deploy a newly designed and developed tether which will provide tether generated drag thrust of approx. 0.4 N. The development and production of very long tethers with specific properties for performance and survivability will be required to enable future tether missions. The ProSEDS tether design and the development process may provide some lessons learned for these future missions. The ProSEDS system requirements drove the design of the tether to have three different sections of tether each serving a specialized purpose. The tether is a total of 15 kilometers long: 10 kilometers of a non-conductive Dyneema lead tether; 5 km of CCOR conductive coated wire; and 220 meters of insulated wire with a protective Kevlar overbraid. Production and joining of long tether lengths involved many development efforts. Extensive testing of tether materials including ground deployment of the full-length ProSEDS tether was conducted to validate the tether design and performance before flight.

GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of thismore » domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm interacts with NP via its N-terminal unfolded region and the NSm–NP complex could in turn interact with the ER membrane via the C-terminal coiled coil domain of NSm to form vesicles that are targeted to PD and there by assist the cell to cell movement of the viral genome complex. - Highlights: • GBNV NSm localizes to plasmodesmata via the C-terminal coiled coil domain. • GBNV NSm interacts with endoplasmic reticulum network and remodels it to vesicles. • The C-terminal coiled domain alone is responsible for vesicle formation. • The N-terminal unfolded region of NSm is involved in the re-localization of NP to PD.« less

Tuning peptide self-assembly by an in-tether chiral center

PubMed Central

Hu, Kuan; Xiong, Wei; Li, Hu; Zhang, Pei-Yu; Yin, Feng; Zhang, Qianling; Jiang, Fan; Li, Zigang

2018-01-01

The self-assembly of peptides into ordered nanostructures is important for understanding both peptide molecular interactions and nanotechnological applications. However, because of the complexity and various self-assembling pathways of peptide molecules, design of self-assembling helical peptides with high controllability and tunability is challenging. We report a new self-assembling mode that uses in-tether chiral center-induced helical peptides as a platform for tunable peptide self-assembly with good controllability. It was found that self-assembling behavior was governed by in-tether substitutional groups, where chirality determined the formation of helical structures and aromaticity provided the driving force for self-assembly. Both factors were essential for peptide self-assembly to occur. Experiments and theoretical calculations indicate long-range crystal-like packing in the self-assembly, which was stabilized by a synergy of interpeptide π-π and π-sulfur interactions and hydrogen bond networks. In addition, the self-assembled peptide nanomaterials were demonstrated to be promising candidate materials for applications in biocompatible electrochemical supercapacitors.

Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Benoit, Danielle S. W.; Schwartz, Michael P.; Durney, Andrew R.; Anseth, Kristi S.

2008-10-01

Cell-matrix interactions have critical roles in regeneration, development and disease. The work presented here demonstrates that encapsulated human mesenchymal stem cells (hMSCs) can be induced to differentiate down osteogenic and adipogenic pathways by controlling their three-dimensional environment using tethered small-molecule chemical functional groups. Hydrogels were formed using sufficiently low concentrations of tether molecules to maintain constant physical characteristics, encapsulation of hMSCs in three dimensions prevented changes in cell morphology, and hMSCs were shown to differentiate in normal growth media, indicating that the small-molecule functional groups induced differentiation. To our knowledge, this is the first example where synthetic matrices are shown to control induction of multiple hMSC lineages purely through interactions with small-molecule chemical functional groups tethered to the hydrogel material. Strategies using simple chemistry to control complex biological processes would be particularly powerful as they could make production of therapeutic materials simpler, cheaper and more easily controlled.

Space Tethers: Design Criteria

NASA Technical Reports Server (NTRS)

Tomlin, D. D.; Faile, G. C.; Hayashida, K. B.; Frost, C. L.; Wagner, C. Y.; Mitchell, M. L.; Vaughn, J. A.; Galuska, M. J.

1997-01-01

This document is prepared to provide a systematic process for the selection of tethers for space applications. Criteria arc provided for determining the strength requirement for tether missions and for mission success from tether severing due to micrometeoroids and orbital debris particle impacts. Background information of materials for use in space tethers is provided, including electricity-conducting tethers. Dynamic considerations for tether selection is also provided. Safety, quality, and reliability considerations are provided for a tether project.

Transfer Hydrogenation and Antiproliferative Activity of Tethered Half-Sandwich Organoruthenium Catalysts

PubMed Central

2018-01-01

We report the synthesis and characterization of four neutral organometallic tethered complexes, [Ru(η6-Ph(CH2)3-ethylenediamine-N-R)Cl], where R = methanesulfonyl (Ms, 1), toluenesulfonyl (Ts, 2), 4-trifluoromethylbenzenesulfonyl (Tf, 3), and 4-nitrobenzenesulfonyl (Nb, 4), including their X-ray crystal structures. These complexes exhibit moderate antiproliferative activity toward human ovarian, lung, hepatocellular, and breast cancer cell lines. Complex 2 in particular exhibits a low cross-resistance with cisplatin. The complexes show potent catalytic activity in the transfer hydrogenation of NAD+ to NADH with formate as hydride donor in aqueous solution (310 K, pH 7). Substituents on the chelated ligand decreased the turnover frequency in the order Nb > Tf > Ts > Ms. An enhancement of antiproliferative activity (up to 22%) was observed on coadministration with nontoxic concentrations of sodium formate (0.5–2 mM). Complex 2 binds to nucleobase guanine (9-EtG), but DNA appears not to be the target, as little binding to calf thymus DNA or bacterial plasmid DNA was observed. In addition, complex 2 reacts rapidly with glutathione (GSH), which might hamper transfer hydrogenation reactions in cells. Complex 2 induced a dose-dependent G1 cell cycle arrest after 24 h exposure in A2780 human ovarian cancer cells while promoting an increase in reactive oxygen species (ROS), which is likely to contribute to its antiproliferative activity.

Evidence That Rhesus Macaques Self-Cure from a Schistosoma japonicum Infection by Disrupting Worm Esophageal Function: A New Route to an Effective Vaccine?

PubMed Central

Li, Xiao-Hong; Xu, Yu-Xin; Vance, Gill; Wang, Yun; Lv, Long-Bao; van Dam, Govert J.; Cao, Jian-Ping; Wilson, R. Alan

2015-01-01

Background Rhesus macaques are unusual among schistosome hosts, self-curing from an established infection and thereafter manifesting solid immunity against a challenge, an ideal model for vaccine development. Previously, the immunological basis of self-cure was confirmed; surviving worms had ceased feeding but how immunological pressure achieved this was unclear. The schistosome esophagus is not simply a conduit for blood but plays a central role in its processing. Secretions from the anterior and posterior esophageal glands mix with incoming blood causing erythrocyte lysis and tethering and killing of leucocytes. Methodology/Principal Findings We have analysed the self-cure process in rhesus macaques infected with Schistosoma japonicum. Faecal egg output and circulating antigen levels were used to chart the establishment of a mature worm population and its subsequent demise. The physiological stress of surviving females at perfusion was especially evident from their pale, shrunken appearance, while changes in the structure and function of the esophagus were observed in both sexes. In the anterior region electron microscopy revealed that the vesicle secretory process was disrupted, the tips of lining corrugations being swollen by greatly enlarged vesicles and the putative sites of vesicle release obscured by intense deposits of IgG. The lumen of the posterior esophagus in starving worms was occluded by cellular debris and the lining cytoplasmic plates were closely adherent, also potentially preventing secretion. Seven proteins secreted by the posterior gland were identified and IgG responses were detected to some or all of them. Intrinsic rhesus IgG colocalized with secreted SjMEGs 4.1, 8.2, 9, 11 and VAL-7 on cryosections, suggesting they are potential targets for disruption of function. Conclusions/Significance Our data suggest that rhesus macaques self-cure by blocking esophagus function with antibody; the protein products of the glands provide a new class of potential vaccine targets. PMID:26161644

Applications of Tethers in Space

NASA Technical Reports Server (NTRS)

Cron, A. C.

1985-01-01

The proceedings of the first workshop on applications of tethers in space are summarized. The workshop gathered personalities from industry, academic institutions and government to discuss the relatively new area of applied technology of very long tethers in space to a broad spectrum of future space missions. A large number of tethered concepts and configurations was presented covering electrodynamic interaction tethers, tethered transportation through angular momentum exchange, tethered constellations, low gravity utilization, applicable technology, and tethered test facilities. Specific recommendations were made to NASA in each area.

Complexin and Ca2+ stimulate SNARE-mediated membrane fusion

PubMed Central

Yoon, Tae-Young; Lu, Xiaobind; Diao, Jiajie; Lee, Soo-Min; Ha, Taekjip; Shin, Yeon-Kyun

2008-01-01

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release. PMID:18552825

Tether dynamics and control results for tethered satellite system's initial flight

NASA Astrophysics Data System (ADS)

Chapel, Jim D.; Flanders, Howard

The recent Tethered Satellite System-1 (TSS-1) mission has provided a wealth of data concerning the dynamics of tethered systems in space and has demonstrated the effectiveness of operational techniques designed to control these dynamics. In this paper, we review control techniques developed for managing tether dynamics, and discuss the results of using these techniques for the Tethered Satellite System's maiden flight on STS-46. In particular, the flight results of controlling libration dynamics, string dynamics, and slack tether are presented. These results show that tether dynamics can be safely managed. The overall stability of the system was found to be surprisingly good even at relatively short tether lengths. In fact, the system operated in passive mode at a tether length of 256 meters for over 9 hours. Only monitoring of the system was required during this time. Although flight anomalies prevented the planned deployment to 20 km, the extended operations at shorter tether lengths have proven the viability of using tethers in space. These results should prove invaluable in preparing for future missions with tethered objects in space.

Tether dynamics and control results for tethered satellite system's initial flight

NASA Technical Reports Server (NTRS)

Chapel, Jim D.; Flanders, Howard

1993-01-01

The recent Tethered Satellite System-1 (TSS-1) mission has provided a wealth of data concerning the dynamics of tethered systems in space and has demonstrated the effectiveness of operational techniques designed to control these dynamics. In this paper, we review control techniques developed for managing tether dynamics, and discuss the results of using these techniques for the Tethered Satellite System's maiden flight on STS-46. In particular, the flight results of controlling libration dynamics, string dynamics, and slack tether are presented. These results show that tether dynamics can be safely managed. The overall stability of the system was found to be surprisingly good even at relatively short tether lengths. In fact, the system operated in passive mode at a tether length of 256 meters for over 9 hours. Only monitoring of the system was required during this time. Although flight anomalies prevented the planned deployment to 20 km, the extended operations at shorter tether lengths have proven the viability of using tethers in space. These results should prove invaluable in preparing for future missions with tethered objects in space.

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

PI(4,5)P2-binding effector proteins for vesicle exocytosis

PubMed Central

Martin, Thomas F. J.

2014-01-01

PI(4,5)P2 participates directly in priming and possibly fusion steps of Ca2+-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P2 reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P2 domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P2 directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P2-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P2 effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P2, which promotes clustering, but an activating role for PI(4,5)P2 in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P2-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P2, which serves to recruit and/or activate multifunctional PI(4,5)P2-binding proteins. PMID:25280637

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

Atmospheric Electricity and Tethered Aerostats, Volume 2

DTIC Science & Technology

1976-05-11

vs Altitude (Non- conducting or Conducting Tethers...Effect of Corona Charge Plume 15 3.1 Tether Current vs Balloon Altitude , BJ+3 - 25 Sep 73 20 3.2 Tether Current vs Balloon Altitude , Baldy - 17 Oct 73 21...3.3 Tether Current vs Balloon Altitude , Baldy - 31 Oct 73 22 3.4 Tether Current vs Balloon Altitude , Baldy - 2 Nov 73 23 3.5 Tether Current vs

Cell-mediated lysis of lipid vesicles containing eye muscle protein: Implications regarding pathogenesis of Graves ophthalmopathy*

PubMed Central

Kriss, Joseph P.; Mehdi, S. Qasim

1979-01-01

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular 99mTc marker. Injury to the vesicular membrane was assessed by measurement of 99mTc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in 99mTc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven. PMID:88053

A novel mechanism of vascular endothelial growth factor, leptin and transforming growth factor-beta2 sequestration in a subpopulation of human ovarian follicle cells.

PubMed

Antczak, M; Van Blerkom, J; Clark, A

1997-10-01

This study describes the occurrence of a highly specialized subpopulation of granulosa and cumulus oophorus cells that accumulate and sequester specific growth factors by a novel mechanism. These cells are characterized by multiple balloon-like processes tethered to the cell by means of a slender stalk of plasma membrane. Time-lapse analyses demonstrate that these tethered structures (TS) form in minutes and frequently detach from the cell with the bulbous portion remaining motile on the cell surface. Serial section reconstruction of transmission electron microscopic images shows a specific and stable intracellular organization in which an apparent secretory compartment composed of densely packed vacuoles, vesicles, and cisternae is separated by a thick filamentous network from a nuclear compartment containing mitochondria, polyribosomes, lipid inclusions, and rough-surfaced endoplasmic reticulum. Immunofluorescent analysis performed during the formation of these structures showed a progressive accumulation of vascular endothelial growth factor, leptin, and transforming growth factor-beta2 in the bulbous region. TS were identified in newly aspirated masses of granulosa and cumulus oophorus, and their production persists for months in culture. Observations of TS-forming cells made over several days of culture indicates that their production is episodic and factor release from these cells may be pulsatile. The findings suggest that a novel method of growth factor storage and release by an apparent apocrine-like mechanism occurs in the human ovarian follicle. The results are discussed with respect to possible roles in pre- and post-ovulatory follicular development.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

PubMed

De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie

2014-01-01

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Use of top tethers with forward-facing child restraints: observations and driver interviews.

PubMed

Eichelberger, Angela H; Decina, Lawrence E; Jermakian, Jessica S; McCartt, Anne T

2014-02-01

Despite the safety benefits, many parents do not use top tethers with forward-facing child restraints. Detailed information was collected about why parents are not using tethers. The sample included 479 drivers who had forward-facing child restraints installed in passenger vehicles equipped with tether anchors. The survey was conducted primarily at shopping centers, recreation facilities, child care facilities, car seat check events, and health care facilities in mostly suburban areas surrounding Philadelphia, Washington, DC, Fredericksburg (VA), and Seattle. Drivers were surveyed about their knowledge and use of tethers and experience with child restraints. Tether use was observed to verify whether tethers were being used correctly. Fifty-six percent of forward-facing child restraints were installed with the tether; 39% were installed with the tether used correctly. The tether was used with 71% of LATCH lower anchor installations and 33% of seat belt installations. Drivers who installed child restraints without tethers most often said they did not know about the tether or how to use it. Although the tether use rate was slightly higher in the current research than in previous studies, many parents and caregivers still use forward-facing child restraints without attaching the tether. Because the main problem is lack of awareness of the tether or how to use it, public education should focus specifically on the safety benefits of tethers and how to use them. Information about why caregivers fail to use top tethers is potentially useful to child restraint manufacturers, child passenger safety technicians, and others who work with parents to improve motor vehicle safety. Copyright © 2013 Elsevier Ltd and National Safety Council. All rights reserved.

Ultrastructure of the endolymphatic sac in the larva of the japanese red-bellied newt Cynops pyrrhogaster

NASA Technical Reports Server (NTRS)

Gao, W.; Wiederhold, M.; Hejl, R.

1998-01-01

The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the "floccular" vesicle and the "granular" vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain floccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.

Tethers

NASA Technical Reports Server (NTRS)

Cutler, Andrew Hall; Carroll, Joseph A.

1992-01-01

A tether of sufficient strength, capable of being lengthened or shortened and having appropriate apparatuses for capturing and releasing bodies at its ends, may be useful in propulsion applications. For example, a tether could allow rendezvous between spacecraft in substantially different orbits without using propellant. A tether could also allow co-orbiting spacecraft to exchange momentum and separate. Thus, a reentering spacecraft (such as the Shuttle) could give its momentum to one remaining on orbit (such as the space station). Similarly, a tether facility could gain momentum from a high I(sub sp)/low thrust mechanism (which could be an electrodynamics tether) and transfer than momentum by means of a tether to payloads headed for many different orbits. Such a facility would, in effect, combine high I(sub sp) with high thrust, although only briefly. An electrodynamic tether could propel a satellite from its launch inclination to a higher or lower inclination. Tethers could also allow samples to be taken from bodies such as the Moon. Three types of tether operations are illustrated. The following topics are discussed: (1) tether characteristics; (2) tether propulsion methods--basics, via momentum transfer, and electrodynamic tether propulsion; and (3) their use in planetary exploration.

Observer enhanced control for spin-stabilized tethered formation in earth orbit

NASA Astrophysics Data System (ADS)

Guang, Zhai; Yuyang, Li; Liang, Bin

2018-04-01

This paper addresses the issues relevant to control of spin-stabilized tethered formation in circular orbit. Due to the dynamic complexities and nonlinear perturbations, it is challenging to promote the control precision for the formation deployment and maintenance. In this work, the formation dynamics are derived with considering the spinning rate of the central body, then major attention is dedicated to develop the nonlinear disturbance observer. To achieve better control performance, the observer-enhanced controller is designed by incorporating the disturbance observer into the control loop, benefits from the disturbance compensation are demonstrated, and also, the dependences of the disturbance observer performance on some important parameters are theoretically and numerically analyzed.

Applications of Tethers in Space: Workshop Proceedings, Volume 2

NASA Technical Reports Server (NTRS)

Baracat, W. A. (Compiler)

1986-01-01

Topics addressed include: tethered orbital transfer vehicle operations, Centaur and Shuttle tether technology; tethered constellations, gravitational effects; Shuttle continuous open wind tunnel; optimal control laws, electrodynamic tether technology; and space station facilities.

Plasma Interactions With a Negative Biased Electrodynamic Tether

NASA Technical Reports Server (NTRS)

Vaughn, Jason A.; Curtis, Leslie; Welzyn, Ken J.

2004-01-01

The ProSEDS conductive tether design incorporates two distinct types of tethers from a plasma interaction viewpoint. The 200 m closest to the Delta II spacecraft is insulated from the plasma, and the remaining 4800 m is semi-bare. This latter portion is considered semi-bare because a conductive coating, which is designed to collect electrons from the plasma, was applied to the wires to regulate the overall tether temperature. Because the tether has both insulating and conductive tether sections, a transition point exists between the two that forms a triple point with the space plasma. Also, insulated tethers can arc to the space plasma if the insulation is weakened or breached by pinholes caused by either improper handling or small meteoroid and orbital debris strikes. Because electrodynamic tethers are typically long, they have a high probability of these impacts. The particles, which strike the tether, may not have sufficient size to severe the tether, but they can easily penetrate the tether insulation producing a plasma discharge to the ambient plasma. Samples of both the ProSEDS tether transition region and the insulated tether section with various size of pinholes were placed into the MSFC plasma chamber and biased to typical ProSEDS open circuit tether potentials (-500 V to -1600 V). The results of the testing showed that the transition region of the tether (i.e. the triple point) arced to the ambient plasma at -900 V, and the tethers damaged by a pinhole or simulated debris strike arced to the plasma between -700 V and -900 V. Specific design steps were taken to eliminate the triple point issue in the ProSEDS tether design and make it ready for flight. To reduce the pinhole arcing risk, ProSEDS mission operations were changed to eliminate the high negative potential on the insulated tether. The results of the testing campaign and the design changes implemented to ensure a successful flight are described.

Multiphysics elastodynamic finite element analysis of space debris deorbit stability and efficiency by electrodynamic tethers

NASA Astrophysics Data System (ADS)

Li, Gangqiang; Zhu, Zheng H.; Ruel, Stephane; Meguid, S. A.

2017-08-01

This paper developed a new multiphysics finite element method for the elastodynamic analysis of space debris deorbit by a bare flexible electrodynamic tether. Orbital motion limited theory and dynamics of flexible electrodynamic tethers are discretized by the finite element method, where the motional electric field is variant along the tether and coupled with tether deflection and motion. Accordingly, the electrical current and potential bias profiles of tether are solved together with the tether dynamics by the nodal position finite element method. The newly proposed multiphysics finite element method is applied to analyze the deorbit dynamics of space debris by electrodynamic tethers with a two-stage energy control strategy to ensure an efficient and stable deorbit process. Numerical simulations are conducted to study the coupled effect between the motional electric field and the tether dynamics. The results reveal that the coupling effect has a significant influence on the tether stability and the deorbit performance. It cannot be ignored when the libration and deflection of the tether are significant.

Guidebook for analysis of tether applications

NASA Technical Reports Server (NTRS)

Carroll, J. A.

1985-01-01

This guidebook is intended as a tool to facilitate initial analyses of proposed tether applications in space. Topics disscussed include: orbit and orbit transfer equations; orbital perturbations; aerodynamic drag; thermal balance; micrometeoroids; gravity gradient effects; tether control strategies; momentum transfer; orbit transfer by tethered release/rendezvous; impact hazards for tethers; electrodynamic tether principles; and electrodynamic libration control issues.

Selected tether applications in space: Phase 2. Executive summary

NASA Technical Reports Server (NTRS)

Thorson, M. H.; Lippy, L. J.

1985-01-01

The application of tether technology has the potential to increase the overall performance efficiency and capability of the integrated space operations and transportation systems through the decade of the 90s. The primary concepts for which significant economic benefits were identified are dependent on the space station as a storage device for angular momentum and as an operating base for the tether system. Concepts examined include: (1) tether deorbit of shuttle from space station; (2) tethered orbit insertion of a spacecraft from shuttle; (3) tethered platform deployed from space station; (4) tether-effected rendezvous of an OMV with a returning OTV; (5) electrodynamic tether as an auxiliary power source for space station; and (6) tether assisted launch of an OTV mission from space station.

Molecular engineering of polymersome surface topology

PubMed Central

Ruiz-Pérez, Lorena; Messager, Lea; Gaitzsch, Jens; Joseph, Adrian; Sutto, Ludovico; Gervasio, Francesco Luigi; Battaglia, Giuseppe

2016-01-01

Biological systems exploit self-assembly to create complex structures whose arrangements are finely controlled from the molecular to mesoscopic level. We report an example of using fully synthetic systems that mimic two levels of self-assembly. We show the formation of vesicles using amphiphilic copolymers whose chemical nature is chosen to control both membrane formation and membrane-confined interactions. We report polymersomes with patterns that emerge by engineering interfacial tension within the polymersome surface. This allows the formation of domains whose topology is tailored by chemical synthesis, paving the avenue to complex supramolecular designs functionally similar to those found in viruses and trafficking vesicles. PMID:27152331

Tethers as Debris: Hydrocode Simulation of Impacts of Polymer Tether Fragments on Aluminum Plates

NASA Technical Reports Server (NTRS)

Evans, Steven W.

2003-01-01

Tethers promise to find use in a variety of space applications. Despite being narrow objects, their great lengths result in them having large total areas. Consequently, tethers are very susceptible to being severed by orbital debris. Extensive work has been done designing tethers that resist severs by small debris objects, in order to lengthen their working lives. It is from this perspective that most recent work has considered the tether - debris question. The potential of intact tethers, or severed tether fragments, as debris, to pose a significant collision risk to other spacecraft has been less well studied. Understanding the consequences of such collisions is important in assessing the risks tethers pose to other spacecraft. This paper discusses the damage that polymer tethers may produce on aluminum plates, as revealed by hypervelocity impact simulations using the SPHC hydrodynamic code.

Enhancement of sensitivity using hybrid stimulus for the diagnosis of prostate cancer based on polydiacetylene (PDA) supramolecules.

PubMed

Kwon, Il Kyoung; Kim, Jun Pyo; Sim, Sang Jun

2010-12-15

In this study, hybrid stimulus was initially introduced to improve the sensitivity of PDA vesicle chip for detection of prostate-specific antigen-α1-antichymotrypsin (PSA-ACT) complex. The strategy of hybrid stimulus on PDA vesicle chip offers the amplification method of fluorescent signal which combines a primary response by the immune reaction of antigen-antibody and a secondary response by the mechanical pressure of pAb-conjugated magnetic beads. As the primary response result on PDA vesicle chip, the PSA-ACT complex in PBS buffer was detected at 10 ng/mL. However, this detection sensitivity was insufficient for diagnosis of prostate cancer because the normal human PSA concentration is less than 4.0 ng/mL. To solve this problem, polyclonal PSA antibody-conjugated magnetic beads were used as an amplifying agent after primary immunoresponse. As a result, the PSA-ACT complex concentrations (as low as 0.1 ng/mL) could be detected in the PBS buffer sample. Therefore, this result can be applied to various fields, such as the detection of cells, proteins, and DNA for sensitive and specific biosensing based on PDA supramolecules. Copyright © 2010 Elsevier B.V. All rights reserved.

A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position, Movement, and Function of Woronin Bodies in Aspergillus oryzae

PubMed Central

Han, Pei; Jin, Feng Jie; Kitamoto, Katsuhiko

2014-01-01

The Woronin body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies. PMID:24813188

Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition.

PubMed

Shen, Donglai; Skibbens, Robert V

2017-01-01

Chl1 DNA helicase promotes sister chromatid cohesion and associates with both the cohesion establishment acetyltransferase Eco1/Ctf7 and the DNA polymerase processivity factor PCNA that supports Eco1/Ctf7 function. Mutation in CHL1 results in precocious sister chromatid separation and cell aneuploidy, defects that arise through reduced levels of chromatin-bound cohesins which normally tether together sister chromatids (trans tethering). Mutation of Chl1 family members (BACH1/BRIP/FANCJ and DDX11/ChlR1) also exhibit genotoxic sensitivities, consistent with a role for Chl1 in trans tethering which is required for efficient DNA repair. Chl1 promotes the recruitment of Scc2 to DNA which is required for cohesin deposition onto DNA. There is limited evidence, however, that Scc2 also directs the deposition onto DNA of condensins which promote tethering in cis (intramolecular DNA links). Here, we test the ability of Chl1 to promote cis tethering and the role of both Chl1 and Scc2 to promote condensin recruitment to DNA. The results reveal that chl1 mutant cells exhibit significant condensation defects both within the rDNA locus and genome-wide. Importantly, chl1 mutant cell condensation defects do not result from reduced chromatin binding of condensin, but instead through reduced chromatin binding of cohesin. We tested scc2-4 mutant cells and similarly found no evidence of reduced condensin recruitment to chromatin. Consistent with a role for Scc2 specifically in cohesin deposition, scc2-4 mutant cell condensation defects are irreversible. We thus term Chl1 a novel regulator of both chromatin condensation and sister chromatid cohesion through cohesin-based mechanisms. These results reveal an exciting interface between DNA structure and the highly conserved cohesin complex.

Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition

PubMed Central

Shen, Donglai

2017-01-01

Chl1 DNA helicase promotes sister chromatid cohesion and associates with both the cohesion establishment acetyltransferase Eco1/Ctf7 and the DNA polymerase processivity factor PCNA that supports Eco1/Ctf7 function. Mutation in CHL1 results in precocious sister chromatid separation and cell aneuploidy, defects that arise through reduced levels of chromatin-bound cohesins which normally tether together sister chromatids (trans tethering). Mutation of Chl1 family members (BACH1/BRIP/FANCJ and DDX11/ChlR1) also exhibit genotoxic sensitivities, consistent with a role for Chl1 in trans tethering which is required for efficient DNA repair. Chl1 promotes the recruitment of Scc2 to DNA which is required for cohesin deposition onto DNA. There is limited evidence, however, that Scc2 also directs the deposition onto DNA of condensins which promote tethering in cis (intramolecular DNA links). Here, we test the ability of Chl1 to promote cis tethering and the role of both Chl1 and Scc2 to promote condensin recruitment to DNA. The results reveal that chl1 mutant cells exhibit significant condensation defects both within the rDNA locus and genome-wide. Importantly, chl1 mutant cell condensation defects do not result from reduced chromatin binding of condensin, but instead through reduced chromatin binding of cohesin. We tested scc2-4 mutant cells and similarly found no evidence of reduced condensin recruitment to chromatin. Consistent with a role for Scc2 specifically in cohesin deposition, scc2-4 mutant cell condensation defects are irreversible. We thus term Chl1 a novel regulator of both chromatin condensation and sister chromatid cohesion through cohesin-based mechanisms. These results reveal an exciting interface between DNA structure and the highly conserved cohesin complex. PMID:29186203

AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

PubMed Central

Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

2013-01-01

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001 PMID:23482940

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

2016-10-01

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Cdon acts as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle

PubMed Central

Cardozo, Marcos Julián; Sánchez-Arrones, Luisa; Sandonis, África; Sánchez-Camacho, Cristina; Gestri, Gaia; Wilson, Stephen W.; Guerrero, Isabel; Bovolenta, Paola

2014-01-01

Patterning of the vertebrate optic vesicle into proximal/optic stalk and distal/neural retina involves midline-derived Hedgehog (Hh) signalling, which promotes stalk specification. In the absence of Hh signalling, the stalks are not specified, causing cyclopia. Recent studies showed that the cell adhesion molecule Cdon forms a heteromeric complex with the Hh receptor Patched 1 (Ptc1). This receptor complex binds Hh and enhances signalling activation, indicating that Cdon positively regulates the pathway. Here we show that in the developing zebrafish and chick optic vesicle, in which cdon and ptc1 are expressed with a complementary pattern, Cdon acts as a negative Hh signalling regulator. Cdon predominantly localizes to the basolateral side of neuroepithelial cells, promotes the enlargement of the neuroepithelial basal end-foot and traps Hh protein, thereby limiting its dispersion. This Ptc-independent function protects the retinal primordium from Hh activity, defines the stalk/retina boundary and thus the correct proximo-distal patterning of the eye. PMID:25001599

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

PubMed

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Tether Technology Interchange Meeting

NASA Technical Reports Server (NTRS)

Harrison, James K. (Compiler)

1998-01-01

This is a compilation of 25 papers presented at a tether technical interchange meeting in Huntsville, AL, on September 9-10, 1997. After each presentation, a technical discussion was held to clarify and expand the salient points. A wide range of subjects was covered including tether dynamics, electrodynamics, space power generation, plasma physics, ionospheric physics, towing tethers, tethered reentry schemes, and future tether missions.

Tethered satellite control mechanism

NASA Technical Reports Server (NTRS)

Kyrias, G. M.

1983-01-01

The tethered satellite control mechanisms consist of four major subsystems. The reel drive mechanism stores the tether. It is motor driven and includes a level wind to uniformly feed the tether to the reel. The lower boom mechanism serves two primary functions: (1) it measures tether length and velocity as the tether runs through the mechanism, and (2) it reads the tether tension at the reel. It also provides change the direction for the tether from the reel to the upper boom mechanism. The deployment boom positions the upper boom mechanism with satellite out of the cargo bay. The deployment function places the 500-kg satellite 20 m away from the Space Shuttle (producing a small natural gravity gradient force), impacts an initial velocity to the satellite for deployment, and allows for satellite docking at a safe distance from the body of the Space Shuttle. The upper boom mechanism (UBM) services three functions: (1) it provides tether control to the satellite as the satellite swings in and out of plane; (2) it reads tether tension in the low range during the early deployment and final retrieval parts of the mission; and (3) it produces additional tether tension at the reel when tether tension to the satellite is in the low range.

Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

PubMed

Simionescu, N; Lupu, F; Simionescu, M

1983-11-01

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.

Dynactin functions as both a dynamic tether and brake during dynein-driven motility

NASA Astrophysics Data System (ADS)

Ayloo, Swathi; Lazarus, Jacob E.; Dodda, Aditya; Tokito, Mariko; Ostap, E. Michael; Holzbaur, Erika L. F.

2014-09-01

Dynactin is an essential cofactor for most cellular functions of the microtubule motor cytoplasmic dynein, but the mechanism by which dynactin activates dynein remains unclear. Here we use single molecule approaches to investigate dynein regulation by the dynactin subunit p150Glued. We investigate the formation and motility of a dynein-p150Glued co-complex using dual-colour total internal reflection fluorescence microscopy. p150Glued recruits and tethers dynein to the microtubule in a concentration-dependent manner. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of p150Glued decreases the detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

Brownian motion of tethered nanowires.

PubMed

Ota, Sadao; Li, Tongcang; Li, Yimin; Ye, Ziliang; Labno, Anna; Yin, Xiaobo; Alam, Mohammad-Reza; Zhang, Xiang

2014-05-01

Brownian motion of slender particles near a boundary is ubiquitous in biological systems and in nanomaterial assembly, but the complex hydrodynamic interaction in those systems is still poorly understood. Here, we report experimental and computational studies of the Brownian motion of silicon nanowires tethered on a substrate. An optical interference method enabled direct observation of microscopic rotations of the slender bodies in three dimensions with high angular and temporal resolutions. This quantitative observation revealed anisotropic and angle-dependent hydrodynamic wall effects: rotational diffusivity in inclined and azimuth directions follows different power laws as a function of the length, ∼ L(-2.5) and ∼ L(-3), respectively, and is more hindered for smaller inclined angles. In parallel, we developed an implicit simulation technique that takes the complex wire-wall hydrodynamic interactions into account efficiently, the result of which agreed well with the experimentally observed angle-dependent diffusion. The demonstrated techniques provide a platform for studying the microrheology of soft condensed matters, such as colloidal and biological systems near interfaces, and exploring the optimal self-assembly conditions of nanostructures.

Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oglęcka, Kamila; Rangamani, Padmini; Liedberg, Bo

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressuremore » and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment.« less

Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials

DOE PAGES

Oglęcka, Kamila; Rangamani, Padmini; Liedberg, Bo; ...

2014-10-15

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressuremore » and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment.« less

Tethered Satellites as Enabling Platforms for an Operational Space Weather Monitoring System

NASA Technical Reports Server (NTRS)

Krause, L. Habash; Gilchrist, B. E.; Bilen, S.; Owens, J.; Voronka, N.; Furhop, K.

2013-01-01

Space weather nowcasting and forecasting models require assimilation of near-real time (NRT) space environment data to improve the precision and accuracy of operational products. Typically, these models begin with a climatological model to provide "most probable distributions" of environmental parameters as a function of time and space. The process of NRT data assimilation gently pulls the climate model closer toward the observed state (e.g. via Kalman smoothing) for nowcasting, and forecasting is achieved through a set of iterative physics-based forward-prediction calculations. The issue of required space weather observatories to meet the spatial and temporal requirements of these models is a complex one, and we do not address that with this poster. Instead, we present some examples of how tethered satellites can be used to address the shortfalls in our ability to measure critical environmental parameters necessary to drive these space weather models. Examples include very long baseline electric field measurements, magnetized ionospheric conductivity measurements, and the ability to separate temporal from spatial irregularities in environmental parameters. Tethered satellite functional requirements will be presented for each space weather parameter considered in this study.

System engineering study of electrodynamic tether as a spaceborne generator and radiator of electromagnetic waves in the ULF/ELF frequency band

NASA Technical Reports Server (NTRS)

Estes, Robert D.

1987-01-01

An electrodynamic tether deployed from a satellite in low-Earth orbit can perform, if properly instrumented, as a partially self-powered generator of electromagnetic waves in the ULF/ELF band, potentially at power levels high enough to be of practical use. Two basic problems are examined. The first is that of the level of wave power that the system can be expected to generate in the ULF/ELF radiation band. The second major question is whether an electrodynamic tethered satellite system for transmitting waves can be made partially self-powering so that power requirements for drag compensation can be met within economical constraints of mass, cost, and complexity. The theoretical developments and the system applications study are presented. The basic design criteria, the drag-compensation method, the effects on the propagation paths from orbit to Earth surface of high-altitude nuclear debris patches, and the estimate of masses and sizes are covered. An outline of recommended analytical work, to be performed as a follow-on to the present study, is contained.

Analysis of thermionic bare tether operation regimes in passive mode

NASA Astrophysics Data System (ADS)

Sanmartín, J. R.; Chen, Xin; Sánchez-Arriaga, G.

2017-01-01

A thermionic bare tether (TBT) is a long conductor coated with a low work-function material. In drag mode, a tether segment extending from anodic end A to a zero-bias point B, with the standard Orbital-motion-limited current collection, is followed by a complex cathodic segment. In general, as bias becomes more negative in moving from B to cathodic end C, one first finds space-charge-limited (SCL) emission covering up to some intermediate point B*, then full Richardson-Dushman (RD) emission reaching from B* to end C. An approximate analytical study, which combines the current and voltage profile equations with results from asymptotic studies of the Vlasov-Poisson system for emissive probes, is carried out to determine the parameter domain covering two limit regimes, which are effectively controlled by just two dimensionless parameters involving ambient plasma and TBT material properties. In one such limit regime, no point B* is reached and thus no full RD emission develops. In an opposite regime, SCL segment BB* is too short to contribute significantly to the current balance.

Sensory-Neuropathy-Causing Mutations in ATL3 Cause Aberrant ER Membrane Tethering.

PubMed

Krols, Michiel; Detry, Sammy; Asselbergh, Bob; Almeida-Souza, Leonardo; Kremer, Anna; Lippens, Saskia; De Rycke, Riet; De Winter, Vicky; Müller, Franz-Josef; Kurth, Ingo; McMahon, Harvey T; Savvides, Savvas N; Timmerman, Vincent; Janssens, Sophie

2018-05-15

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Space Tethers Design Criteria

NASA Technical Reports Server (NTRS)

Tomlin, Donald D.; Faile, Gwyn C.; Hayashida, Kazuo B.; Frost, Cynthia L.; Wagner, Carole Y.; Mitchell, Michael L.; Vaughn, Jason A.; Galuska, Michael J.

1998-01-01

The small expendable deployable system and tether satellite system programs did not have a uniform written criteria for tethers. The JSC safety panel asked what criteria was used to design the tethers. Since none existed, a criteria was written based on past experience for future tether programs.

Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells

PubMed Central

Pujol, Remy; Cunningham, Dale E.; Hailey, Dale W.; Prendergast, Andrew; Rubel, Edwin W.; Raible, David W.

2016-01-01

ABSTRACT Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. PMID:27103160

Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

PubMed

Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

2016-06-01

Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. © 2016. Published by The Company of Biologists Ltd.

Selected tether applications in space: An analysis of five selected concepts

NASA Technical Reports Server (NTRS)

1984-01-01

Ground rules and assumptions; operations; orbit considerations/dynamics; tether system design and dynamics; functional requirements; hardware concepts; and safety factors are examined for five scenarios: tethered effected separation of an Earth bound shuttle from the space station; tether effected orbit boost of a spacecraft (AXAF) into its operational orbit from the shuttle; an operational science/technology platform tether deployed from space station; a tether mediated rendezvous involving an OMV tether deployed from space station to rendezvous with an aerobraked OTV returning to geosynchronous orbit from a payload delivery mission; and an electrodynamic tether used in a dual motor/generator mode to serve as the primary energy storage facility for space station.

Mechanics and stability of vesicles and droplets in confined spaces

PubMed Central

Benet, Eduard; Vernerey, Franck J.

2017-01-01

The permeation and trapping of soft colloidal particles in the confined space of porous media are of critical importance in cell migration studies, design of drug delivery vehicles, and colloid separation devices. Our current understanding of these processes is however limited by the lack of quantitative models that can relate how the elasticity, size, and adhesion properties of the vesicle-pore complex affect colloid transport. We address this shortcoming by introducing a semianalytical model that predicts the equilibrium shapes of a soft vesicle driven by pressure in a narrow pore. Using this approach, the problem is recast in terms of pressure and energy diagrams that characterize the vesicle stability and permeation pressures in different conditions. We particularly show that the critical permeation pressure for a vesicle arises from a compromise between the critical entry pressure and exit pressure, both of which are sensitive to geometrical features, mechanics, and adhesion. We further find that these results can be leveraged to rationally design microfluidic devices and diodes that can help characterize, select, and separate colloids based on physical properties. PMID:28085314

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

PubMed Central

Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

2014-01-01

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

Analytical investigation of the dynamics of tethered constellations in Earth orbit, phase 2

NASA Technical Reports Server (NTRS)

Lorenzini, Enrico C.; Gullahorn, Gordon E.; Cosmo, Mario L.; Estes, Robert D.; Grossi, Mario D.

1994-01-01

This final report covers nine years of research on future tether applications and on the actual flights of the Small Expendable Deployment System (SEDS). Topics covered include: (1) a description of numerical codes used to simulate the orbital and attitude dynamics of tethered systems during station keeping and deployment maneuvers; (2) a comparison of various tethered system simulators; (3) dynamics analysis, conceptual design, potential applications and propagation of disturbances and isolation from noise of a variable gravity/microgravity laboratory tethered to the Space Station; (4) stability of a tethered space centrifuge; (5) various proposed two-dimensional tethered structures for low Earth orbit for use as planar array antennas; (6) tethered high gain antennas; (7) numerical calculation of the electromagnetic wave field on the Earth's surface on an electrodynamically tethered satellite; (8) reentry of tethered capsules; (9) deployment dynamics of SEDS-1; (10) analysis of SEDS-1 flight data; and (11) dynamics and control of SEDS-2.

Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs Glycymeris sp. (Pteriomorphia) and Eurhomalea rufa (Heterodonta)

NASA Astrophysics Data System (ADS)

Guerra, Rosa; Sousa, Mário; Torres, Artur; Oliveira, Elsa; Baldaia, Luis

2003-03-01

Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30-40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae.

A Role for an Hsp70 Nucleotide Exchange Factor in the Regulation of Synaptic Vesicle Endocytosis

PubMed Central

Morgan, Jennifer R.; Jiang, Jianwen; Oliphint, Paul A.; Jin, Suping; Gimenez, Luis E.; Busch, David J.; Foldes, Andrea E.; Zhuo, Yue; Sousa, Rui; Lafer, Eileen M.

2013-01-01

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. Following the internalization of a clathrin coated vesicle (CCV), the vesicle must uncoat to replenish the pool of SVs. CCV uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo, and whether they play such roles in SV endocytosis in particular is unknown. To address this question we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:cochaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so it could no longer bind and inhibit Hsp110--a NEF that we find to be highly abundant in brain cytosol--its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis. PMID:23637191

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

PubMed

Mossberg, Ann-Kristin; Puchades, Maja; Halskau, Øyvind; Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

2010-02-23

Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

Visco-Elastic Membrane Tethers Extracted from Escherichia coli by Optical Tweezers

PubMed Central

Jauffred, Liselotte; Callisen, Thomas Hønger; Oddershede, Lene Broeng

2007-01-01

Tethers were created between a living Escherichia coli bacterium and a bead by unspecifically attaching the bead to the outer membrane and pulling it away using optical tweezers. Upon release, the bead returned to the bacterium, thus showing the existence of an elastic tether between the bead and the bacterium. These tethers can be tens of microns long, several times the bacterial length. Using mutants expressing different parts of the outer membrane structure, we have shown that an intact core lipopolysaccharide is a necessary condition for tether formation, regardless of whether the beads were uncoated polystyrene or beads coated with lectin. A physical characterization of the tethers has been performed yielding visco-elastic tether force-extension relationships: for first pull tethers, a spring constant of 10–12 pN/μm describes the tether visco-elasticity, for subsequent pulls the spring constant decreases to 6–7 pN/μm, and typical relaxation timescales of hundreds of seconds are observed. Studies of tether stability in the presence of proteases, lipases, and amylases lead us to propose that the extracted tether is primarily composed of the asymmetric lipopolysaccharide containing bilayer of the outer membrane. This unspecific tethered attachment mechanism could be important in the initiation of bacterial adhesion. PMID:17704145

The dynamic phenomena of a tethered satellite: NASA's first Tethered Satellite Mission, TSS-1

NASA Technical Reports Server (NTRS)

Ryan, R. S.; Mowery, D. K.; Tomlin, D. D.

1993-01-01

The tethered satellite system (TSS) was envisioned as a means of extending a satellite from its base (space shuttle, space station, space platform) into a lower or higher altitude in order to more efficiently acquire data and perform science experiments. This is accomplished by attaching the satellite to a tether, deploying it, then reeling it in. When its mission is completed, the satellite can be returned to its base for reuse. If the tether contains a conductor, it can also be used as a means to generate and flow current to and from the satellite to the base. When current is flowed, the tether interacts with the Earth's magnetic field, deflecting the tether. When the current flows in one direction, the system becomes a propulsive system that can be used to boost the orbiting system. In the other direction, it is a power generating system. Pulsing the current sets up a dynamic oscillation in the tether, which can upset the satellite attitude and preclude docking. A basic problem occurs around 400-m tether length, during satellite retrieval when the satellite's pendulous (rotational) mode gets in resonance with the first lateral tether string mode. The problem's magnitude is determined by the amount of skiprope present coming into this resonance condition. This paper deals with the tethered satellite, its dynamic phenomena, and how the resulting problems were solved for the first tethered satellite mission (TSS-1). Proposals for improvements for future tethered satellite missions are included. Results from the first tethered satellite flight are summarized.

Disruption of the Abdominal-B Promoter Tethering Element Results in a Loss of Long-Range Enhancer-Directed Hox Gene Expression in Drosophila

PubMed Central

Ho, Margaret C. W.; Schiller, Benjamin J.; Akbari, Omar S.; Bae, Esther; Drewell, Robert A.

2011-01-01

There are many examples within gene complexes of transcriptional enhancers interacting with only a subset of target promoters. A number of molecular mechanisms including promoter competition, insulators and chromatin looping are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C), the IAB5 enhancer specifically drives gene expression only from the Abdominal-B (Abd-B) promoter, even though the enhancer and promoter are 55 kb apart and are separated by at least three insulators. In previous studies, we discovered that a 255 bp cis-regulatory module, the promoter tethering element (PTE), located 5′ of the Abd-B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. In this study we examine the functional role of the PTE at the endogenous BX-C using transposon-mediated mutagenesis. Disruption of the PTE by P element insertion results in a loss of enhancer-directed Abd-B expression during embryonic development and a homeotic transformation of abdominal segments. A partial deletion of the PTE and neighboring upstream genomic sequences by imprecise excision of the P element also results in a similar loss of Abd-B expression in embryos. These results demonstrate that the PTE is an essential component of the regulatory network at the BX-C and is required in vivo to mediate specific long-range enhancer-promoter interactions. PMID:21283702

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

Turnip mosaic virus Moves Systemically through Both Phloem and Xylem as Membrane-Associated Complexes1

PubMed Central

Zheng, Huanquan

2015-01-01

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K2, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K2-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem. PMID:25717035

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Daohong; Chen, Xi; Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003

Highlights: ► CCCP-induced LC3 lipidation can be independent of initiation/nucleation molecules. ► Atg9-mediated trafficking is critically required for CCCP-induced LC3 lipidation. ► CCCP-induced mitophagy may thus be more dependent on Atg9-positive vesicles. -- Abstract: Treatment of cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial proton gradient uncoupler, can result in mitochondrial damage and autophagy activation, which in turn eliminates the injured mitochondria in a Parkin-dependent way. How CCCP mobilizes the autophagy machinery is not fully understood. By analyzing a key autophagy step, LC3 lipidation, we examined the roles of two kinase complexes typically involved in the initiation and nucleation phasesmore » of autophagy, namely the ULK kinase complex (UKC) and the Beclin 1/Atg14 complex. We found that CCCP-induced LC3 lipidation could be independent of Beclin 1 and Atg14. In addition, deletion or knockdown of the UKC component FIP200 or Atg13 only led to a partial reduction in LC3 lipidation, indicating that UKC could be also dispensable for this step during CCCP treatment. In contrast, Atg9, which is important for transporting vesicles to early autophagosomal structure, was required for CCCP-induced LC3 lipidation. Taken together, these data suggest that CCCP-induced autophagy and mitophagy depends more critically on Atg9 vesicles than on UKC and Beclin 1/Atg14 complex.« less

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

PubMed

Yang, Yoosoo; Kong, Byoungjae; Jung, Younghoon; Park, Joon-Bum; Oh, Jung-Mi; Hwang, Jaesung; Cho, Jae Youl; Kweon, Dae-Hyuk

2018-01-01

Vesicle-associated V-soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans -SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Niosomes encapsulating Ibuprofen-cyclodextrin complexes: preparation and characterization.

PubMed

Marianecci, Carlotta; Rinaldi, Federica; Esposito, Sara; Di Marzio, Luisa; Carafa, Maria

2013-08-01

A new delivery system based on ibuprofen-β-cyclodextrin (βCd) complexation and its loading into non-ionic surfactant vesicles (NSVs) was developed to improve ibuprofen therapeutic efficacy in topical formulations. The proposed strategy exploits the well known solubilizing and stabilizing properties of cyclodextrins together with the high tolerability and percutaneous absorption enhancing properties of NSVs. The complexing capacity of Cds in the presence of Ibuprofen in aqueous solution was evaluated by means of phase solubility studies. The technique used to obtain solid ibuprofen-βCd complexes was the co-lyophilization method. The influence of the preparation method on the physicochemical properties of the final product was evaluated by means of Fourier Transform Infrared Spectroscopy and Differential scanning calorimetry studies. Ibuprofen-βCd complexes were included in Tween 20/Cholesterol vesicles and characterized in terms of size, zeta (ζ)-potential, stability, drug entrapment efficiency and drug release. The best ibuprofen-βCd-NSV system exhibited in vitro drug permeation properties significantly improved with respect to those of the plain drug suspension.

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

PubMed Central

Schluter, Cayetana; Lam, Karen K.Y.; Brumm, Jochen; Wu, Bella W.; Saunders, Matthew; Stevens, Tom H.

2008-01-01

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. PMID:18216282

The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle.

PubMed

Rawat, Anamika; Brejšková, Lucie; Hála, Michal; Cvrčková, Fatima; Žárský, Viktor

2017-10-01

The exocyst, an evolutionarily conserved secretory vesicle-tethering complex, spatially controls exocytosis and membrane turnover in fungi, metazoans and plants. The exocyst subunit EXO70 exists in multiple paralogs in land plants, forming three conserved clades with assumed distinct roles. Here we report functional analysis of the first moss exocyst subunit to be studied, Physcomitrella patens PpEXO70.3d (Pp1s97_91V6), from the, as yet, poorly characterized EXO70.3 clade. Following phylogenetic analysis to confirm the presence of three ancestral land plant EXO70 clades outside angiosperms, we prepared and phenotypically characterized loss-of-function Ppexo70.3d mutants and localized PpEXO70.3d in vivo using green fluorescent protein-tagged protein expression. Disruption of PpEXO70.3d caused pleiotropic cell elongation and differentiation defects in protonemata, altered response towards exogenous auxin, increased endogenous IAA concentrations, along with defects in bud and gametophore development. During mid-archegonia development, an abnormal egg cell is formed and subsequently collapses, resulting in mutant sterility. Mutants exhibited altered cell wall and cuticle deposition, as well as compromised cytokinesis, consistent with the protein localization to the cell plate. Despite some functional redundancy allowing survival of moss lacking PpEXO70.3d, this subunit has an essential role in the moss life cycle, indicating sub-functionalization within the moss EXO70 family. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

Tether fundamentals

NASA Technical Reports Server (NTRS)

Carroll, J. A.

1986-01-01

Some fundamental aspects of tethers are presented and briefly discussed. The effects of gravity gradients, dumbbell libration in circular orbits, tether control strategies and impact hazards for tethers are among those fundamentals. Also considered are aerodynamic drag, constraints in momentum transfer applications and constraints with permanently deployed tethers. The theoretical feasibility of these concepts are reviewed.

Atmospheric tether mission analyses

NASA Technical Reports Server (NTRS)

1996-01-01

NASA is considering the use of tethered satellites to explore regions of the atmosphere inaccessible to spacecraft or high altitude research balloons. This report summarizes the Lockheed Martin Astronautics (LMA) effort for the engineering study team assessment of an Orbiter-based atmospheric tether mission. Lockheed Martin responsibilities included design recommendations for the deployer and tether, as well as tether dynamic analyses for the mission. Three tether configurations were studied including single line, multistrand (Hoytether) and tape designs.

Tethered body problems and relative motion orbit determination

NASA Technical Reports Server (NTRS)

Eades, J. B., Jr.; Wolf, H.

1972-01-01

Selected problems dealing with orbiting tethered body systems have been studied. In addition, a relative motion orbit determination program was developed. Results from these tasks are described and discussed. The expected tethered body motions were examined, analytically, to ascertain what influence would be played by the physical parameters of the tether, the gravity gradient and orbit eccentricity. After separating the motion modes these influences were determined; and, subsequently, the effects of oscillations and/or rotations, on tether force, were described. A study was undertaken, by examining tether motions, to see what type of control actions would be needed to accurately place a mass particle at a prescribed position relative to a main vehicle. Other applications for tethers were studied. Principally these were concerned with the producing of low-level gee forces by means of stabilized tether configurations; and, the initiation of free transfer trajectories from tether supported vehicle relative positions.

Precision tethered satellite attitude control. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Kline-Schoder, Robert J.

1990-01-01

Tethered spacecraft possess unique dynamic characteristics which make them advantageous for certain classes of experiments. One use for which tethers are particularly well suited is to provide an isolated platform for spaceborne observatories. The advantages of tethering a pointing platform 1 or 2 km from a space shuttle or space station are that, compared to placing the observatory on the parent spacecraft, vibrational disturbances are attenuated and contamination is eliminated. In practice, all satellites have some requirement on the attitude control of the spacecraft, and tethered satellites are no exception. It has previously been shown that conventional means of performing attitude control for tethered satellites are insufficient for any mission with pointing requirements more stringent than about 1 deg. This is due mainly to the relatively large force applied by the tether to the spacecraft. A particularly effective method of implementing attitude control for tethered satellites is to use this tether tension force to generate control torques by moving the tether attach point relative to the subsatellite center of mass. A demonstration of this attitude control technique on an astrophysical pointing platform has been proposed for a space shuttle flight test project and is referred to as the Kinetic Isolation Tether Experiment (KITE).

Dynamics and offset control of tethered space-tug system

NASA Astrophysics Data System (ADS)

Zhang, Jingrui; Yang, Keying; Qi, Rui

2018-01-01

Tethered space-tug system is regarded as one of the most promising active debris removal technologies to effectively decrease the steep increasing population of space debris. In order to suppress the spin of space debris, single-tethered space-tug system is employed by regulating the tether. Unfortunately, this system is underactuated as tether length is the only input, and there are two control objectives: the spinning debris and the vibration of tether. Thus, it may suffer great oscillations and result in failure in space debris removal. This paper presents the study of attitude stabilization of the single-tethered space-tug system using not only tether length but also the offset of tether attachment point to suppress the spin of debris, so as to accomplish the space debris removal mission. Firstly, a precise 3D mathematical model in which the debris and tug are both treated as rigid bodies is developed to study the dynamical evolution of the tethered space-tug system. The relative motion equation of the system is described using Lagrange method. Secondly, the dynamic characteristic of the system is analyzed and an offset control law is designed to stabilize the spin of debris by exploiting the variation of tether offset and the regulation of tether length. Besides, an estimation formula is proposed to evaluate the capability of tether for suppressing spinning debris. Finally, the effectiveness of attitude stabilization by the utilization of the proposed scheme is demonstrated via numerical case studies.

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

PubMed Central

Ran, Yong; Fanucci, Gail E.

2009-01-01

Abstract The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles. PMID:19580763

Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.

PubMed

Ran, Yong; Fanucci, Gail E

2009-07-08

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.

Flow around a tethered cylinder, the effect of tether length at high layover angles

NASA Astrophysics Data System (ADS)

Ryan, Kris

2011-07-01

Tethered cylinder systems constitute a natural extension of the lightly damped, hydro-elastically mounted cylinder. In this case, the cylinder is constrained to travel along an arc prescribed by the tether length. The analysis of the tethered cylinder system is hampered by the dependence of the natural frequency of the system on both the fluid forces acting on the system and the curved motion (which in turn alters the added mass coefficient away from unity). These difficulties have precluded prior studies considering the natural frequency or reduced velocity as a controlling parameter, making direct comparison with the hydro-elastically mounted cylinder system difficult.This investigation considers the case of a tethered cylinder at low Reynolds number (Re=200) for a mass ratio m*=0.2. It notes a local maximum in the amplitude of oscillation when the normalized tether length L*≃2.0, in agreement with prior studies. By instead considering the amplitude of oscillation in a rotational framework, we are able to explain the existence of this peak, and identify two regions of amplitude response, the first region exists for very small tether lengths (L*≲0.3), while the second exists for larger tether lengths. The transition from small tether lengths to large tether lengths exhibits the highest amplitude angular oscillations.Several wake states are also considered for a tethered cylinder which is oscillating about a horizontal mean layover angle. By considering these wake states, coupled with the definition of the natural frequency, an estimate of the added mass coefficient is made. Here we predict that CA≃0.5 for a tether length of L*=1.5. This prediction is based not only on the tether length, but also on the amplitude of oscillation, and hence is Reynolds number dependent.

Configuration maintaining control of three-body ring tethered system based on thrust compensation

NASA Astrophysics Data System (ADS)

Huang, Panfeng; Liu, Binbin; Zhang, Fan

2016-06-01

Space multi-tethered systems have shown broad prospects in remote observation missions. This paper mainly focuses on the dynamics and configuration maintaining control of space spinning three-body ring tethered system for such mission. Firstly, we establish the spinning dynamic model of the three-body ring tethered system considering the elasticity of the tether using Newton-Euler method, and then validate the suitability of this model by numerical simulation. Subsequently, LP (Likins-Pringle) initial equilibrium conditions for the tethered system are derived based on rigid body's equilibrium theory. Simulation results show that tether slack, snapping and interaction between the tethers exist in the three-body ring system, and its' configuration can not be maintained without control. Finally, a control strategy based on thrust compensation, namely thrust to simulate tether compression under LP initial equilibrium conditions is designed to solve the configuration maintaining control problem. Control effects are verified by numerical simulation compared with uncontrolled situation. Simulation results show that the configuration of the three-body ring tethered system could maintain under this active control strategy.

The role of microvesicles in malignancies.

PubMed

Pap, Erna

2011-01-01

Microvesicles are membrane-covered cell fragments whose size varies between 30 and 1,000 nm. They are generated by all cell types, constituvely and in response to activation signals. Their importance in intercellular communication has been only recently discovered. They seem to enhance the potential of information transfer between cells, displaying a large number of proteins and lipids as membrane constituents and as components of the inner vesicular content. The content reflects the phenotype of the donor cell and allows the identification of the microvesicular origine as well. Complex "packets" of molecules are transmitted to the target cells this way, modifying their cellular physiology. Additionally, epigenetic changes may be induced by transmitted DNA and RNAs, that have also been identified in these vesicles. The vesicles can act in close and far distances as well. Microvesicles have been implicated in several physiological and pathological processes. There is an increasing evidence, that they play a pivotal role in tumorigenesis. Vesicles shedding from tumor cells reflect the special potential of the tumor for survival and expansion, independently from cell-to-cell contact. Tumor derived vesicles are fully equipped to facilitate the escape of tumor cells from immune surveillance through their protein and RNA content, at the same time they are involved in the establishment of an optimal environment for newly formed and metastatic tumor cells, influencing angiogenesis and the reorganization of the extracellular matrix. As immune cells, endothels, platelets and stem cells also release microvesicles, a multilevel communication network draws up, allowing a complex interplay between the cells. The concentration of tumor derived vesicles increases in blood plasma and other body fluids with the progression of the disease; therefor they may serve as prognostic markers. The microvesicular approach can offer new perspectives: interfering with the formation, release and propagation of these vesicles, they can be considered as new targets in tumor therapy.

Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly.

PubMed

Munson, M; Chen, X; Cocina, A E; Schultz, S M; Hughson, F M

2000-10-01

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.

System protection from atmospheric electricity for aerostats with conducting tethers

NASA Astrophysics Data System (ADS)

Wheeler, M. S.; Beach, G. R.; Jakubowski, P. R.; Fisher, F. A.

1988-04-01

Aerostat power tethers have demonstrated survival of lightning strikes, but they usually have to be reterminated or replaced afterward. Two requirements are given for the prevention of lightning damage to the tether to about 100 kA: installation of a metal-to-metal contact on the outer tether surface to ground the tether at the base flying sheave at typical flying positions; and installation of a shielding band within the outer tether jacket with a weight of about 0.05 lb/ft for a half-inch tether. This determination was made in part by high current tests and in part by electrical modeling.

Energy Transduction Inside of Amphiphilic Vesicles: Encapsulation of Photochemically Active Semiconducting Particles

NASA Astrophysics Data System (ADS)

Summers, David P.; Noveron, Juan; Basa, Ranor C. B.

2009-04-01

Amphiphilic bilayer membrane structures (vesicles) have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth, providing compartmentalization for the origin of life. These vesicles are similar to modern cellular membranes and can serve to contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy in metabolism (i.e. energy transduction) is one of the central issues in the origin of life. This includes such questions as how energy transduction may have occurred before complex enzymatic systems, such as required by contemporary photosynthesis, had developed and how simple a photochemical system is possible. It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has also been shown that pH gradients across the membrane surface can be photochemically created, but coupling these to drive chemical reactions has been difficult. Colloidal semiconducting mineral particles are known to photochemically drive redox chemistry. We propose that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry, and represents a model system for early photosynthesis. In our experiments we show that TiO2 particles, in the ~20 nm size range, can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to concentrate species inside a vesicle.

Dual photo- and pH-responsive supramolecular nanocarriers based on water-soluble pillar[6]arene and different azobenzene derivatives for intracellular anticancer drug delivery.

PubMed

Hu, Xiao-Yu; Jia, Keke; Cao, Yu; Li, Yan; Qin, Shan; Zhou, Fan; Lin, Chen; Zhang, Dongmei; Wang, Leyong

2015-01-12

Two novel types of supramolecular nanocarriers fabricated by the amphiphilic host-guest inclusion complex formed from water-soluble pillar[6]arene (WP6) and azobenzene derivatives G1 or G2 have been developed, in which G1 is structurally similar to G2 but has an extra phenoxy group in its hydrophobic region. Supramolecular micelles can be initially formed by WP6 with G1, which gradually transform into layered structures with liquid-crystalline properties, whereas stable supramolecular vesicles are obtained from WP6 and G2, which exhibit dual photo- and pH-responsiveness. Notably, the resulting WP6⊃G2 vesicles can efficiently encapsulate anticancer drug mitoxantrone (MTZ) to achieve MTZ-loaded vesicles, which maintain good stability in a simulated normal physiological environment, whereas in an acid environment similar to that of tumor cells or with external UV irradiation, the encapsulated drug is promptly released. More importantly, cytotoxicity assay indicates that such vesicles have good biocompatibility and the MTZ-loaded vesicles exhibit comparable anticancer activity to free MTZ, especially with additional UV stimulus, whereas its cytotoxicity for normal cells was remarkably reduced. Flow cytometric analysis further confirms that the cancer cell death caused by MTZ-loaded vesicles is associated with apoptosis. Therefore, the dual pH- and UV-responsive supramolecular vesicles are a potential platform for controlled release and targeted anticancer drug delivery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Energy transduction inside of amphiphilic vesicles: encapsulation of photochemically active semiconducting particles.

PubMed

Summers, David P; Noveron, Juan; Basa, Ranor C B

2009-04-01

Amphiphilic bilayer membrane structures (vesicles) have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth, providing compartmentalization for the origin of life. These vesicles are similar to modern cellular membranes and can serve to contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy in metabolism (i.e. energy transduction) is one of the central issues in the origin of life. This includes such questions as how energy transduction may have occurred before complex enzymatic systems, such as required by contemporary photosynthesis, had developed and how simple a photochemical system is possible. It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has also been shown that pH gradients across the membrane surface can be photochemically created, but coupling these to drive chemical reactions has been difficult. Colloidal semiconducting mineral particles are known to photochemically drive redox chemistry. We propose that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry, and represents a model system for early photosynthesis. In our experiments we show that TiO2 particles, in the approximately 20 nm size range, can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to concentrate species inside a vesicle.

Shuttle/tethered satellite system conceptual design study

NASA Technical Reports Server (NTRS)

1976-01-01

A closed-loop control system was added to the tether reel which improves control over the tethered satellite. In addition to increasing the stability of the tethered satellite along local vertical, this control system is used for deployment and retrieval of tethered satellites. This conceptual design study describes a tether system for suspending a science payload at an altitude of 120 km from space shuttle orbiter flying at an altitude of 200 km. In addition to the hardware conceptual designs, various aspects concerning Orbiter accommodations are discussed.

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

PubMed

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Proceedings of a Workshop on Applications of Tethers in Space, Executive Summary

NASA Technical Reports Server (NTRS)

1983-01-01

The objectives were to identify potential applications for tethers in space; develop a first order assessment of the feasibility and benefits of tether applications; recommend future actions necessary to enable tether applications, including required technology advancements; and stimulate industry and government planners to consider the unique properties of tethers in designs for future missions.

Insulin-regulated Glut4 Translocation

PubMed Central

Brewer, Paul Duffield; Habtemichael, Estifanos N.; Romenskaia, Irina; Mastick, Cynthia Corley; Coster, Adelle C. F.

2014-01-01

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min−1 versus 0.6 min−1). Differentiation decreases Glut4 ken 40% (ken = 0.12 min−1). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025–0.038 min−1), not through the fast Tf receptor pathway (kex = 0.2 min−1). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min−1). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs. PMID:24778187

Characterization of a Biomimetic Polymeric-Lipid Bilayer by Phase Sensitive Neutron Reflectivity

NASA Astrophysics Data System (ADS)

Perez-Salas, Ursula A.; Krueger, Susan; Majkrzak, Charles F.; Berk, Norman F.; Faucher, Keith M.; Chaikof, Elliot L.

2003-03-01

Lipid membranes, the boundaries for cellular and intracellular structures, regulate many crucial biological processes. Planar supported mimics of cell membranes are of great interest as model systems for the study of membrane structure/function phenomena in fundamental biophysics research. We studied a supported biomedically relevant membrane-mimetic system composed of a polyelectrolyte cushion, a terpolymer and a self-assembled phospholipid monolayer and obtained a detailed profile characterization of the system by neutron reflectometry. The water-swellable hydrophilic polyelectrolyte acts as a support for the biomembrane, not unlike the cytoskeletal support found in actual mammalian cell membranes. The "cushion" polymers are fixed to the flat, hard surface by having the polymer interact with it electrostatically. The terpolymer has the following desirable features: it tethers to the polyelectrolyte layer and it creates a hydrophilic and a hydrophobic region. Unilamellar phospholipid vesicle fusion on to the hydrophobic region of the terpolymer creates the hybrid tethered membrane. For added stability to external force fields (such as shear flow), the phospholipid monolayer is then polymerized in situ, effectively anchoring the lipid layer to the hydrophobic region of the terpolymer. Neutron reflectivity measurements were done on the polyelectrolyte layer, the polyelectrolyte layer plus terpolymer and the polylectrolyte layer plus terpolymer plus phospholipid. The layers were studied dry and hydrated and under 95α D_2O and 50% \\ 50% α H_2O \\ α D_2O) on the polyelectrolyte layer plus terpolymer and the polylectrolyte layer plus terpolymer plus phospholipid the distribution of water in the layers was obtained. The results will be correlated to impedance measurements flourescence measurements and infrared spectroscopy measurements made on equivalent samples.

Formation of HDL-like complexes from apolipoprotein A-I(M) and DMPC.

PubMed

Suurkuusk, M; Singh, S K

2000-01-20

Conditions for the preparation of reconstituted high density lipoproteins (HDLs) by incubation of the synthetic lipid dimyristoylphosphatidylcholine (DMPC) and recombinant apolipoprotein A-I(M) have been investigated as a function of ratio of incubation lipid to protein, incubation temperature and the lipid form (multilamellar (MLV) or small unilamellar (SUV) vesicles). The size distributions of the resultant lipid-protein complex particles from various incubations have been evaluated by native gel electrophoresis. Structural changes of the protein after incorporation into these complex particles have been estimated by CD. Thermal characteristics of the particles has been examined by DSC and correlated with CD results. Titration calorimetry has been used to obtain interaction parameters based on a simplified binding model. It is hypothesized that the major enthalpic step in the production of rHDLs is the primary association step between protein and lipid vesicles. It has been shown that by raising the temperature and incubation ratio, the formation of rHDL particles can be directed towards smaller size and a narrower size distribution. The results have been described on the basis of a model where formation of discoidal particles requires prior saturation of vesicle surface area by adsorbed protein, thus explaining differences between particles formed from MLVs and SUVs.

Investigation of electrodynamic stabilization and control of long orbiting tethers

NASA Technical Reports Server (NTRS)

Colombo, G.; Arnold, D.

1984-01-01

The state-of-the-art in tether modelling among participants in the Tethered Satellite System (TSS) Program, the slack tether and its behavior, and certain advanced applications of the tether to problems in orbital mechanics are identified. The features and applications of the TSS software set are reviewed. Modelling the slack tether analytically with as many as 50 mass points and the application of this new model to a study of the behavior of a broken tether near the Shuttle are described. A reel control algorithm developed by SAO and examples of its use are described, including an example which also demonstrates the use of the tether in transferring a heavy payload from a low-orbiting Shuttle to a high circular orbit. Capture of a low-orbiting payload by a Space Station in high circular orbit is described. Energy transfer within a dumbbell-type spacecraft by cyclical reeling operations or gravitational effects on the natural elasticity of the connecting tether, it is shown, can circularize the orbit of the spacecraft.

Impact of mitral valve geometry on hemodynamic efficacy of surgical repair in secondary mitral regurgitation.

PubMed

Padala, Muralidhar; Gyoneva, Lazarina I; Thourani, Vinod H; Yoganathan, Ajit P

2014-01-01

Mitral valve geometry is significantly altered secondary to left ventricular remodeling in non-ischemic and ischemic dilated cardiomyopathies. Since the extent of remodeling and asymmetry of dilatation of the ventricle differ significantly between individual patients, the valve geometry and tethering also differ. The study aim was to determine if mitral valve geometry has an impact on the efficacy of surgical repairs to eliminate regurgitation and restore valve closure in a validated experimental model. Porcine mitral valves (n = 8) were studied in a pulsatile heart simulator, in which the mitral valve geometry can be precisely altered and controlled throughout the experiment. Baseline hemodynamics for each valve were measured (Control), and the valves were tethered in two distinct ways: annular dilatation with 7 mm apical papillary muscle (PM) displacement (Tether 1, symmetric), and annular dilatation with 7 mm apical, 7 mm posterior and 7 mm lateral PM displacement (Tether 2, asymmetric). Mitral annuloplasty was performed on each valve (Annular Repair), succeeded by anterior leaflet secondary chordal cutting (Sub-annular Repair). The efficacy of each repair in the setting of a given valve geometry was quantified by measuring the changes in mitral regurgitation (MR), leaflet coaptation length, tethering height and area. At baseline, none of the valves was regurgitant. Significant leaflet tethering was measured in Tether 2 over Tether 1, but both groups were significantly higher compared to baseline (60.9 +/- 31 mm2 for Control versus 129.7 +/- 28.4 mm2 for Tether 1 versus 186.4 +/- 36.3 mm2 for Tether 2). Consequently, the MR fraction was higher in Tether 2 group (23.0 +/- 5.7%) than in Tether 1 (10.5 +/- 5.5%). Mitral annuloplasty reduced MR in both groups, but remnant regurgitation after the repair was higher in Tether 2. After chordal cutting a similar trend was observed with trace regurgitation in Tether 1 group at 3.6 +/- 2.8%, in comparison to 18.6 +/- 4.2% in the Tether 2 group. In this experimental model, the tethering geometry of the mitral valve impacts the valve hemodynamics after annuloplasty and chordal cutting. The quantitative assessment of valve geometry may help in tailoring a repair to the specific tethering pattern.

Study of tethered satellite active attitude control

NASA Technical Reports Server (NTRS)

Colombo, G.

1982-01-01

Existing software was adapted for the study of tethered subsatellite rotational dynamics, an analytic solution for a stable configuration of a tethered subsatellite was developed, the analytic and numerical integrator (computer) solutions for this "test case' was compared in a two mass tether model program (DUMBEL), the existing multiple mass tether model (SKYHOOK) was modified to include subsatellite rotational dynamics, the analytic "test case,' was verified, and the use of the SKYHOOK rotational dynamics capability with a computer run showing the effect of a single off axis thruster on the behavior of the subsatellite was demonstrated. Subroutines for specific attitude control systems are developed and applied to the study of the behavior of the tethered subsatellite under realistic on orbit conditions. The effect of all tether "inputs,' including pendular oscillations, air drag, and electrodynamic interactions, on the dynamic behavior of the tether are included.

Space experiments on basic technologies for a space elevator using microsatellites

NASA Astrophysics Data System (ADS)

Yamagiwa, Yoshiki; Nohmi, Masahiro; Aoki, Yoshio; Momonoi, Yu; Nanba, Hirotaka; Aiga, Masanori; Kumao, Takeru; Watahiki, Masahito

2017-09-01

We attempt to verify two basic technologies required for a space elevator using microsatellites; the tether (cable) deployment technology and the climber operation along the tether in space. Tether deployment is performed by a CubeSat called STARS-C (Space Tethered Autonomous Robotic Satellite - Cube) which will be released from the Japanese experimental module Kibo on ISS early in 2017. STARS-C consists of a mother satellite (MS) and daughter satellite (DS) connected by a 100-m tether. Its mission is focused on the tether deployment for studying the tether dynamics during the deployment with the goal of improving the smoothness of such deployment in future tether missions including space elevator. The MS and DS have common subsystems, including power, communication, and command and data handling systems. They also have a tether unit with spool and reel mechanisms as a mission system. In addition, we have been designing the next-step microsatellite called STARS-E (Space Tethered Autonomous Robotic Satellite - Elevator) under a Grant-in-Aid for Scientific Research. STARS-E is a 500-mm size satellite intended to verify the climber operation in space. It consists of a MS and DS jointed by a 2-km tether, and a climber that moves along the tether. STARS-C was launched on December 9 in 2016 and will be performed its mission early in 2017. STARS-E is in the BBM phase, and some designs are currently being fixed.

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.

PubMed

Campoy, Irene; Lanau, Lucia; Altadill, Tatiana; Sequeiros, Tamara; Cabrera, Silvia; Cubo-Abert, Montserrat; Pérez-Benavente, Assumpción; Garcia, Angel; Borrós, Salvador; Santamaria, Anna; Ponce, Jordi; Matias-Guiu, Xavier; Reventós, Jaume; Gil-Moreno, Antonio; Rigau, Marina; Colas, Eva

2016-06-18

Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology.

A Novel Synaptic Vesicle Fusion Path in the Rat Cerebral Cortex: The “Saddle” Point Hypothesis

PubMed Central

Zampighi, Guido A.; Serrano, Raul; Vergara, Julio L.

2014-01-01

We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that “rods” assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We equated these “connector rods” to protein complexes involved in “docking” and “priming” vesicles to the active zone. Depending on their orientation, the “rods” define two synaptic vesicle-fusion paths: When parallel to the plasma membrane, the vesicles hemi-fuse anywhere (“randomly”) in the active zone following the conventional path anticipated by the SNARE hypothesis. When perpendicular to the plasma membrane, the vesicles hemi-fuse at the base of sharp crooks, called “indentations,” that are spaced 75–85 nm center-to-center, arranged in files and contained within gutters. They result from primary and secondary membrane curvatures that intersect at stationary inflection (“saddle”) points. Computer simulations indicate that this novel vesicle-fusion path evokes neurotransmitter concentration domains on the post-synaptic spine that are wider, shallower, and that reach higher average concentrations than the more conventional vesicle fusion path. In the post-synaptic spine, large (∼9× ∼15 nm) rectangular particles at densities of 72±10/ µm2 (170–240/spine) match the envelopes of the homotetrameric GluR2 AMPA-sensitive receptor. While these putative receptors join clusters, called the “post-synaptic domains,” the overwhelming majority of the rectangular particles formed bands in the “non-synaptic” plasma membrane of the spine. In conclusion, in the neuropil of the rat cerebral cortex, curvatures of the plasma membrane define a novel vesicle-fusion path that preconditions specific regions of the active zone for neurotransmitter release. We hypothesize that a change in the hybridization of the R-SNARE synaptobrevin from parallel to antiparallel swings the synapse into this novel vesicle-fusion path. PMID:24959848

Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles.

PubMed

Alves, Nathan J; Turner, Kendrick B; Medintz, Igor L; Walper, Scott A

2015-07-01

Nanoparticle-based therapeutics are poised to play a critical role in treating disease. These complex multifunctional drug delivery vehicles provide for the passive and active targeted delivery of numerous small molecule, peptide and protein-derived pharmaceuticals. This article will first discuss some of the current state of the art nanoparticle classes (dendrimers, lipid-based, polymeric and inorganic), highlighting benefits/drawbacks associated with their implementation. We will then discuss an emerging class of nanoparticle therapeutics, bacterial outer membrane vesicles, that can provide many of the nanoparticle benefits while simplifying assembly. Through molecular biology techniques; outer membrane vesicle hijacking potentially allows for stringent control over nanoparticle production allowing for targeted protein packaged nanoparticles to be fully synthesized by bacteria.

System engineering study of electrodynamic tether as a spaceborne generator and radiator of electromagnetic waves in the ULF/ELF frequency band

NASA Technical Reports Server (NTRS)

Estes, R. D.; Grossi, M. D.; Lorenzini, E. C.

1986-01-01

The transmission and generation by orbiting tethered satellite systems of information carrying electromagnetic waves in the ULF/ELF frequency band to the Earth at suitably high signal intensities was examined and the system maintaining these intensities in their orbits for long periods of time without excessive onboard power requirements was investigated. The injection quantity power into electromagnetic waves as a function of system parameters such as tether length and orbital height was estimated. The basic equations needed to evaluate alternataing current tethered systems for external energy requirements are presented. The energy equations to tethered systems with various lengths, tether resistances, and radiation resistances, operating at different current values are applied. Radiation resistance as a function of tether length and orbital height is discussed. It is found that ULF/ELF continuously radiating systems could be maintained in orbit with moderate power requirements. The effect of tether length on the power going into electromagnetic waves and whether a single or dual tether system is preferable for the self-driven mode is discussed. It is concluded that the single tether system is preferable over the dual system.

The organization of LH2 complexes in membranes from Rhodobacter sphaeroides.

PubMed

Olsen, John D; Tucker, Jaimey D; Timney, John A; Qian, Pu; Vassilev, Cvetelin; Hunter, C Neil

2008-11-07

The mapping of the photosynthetic membrane of Rhodobacter sphaeroides by atomic force microscopy (AFM) revealed a unique organization of arrays of dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) core complexes surrounded and interconnected by light-harvesting LH2 complexes (Bahatyrova, S., Frese, R. N., Siebert, C. A., Olsen, J. D., van der Werf, K. O., van Grondelle, R., Niederman, R. A., Bullough, P. A., Otto, C., and Hunter, C. N. (2004) Nature 430, 1058-1062). However, membrane regions consisting solely of LH2 complexes were under-represented in these images because these small, highly curved areas of membrane rendered them difficult to image even using gentle tapping mode AFM and impossible with contact mode AFM. We report AFM imaging of membranes prepared from a mutant of R. sphaeroides, DPF2G, that synthesizes only the LH2 complexes, which assembles spherical intracytoplasmic membrane vesicles of approximately 53 nm diameter in vivo. By opening these vesicles and adsorbing them onto mica to form small, < or =120 nm, largely flat sheets we have been able to visualize the organization of these LH2-only membranes for the first time. The transition from highly curved vesicle to the planar sheet is accompanied by a change in the packing of the LH2 complexes such that approximately half of the complexes are raised off the mica surface by approximately 1 nm relative to the rest. This vertical displacement produces a very regular corrugated appearance of the planar membrane sheets. Analysis of the topographs was used to measure the distances and angles between the complexes. These data are used to model the organization of LH2 complexes in the original, curved membrane. The implications of this architecture for the light harvesting function and diffusion of quinones in native membranes of R. sphaeroides are discussed.

Learning characteristics of a space-time neural network as a tether skiprope observer

NASA Technical Reports Server (NTRS)

Lea, Robert N.; Villarreal, James A.; Jani, Yashvant; Copeland, Charles

1993-01-01

The Software Technology Laboratory at the Johnson Space Center is testing a Space Time Neural Network (STNN) for observing tether oscillations present during retrieval of a tethered satellite. Proper identification of tether oscillations, known as 'skiprope' motion, is vital to safe retrieval of the tethered satellite. Our studies indicate that STNN has certain learning characteristics that must be understood properly to utilize this type of neural network for the tethered satellite problem. We present our findings on the learning characteristics including a learning rate versus momentum performance table.

Learning characteristics of a space-time neural network as a tether skiprope observer

NASA Technical Reports Server (NTRS)

Lea, Robert N.; Villarreal, James A.; Jani, Yashvant; Copeland, Charles

1992-01-01

The Software Technology Laboratory at JSC is testing a Space Time Neural Network (STNN) for observing tether oscillations present during retrieval of a tethered satellite. Proper identification of tether oscillations, known as 'skiprope' motion, is vital to safe retrieval of the tethered satellite. Our studies indicate that STNN has certain learning characteristics that must be understood properly to utilize this type of neural network for the tethered satellite problem. We present our findings on the learning characteristics including a learning rate versus momentum performance table.

Structural characterization of more potent alternatives to HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

PubMed

Nemashkalova, Ekaterina L; Kazakov, Alexei S; Khasanova, Leysan M; Permyakov, Eugene A; Permyakov, Sergei E

2013-09-10

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and β-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-β-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.

Tethered Satellite System Contingency Investigation Board

NASA Technical Reports Server (NTRS)

1992-01-01

The Tethered Satellite System (TSS-1) was launched aboard the Space Shuttle Atlantis (STS-46) on July 31, 1992. During the attempted on-orbit operations, the Tethered Satellite System failed to deploy successfully beyond 256 meters. The satellite was retrieved successfully and was returned on August 6, 1992. The National Aeronautics and Space Administration (NASA) Associate Administrator for Space Flight formed the Tethered Satellite System (TSS-1) Contingency Investigation Board on August 12, 1992. The TSS-1 Contingency Investigation Board was asked to review the anomalies which occurred, to determine the probable cause, and to recommend corrective measures to prevent recurrence. The board was supported by the TSS Systems Working group as identified in MSFC-TSS-11-90, 'Tethered Satellite System (TSS) Contingency Plan'. The board identified five anomalies for investigation: initial failure to retract the U2 umbilical; initial failure to flyaway; unplanned tether deployment stop at 179 meters; unplanned tether deployment stop at 256 meters; and failure to move tether in either direction at 224 meters. Initial observations of the returned flight hardware revealed evidence of mechanical interference by a bolt with the level wind mechanism travel as well as a helical shaped wrap of tether which indicated that the tether had been unwound from the reel beyond the travel by the level wind mechanism. Examination of the detailed mission events from flight data and mission logs related to the initial failure to flyaway and the failure to move in either direction at 224 meters, together with known preflight concerns regarding slack tether, focused the assessment of these anomalies on the upper tether control mechanism. After the second meeting, the board requested the working group to complete and validate a detailed integrated mission sequence to focus the fault tree analysis on a stuck U2 umbilical, level wind mechanical interference, and slack tether in upper tether control mechanism and to prepare a detailed plan for hardware inspection, test, and analysis including any appropriate hardware disassembly.

Tethered Satellite System Contingency Investigation Board

NASA Astrophysics Data System (ADS)

1992-11-01

The Tethered Satellite System (TSS-1) was launched aboard the Space Shuttle Atlantis (STS-46) on July 31, 1992. During the attempted on-orbit operations, the Tethered Satellite System failed to deploy successfully beyond 256 meters. The satellite was retrieved successfully and was returned on August 6, 1992. The National Aeronautics and Space Administration (NASA) Associate Administrator for Space Flight formed the Tethered Satellite System (TSS-1) Contingency Investigation Board on August 12, 1992. The TSS-1 Contingency Investigation Board was asked to review the anomalies which occurred, to determine the probable cause, and to recommend corrective measures to prevent recurrence. The board was supported by the TSS Systems Working group as identified in MSFC-TSS-11-90, 'Tethered Satellite System (TSS) Contingency Plan'. The board identified five anomalies for investigation: initial failure to retract the U2 umbilical; initial failure to flyaway; unplanned tether deployment stop at 179 meters; unplanned tether deployment stop at 256 meters; and failure to move tether in either direction at 224 meters. Initial observations of the returned flight hardware revealed evidence of mechanical interference by a bolt with the level wind mechanism travel as well as a helical shaped wrap of tether which indicated that the tether had been unwound from the reel beyond the travel by the level wind mechanism. Examination of the detailed mission events from flight data and mission logs related to the initial failure to flyaway and the failure to move in either direction at 224 meters, together with known preflight concerns regarding slack tether, focused the assessment of these anomalies on the upper tether control mechanism. After the second meeting, the board requested the working group to complete and validate a detailed integrated mission sequence to focus the fault tree analysis on a stuck U2 umbilical, level wind mechanical interference, and slack tether in upper tether control mechanism and to prepare a detailed plan for hardware inspection, test, and analysis including any appropriate hardware disassembly.

On equilibrium positions and stabilization of electrodynamic tether system in the orbital frame

NASA Astrophysics Data System (ADS)

Tikhonov, A. A.; Shcherbakova, L. F.

2018-05-01

An electrodynamic tether system (EDTS) in a near-Earth circular orbit is considered. EDTS contains conductive tether with lumped masses attached to it at the ends. Possible equilibrium positions of the stretched tether under the influence of gravity gradient, Ampere and Lorentz forces in orbital frame are investigated. It is shown that in addition to the vertical equilibrium position, the "inclined" equilibrium positions of the tensioned tether are also possible. Conditions are obtained for the EDTS parameters, under which there is only one vertical position of the tether equilibrium. On the basis of nonlinear differential equations of motion, using the Lyapunov functions method, sufficient conditions for the stability of the vertical position of the tether equi-librium are obtained. It is shown that stabilization of the tether in this position is possible in the presence of damping in the EDTS system. The results of numerical simulation are presented.

Simulations of momentum transfer process between solar wind plasma and bias voltage tethers of electric sail thruster

NASA Astrophysics Data System (ADS)

Xia, Guangqing; Han, Yajie; Chen, Liuwei; Wei, Yanming; Yu, Yang; Chen, Maolin

2018-06-01

The interaction between the solar wind plasma and the bias voltage of long tethers is the basic mechanism of the electric sail thruster. The momentum transfer process between the solar wind plasma and electric tethers was investigated using a 2D full particle PIC method. The coupled electric field distribution and deflected ion trajectory under different bias voltages were compared, and the influence of bias voltage on momentum transfer process was analyzed. The results show that the high potential of the bias voltage of long tethers will slow down, stagnate, reflect and deflect a large number of ions, so that ion cavities are formed in the vicinity of the tether, and the ions will transmit the axial momentum to the sail tethers to produce the thrust. Compared to the singe tether, double tethers show a better thrust performance.

Altering surface fluctuations by blending tethered and untethered chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J. K.; Akgun, B.; Jiang, Z.

"Partially tethering" a thin film of a polymer melt by covalently attaching to the substrate a fraction of the chains in an unentangled melt dramatically increases the relaxation time of the surface height fluctuations. This phenomenon is observed even when the film thickness, h, is 20 times the unperturbed chain radius, R g,tethered, of the tethered chains, indicating that partial tethering is more influential than any physical attraction with the substrate. Furthermore, a partially tethered layer of a low average molecular weight of 5k showed much slower surface fluctuations than did a reference layer of pure untethered chains of muchmore » greater molecular weight (48k), so the partial tethering effect is stronger than the effects of entanglement and increase in glass transition temperature, Tg, with molecular weight. Partial tethering offers a means of tailoring these fluctuations which influence wetting, adhesion, and tribology of the surface.« less

Altering surface fluctuations by blending tethered and untethered chains

DOE PAGES

Lee, J. K.; Akgun, B.; Jiang, Z.; ...

2017-10-16

"Partially tethering" a thin film of a polymer melt by covalently attaching to the substrate a fraction of the chains in an unentangled melt dramatically increases the relaxation time of the surface height fluctuations. This phenomenon is observed even when the film thickness, h, is 20 times the unperturbed chain radius, R g,tethered, of the tethered chains, indicating that partial tethering is more influential than any physical attraction with the substrate. Furthermore, a partially tethered layer of a low average molecular weight of 5k showed much slower surface fluctuations than did a reference layer of pure untethered chains of muchmore » greater molecular weight (48k), so the partial tethering effect is stronger than the effects of entanglement and increase in glass transition temperature, Tg, with molecular weight. Partial tethering offers a means of tailoring these fluctuations which influence wetting, adhesion, and tribology of the surface.« less

SPHERES tethered formation flight testbed: advancements in enabling NASA's SPECS mission

NASA Astrophysics Data System (ADS)

Chung, Soon-Jo; Adams, Danielle; Saenz-Otero, Alvar; Kong, Edmund; Miller, David W.; Leisawitz, David; Lorenzini, Enrico; Sell, Steve

2006-06-01

This paper reports on efforts to control a tethered formation flight spacecraft array for NASA's SPECS mission using the SPHERES test-bed developed by the MIT Space Systems Laboratory. Specifically, advances in methodology and experimental results realized since the 2005 SPIE paper are emphasized. These include a new test-bed setup with a reaction wheel assembly, a novel relative attitude measurement system using force torque sensors, and modeling of non-ideal tethers to account for tether vibration modes. The nonlinear equations of motion of multi-vehicle tethered spacecraft with elastic flexible tethers are derived from Lagrange's equations. The controllability analysis indicates that both array resizing and spin-up are fully controllable by the reaction wheels and the tether motor, thereby saving thruster fuel consumption. Based upon this analysis, linear and nonlinear controllers have been successfully implemented on the tethered SPHERES testbed, and tested at the NASA MSFC's flat floor facility using two and three SPHERES configurations.

SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation

PubMed Central

Ahn, Gyeongik; Kim, Hyeran; Kim, Dae Heon; Hanh, Hong; Yoon, Youngdae; Singaram, Indira; Wijesinghe, Kaveesha J.; Johnson, Kristen A.; Liang, Zizhen; Stahelin, Robert V.; Jiang, Liwen; Cho, Wonhwa; Kang, Byung-Ho

2017-01-01

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis. PMID:28584166

Nonmonotonic fluctuation spectra of membranes pinned or tethered discretely to a substrate.

PubMed

Merath, Rolf-Jürgen; Seifert, Udo

2006-01-01

The thermal fluctuation spectrum of a fluid membrane coupled harmonically to a solid support by an array of tethers is calculated. For strong tethers, this spectrum exhibits nonmonotonic, anisotropic behavior with a relative maximum at a wavelength about twice the tether distance. The root-mean-square displacement is evaluated to estimate typical membrane displacements. Possible applications cover pillar-supported or polymer-tethered membranes.

Flight mechanics applications for tethers in space: Cooperative Italian-US programs

NASA Technical Reports Server (NTRS)

Bevilacqua, Franco; Merlina, Pietro; Anderson, John L.

1990-01-01

Since the 1974 proposal by Giuseppe Colombo to fly a tethered subsatellite from the Shuttle Orbiter, the creative thinking of many scientists and engineers from Italy and U.S. has generated a broad range of potential tether applications in space. Many of these applications have promise for enabling innovative research and operational activities relating to flight mechanics in earth orbit and at suborbital altitudes. From a flight mechanics standpoint the most interesting of the currently proposed flight demonstrations are: the second Tethered Satellite System experiment which offers both the potential for aerothermodynamics and hypersonics research and for atmospheric science research; the Tethered Initiated Space Recovery System which would enable orbital deboost and recovery of a re-entry vehicle and waste removal from a space station; and the Tether Elevator/Crawler System which would provide a variable microgravity environment and space station center of mass management. The outer atmospheric and orbital flight mechanics characteristics of these proposed tether flight demonstrations are described. The second Tethered Satellite System mission will deploy the tethered satellite earthward and will bring it as low as 130 km from ground and thus into the transition region between the atmosphere (non-ionized) and the partially ionized ionosphere. The atmospheric flight mechanics of the tethered satellite is discussed and simulation results are presented. The Tether Initiated Space Recovery System experiment will demonstrate the ability of a simple tether system to deboost and recover a reentry vehicle. The main feature of this demonstration is the utilization of a Small Expendable Deployment System (SEDS) and the low-tension deployment assumed to separate the reentry vehicle from the Shuttle. This low-tension deployment maneuver is discussed and its criticalities are outlined. The Tether Elevator/Crawler System is a new space element able to move in a controlled way between the ends of a deployed tethered system. A Shuttle test of an Elevator model is planned to demonstrate the unique capability of this element as a microgravity facility and to test the transfer motion control. The basic dynamical features of the Elevator system are presented and a preliminary assessment of the Elevator-induced tether vibrations is discussed.

Photo-triggered recognition between host and guest compounds in a giant vesicle encapsulating photo-pierceable vesicles.

PubMed

Suzuki, Kentaro; Machida, Kotaro; Yamaguchi, Kazuo; Sugawara, Tadashi

2018-01-01

Here, we used centrifugal precipitation to construct a giant vesicle (GV) encapsulating smaller giant vesicles (GV-in-GV) which comprises a photo-resistant outer GV and a photo-pierceable inner GV; the outer GV contained a fluorescent probe (SYBR Green I) in its inner water pool, and the inner GV contained double-stranded DNA (dsDNA) in its inner water pool. The phospholipid membrane of the inner GV was made photo-pierceable by inclusion of ca. 15mol% of a caged phospholipid in its membrane. Immediately after exposure of the GV-in-GVs to UV irradiation, strong fluorescence was detected in the inner water pool of the outer GV, indicating that dsDNA had been released from the inner GV and had complexed with the fluorescent probe. These dynamics can be recognized as a macroscopic representation of the molecular level function of a caged compound. Copyright © 2017 Elsevier B.V. All rights reserved.

Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials

PubMed Central

Oglęcka, Kamila; Rangamani, Padmini; Liedberg, Bo; Kraut, Rachel S; Parikh, Atul N

2014-01-01

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressure and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment. DOI: http://dx.doi.org/10.7554/eLife.03695.001 PMID:25318069

Remotely controlled fusion of selected vesicles and living cells: a key issue review

NASA Astrophysics Data System (ADS)

Bahadori, Azra; Moreno-Pescador, Guillermo; Oddershede, Lene B.; Bendix, Poul M.

2018-03-01

Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.

Ordering and partitioning in vesicle forming block copolymer thin films

NASA Astrophysics Data System (ADS)

Parnell, Andrew; Kamata, Yohei; Jones, Richard

Cell biology routinely uses encapsulation processes to package a payload and transport it to a location where the payload can then be used. Synthetic polymer based liposomes (Polymersomes) are one possible way in which we can artificially contain a molecule of interest that is protected from its surrounding environment. Encapsulation technologies at present rely on forming a lipid vesicle and then extruding it in a solution containing the target molecule to be encapsulated. Only a small fraction is encapsulated in this process. This is because of the complex structural formation pathway in going from individual isolated amphiphilic molecules into vesicle aggregates. My talk will discuss strategies to overcome the formation pathways, by forming a block copolymer film with the target molecule and then solvent ordering prior to the formation of vesicles. By studying block copolymer thin films with neutron reflectivity and ellipsometry we are able to observe partitioning and ordering which is essential for high encapsulation efficiencies. We acknowledge funding from STFC for use of the ISIS spallation neutron source.

Controlled tether extends satellite's orbital range

NASA Astrophysics Data System (ADS)

Wigotsky, V.

1984-06-01

A low orbit satellite tethered to the Space Shuttle Orbiter's cargo bay would be able to conduct upper atmosphere experiments without fear of orbit deterioration. NASA has in light of this initiated a Tethered Satellite System program aimed at the 1987 deployment of a 1,100-lb, 5 ft-diameter satellite to a distance of 6-12 miles from the Space Shuttle on a Kevlar tether. The distance of the fully developed system will be 62 miles, representing an altitude of 80 miles above the earth. Tether diameters under consideration are in the 0.065-0.1 inch range. The satellite control system will consist of a reel drive, a deployment boom, and a boom-mounted tether control, in order to vary tether tension during gravity gradient changes.

Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy.

PubMed

Zhang, Jinxie; Zhang, Xudong; Liu, Gan; Chang, Danfeng; Liang, Xin; Zhu, Xianbing; Tao, Wei; Mei, Lin

2016-01-01

The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine.

Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles

NASA Astrophysics Data System (ADS)

Sharma, S.; LeClaire, M.; Gimzewski, J. K.

2018-04-01

Over the last 30 years, atomic force microscopy (AFM) has made several significant contributions to the field of biology and medicine. In this review, we draw our attention to the recent applications and promise of AFM as a high-resolution imaging and force sensing technology for probing subcellular vesicles: exosomes and other extracellular vesicles. Exosomes are naturally occurring nanoparticles found in several body fluids such as blood, saliva, cerebrospinal fluid, amniotic fluid and urine. Exosomes mediate cell-cell communication, transport proteins and genetic content between distant cells, and are now known to play important roles in progression of diseases such as cancers, neurodegenerative disorders and infectious diseases. Because exosomes are smaller than 100 nm (about 30-120 nm), the structural and molecular characterization of these vesicles at the individual level has been challenging. AFM has revealed a new degree of complexity in these nanosized vesicles and generated growing interest as a nanoscale tool for characterizing the abundance, morphology, biomechanics, and biomolecular make-up of exosomes. With the recent interest in exosomes for diagnostic and therapeutic applications, AFM-based characterization promises to contribute towards improved understanding of these particles at the single vesicle and sub-vesicular levels. When coupled with complementary methods like optical super resolution STED and Raman, AFM could further unlock the potential of exosomes as disease biomarkers and as therapeutic agents.

Propulsive Small Expendable Deployer System (ProSEDS)

NASA Technical Reports Server (NTRS)

1999-01-01

This Quick Time movie is of NASA's Propulsive Small Expendable Deployer System experiment (ProSEDS). ProSEDS will demonstrate the use of an electrodynamic tether, basically a long, thin wire, for propulsion. An electrodynamic tether uses the same principles as electric motors in toys, appliances and computer disk drives, and generators in automobiles and power plants. When electrical current is flowing through the tether, a magnetic field is produced that pushes against the magnetic field of the Earth. For ProSEDS, the current in the tether results by virtue of the voltage generated when the tether moves through the Earth's magnetic field at more than 17,000 mph. This approach can produce drag thrust generating useable power. Since electrodynamic tethers require no propellant, they could substantially reduce the weight of the spacecraft and provide a cost-effective method of reboosting spacecraft. The tether would be a 3.1-mile (5 kilometer) long, ultrathin base-wire tether connected with a 6.2-mile (10 kilometer) long nonconducting tether. The ProSEDS experiment is managed by the Space Transportation Directorate at the Marshall Space Flight Center.

Dynamics and control of tethered antennas/reflectors in orbit

NASA Astrophysics Data System (ADS)

Liu, Liangdong; Bainum, Peter M.

The system linear equations for the motion of a tethered shallow spherical shell in orbit with its symmetry axis nominally following the local vertical are developed. The shell roll, yaw, tether out-of-plane swing motion and elastic vibrations are decoupled from the shell and tether in-plane pitch motions and elastic vibrations. The neutral gravity stability conditions for the special case of a constant length rigid tether are given for in-plane motion and out-of-plant motion. It is proved that the in-plane motion of the system could be asymptotically stable based on Rupp's tension control law, for a variable length tether. However, the system simulation results indicate that the transient responses can be improved significantly, especially for the damping of the tether and shell pitch motion, by an optimal feedback control law for the rigid variable length tether model. It is also seen that the system could be unstable when the effect of tether flexibility is included if the control gains are not chosen carefully. The transient responses for three different tension control laws are compared during typical station keeping operations.

Dynamics of tether-assisted reentry vehicle systems

NASA Astrophysics Data System (ADS)

Zhu, Renzhang; Misra, A. K.; Lin, Huabao

The dynamics of tether-assisted reentry of a capsule is considered in this paper. A major advantage in tethered-assisted reentry is the ability to replace a retro-rocket by a tether. In this reentry procedure, a capsule is deployed down to a design altitude near the local vertical, and at an appropriate time the capsule is disconnected from the tether and enters into a reentry trajectory. In addition to static release, swing release is also considered in this paper. Three deployment schemes appropriate for swing release are considered. A two-stage accelerated-exponential/decelerated-exponential deployment appears to be the best of the three. In comparison with static release, for the same duration of return, this swing release can lead to about 22 percent reduction in tether length at the cost of an increase in tension in the tether by only 8 to 12 percent, and thus, it could decrease the tether mass launched into space. The paper analyzes the detailed dynamics of the tethered system before release as well as the reentry dynamics of the capsule after release along with the heat generated during reentry.

α-Actinin Anchors PSD-95 at Postsynaptic Sites.

PubMed

Matt, Lucas; Kim, Karam; Hergarden, Anne C; Patriarchi, Tommaso; Malik, Zulfiqar A; Park, Deborah K; Chowdhury, Dhrubajyoti; Buonarati, Olivia R; Henderson, Peter B; Gökçek Saraç, Çiğdem; Zhang, Yonghong; Mohapatra, Durga; Horne, Mary C; Ames, James B; Hell, Johannes W

2018-03-07

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites. Copyright © 2018 Elsevier Inc. All rights reserved.

Use of 3D Printed Bone Plate in Novel Technique to Surgically Correct Hallux Valgus Deformities

PubMed Central

Smith, Kathryn E.; Dupont, Kenneth M.; Safranski, David L.; Blair, Jeremy; Buratti, Dawn; Zeetser, Vladimir; Callahan, Ryan; Lin, Jason; Gall, Ken

2016-01-01

Three-dimensional (3-D) printing offers many potential advantages in designing and manufacturing plating systems for foot and ankle procedures that involve small, geometrically complex bony anatomy. Here, we describe the design and clinical use of a Ti-6Al-4V ELI bone plate (FastForward™ Bone Tether Plate, MedShape, Inc., Atlanta, GA) manufactured through 3-D printing processes. The plate protects the second metatarsal when tethering suture tape between the first and second metatarsals and is a part of a new procedure that corrects hallux valgus (bunion) deformities without relying on doing an osteotomy or fusion procedure. The surgical technique and two clinical cases describing the use of this procedure with the 3-D printed bone plate are presented within. PMID:28337049

Graphene Nanocomposites with High Molecular Weight Poly(ε-caprolactone) Grafts: Controlled Synthesis and Accelerated Crystallization

DOE PAGES

Mondal, Titash; Ashkar, Rana; Butler, Paul; ...

2016-02-08

Grafting of high molecular weight polymers to graphitic nanoplatelets is a critical step toward the development of high performance graphene nanocomposites. However, designing such a grafting route has remained a major impediment. Herein, we report a "grafting to" synthetic pathway by which high molecular weight polymer, poly(e-caprolactone) (PCL), is tethered, at high grafting density, to highly anisotropic graphitic nanoplatelets. The efficacy of this tethering route and the resultant structural arrangements within the composite are confirmed by neutron and X-ray scattering measurements in the melt and solution phase. In the semicrystalline state, Xray analysis indicates that chain tethering onto the graphiticmore » nanoplatelets results in conformational changes of the polymer chains, which enhance the nucleation process and aid formation of PCL crystallites. This is corroborated by the superior thermal properties of the composite, manifested in accelerated crystallization kinetics and a significant increase in the thermal degradation temperature. Lastly, in principle, this synthesis route can be extended to a variety of high molecular weight polymers, which can open new avenues to solution-based processing of graphitic nanomaterials and the fabrication of complex 3D patterned graphitic nanocomposites.« less

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.

PubMed

Pillai, Ramesh S; Artus, Caroline G; Filipowicz, Witold

2004-10-01

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA. Copyright 2004 RNA Society

Modeling meiotic chromosome pairing: nuclear envelope attachment, telomere-led active random motion, and anomalous diffusion

PubMed Central

Marshall, Wallace F.; Fung, Jennifer C.

2016-01-01

The recognition and pairing of homologous chromosomes during meiosis is a complex physical and molecular process involving a combination of polymer dynamics and molecular recognition events. Two highly conserved features of meiotic chromosome behavior are the attachment of telomeres to the nuclear envelope and the active random motion of telomeres driven by their interaction with cytoskeletal motor proteins. Both of these features have been proposed to facilitate the process of homolog pairing, but exactly what role these features play in meiosis remains poorly understood. Here we investigate the roles of active motion and nuclear envelope tethering using a Brownian dynamics simulation in which meiotic chromosomes are represented by a Rouse polymer model subjected to tethering and active forces at the telomeres. We find that tethering telomeres to the nuclear envelope slows down pairing relative to the rates achieved by un-attached chromosomes, but that randomly-directed active forces applied to the telomeres speeds up pairing dramatically in a manner that depends on the statistical properties of the telomere force fluctuations. The increased rate of initial pairing cannot be explained by stretching out of the chromosome conformation but instead seems to correlate with anomalous diffusion of sub-telomeric regions. PMID:27046097

Modeling meiotic chromosome pairing: nuclear envelope attachment, telomere-led active random motion, and anomalous diffusion

NASA Astrophysics Data System (ADS)

Marshall, Wallace F.; Fung, Jennifer C.

2016-04-01

The recognition and pairing of homologous chromosomes during meiosis is a complex physical and molecular process involving a combination of polymer dynamics and molecular recognition events. Two highly conserved features of meiotic chromosome behavior are the attachment of telomeres to the nuclear envelope and the active random motion of telomeres driven by their interaction with cytoskeletal motor proteins. Both of these features have been proposed to facilitate the process of homolog pairing, but exactly what role these features play in meiosis remains poorly understood. Here we investigate the roles of active motion and nuclear envelope tethering using a Brownian dynamics simulation in which meiotic chromosomes are represented by a Rouse polymer model subjected to tethering and active forces at the telomeres. We find that tethering telomeres to the nuclear envelope slows down pairing relative to the rates achieved by unattached chromosomes, but that randomly directed active forces applied to the telomeres speed up pairing dramatically in a manner that depends on the statistical properties of the telomere force fluctuations. The increased rate of initial pairing cannot be explained by stretching out of the chromosome conformation but instead seems to correlate with anomalous diffusion of sub-telomeric regions.

Direct Synthesis of Polymer Nanotubes by Aqueous Dispersion Polymerization of a Cyclodextrin/Styrene Complex.

PubMed

Chen, Xi; Liu, Lei; Huo, Meng; Zeng, Min; Peng, Liao; Feng, Anchao; Wang, Xiaosong; Yuan, Jinying

2017-12-22

A one-step synthesis of nanotubes by RAFT dispersion polymerization of cyclodextrin/styrene (CD/St) complexes directly in water is presented. The resulted amphiphilic PEG-b-PS diblock copolymers self-assemble in situ into nanoparticles with various morphologies. Spheres, worms, lamellae, and nanotubes were controllably obtained. Because of the complexation, the swelling degree of polystyrene (PS) blocks by free St is limited, resulting in limited mobility of PS chains. Consequently, kinetically trapped lamellae and nanotubes were obtained instead of spherical vesicles. During the formation of nanotubes, small vesicles first formed at the ends of the tape-like lamellae, then grew and fused into nanotubes with a limited chain rearrangement. The introduction of a host-guest interaction based on CDs enables the aqueous dispersion polymerization of water-immiscible monomers, and produces kinetically trapped nanostructures, which could be a powerful technique for nanomaterials synthesis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty-seeking behavior

PubMed Central

Pilo Boyl, Pietro; Di Nardo, Alessia; Mulle, Christophe; Sassoè-Pognetto, Marco; Panzanelli, Patrizia; Mele, Andrea; Kneussel, Matthias; Costantini, Vivian; Perlas, Emerald; Massimi, Marzia; Vara, Hugo; Giustetto, Maurizio; Witke, Walter

2007-01-01

Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior. PMID:17541406

Controlled Redox Chemistry at Cerium within a Tripodal Nitroxide Ligand Framework

DOE PAGES

Bogart, Justin A.; Lippincott, Connor A.; Carroll, Patrick J.; ...

2015-10-27

Ligand reorganization has been shown to have a profound effect on the outcome of cerium redox chemistry. Through the use of a tethered, tripodal, trianionic nitroxide ligand, [((2-tBuNOH)C 6 H 4 CH 2 ) 3 N] 3- (TriNO x 3- ), controlled redox chemistry at cerium was accomplished, and typically reactive complexes of tetravalent cerium were isolated. These included rare cationic complexes [Ce(TriNO x )thf][BAr F 4 ], in which Ar F =3,5-(CF 3 ) 2 -C 6 H 3 , and [Ce(TriNO x )py][OTf] . A rare complete Ce-halide series, Ce(TriNO x )X, in which X=F - , Clmore » - , Br - , I - , was also synthesized. We explored the solution chemistry of these complexes through detailed solution-phase electrochemistry and 1 H NMR experiments and showed a unique shift in the ratio of species with inner- and outer-sphere anions with size of the anionic X - group. DFT calculations on the series of calculations corroborated the experimental findings. Also, the use of a bulky and strongly donating tethered tripodal nitroxide ligand allowed the controlled redox chemistry at cerium. As a result, rare examples of cationic Ce IV complexes were synthesized and fully characterized. The full Ce-halide series supported by the tripodal ligand framework is also reported (see scheme).« less

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion*

PubMed Central

Lürick, Anna; Kuhlee, Anne; Bröcker, Cornelia; Kümmel, Daniel; Raunser, Stefan; Ungermann, Christian

2015-01-01

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion. PMID:25564619

HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir

HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 ismore » a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and perturb the mannose 6 phosphate receptor recycling. •Our study identified novel Nef interacting human GCC185 protein and their role regulation of M6P receptor recycling.« less

The use of tethers for payload orbital transfer. Continuation of investigation of electrodynamic stabilization and control of long orbiting tethers, volume 2

NASA Technical Reports Server (NTRS)

Colombo, G.; Martinez-Sanchez, M.; Arnold, D.

1982-01-01

The SKYHOOK program was used to do simulations of two cases of the use of the tether for payload orbital transfer. The transport of a payload along the tether from a heavy lower platform to an upper launching platform is considered. A numerical example of the Shuttle launching a payload using an orbital tether facility is described.

The investigation of tethered satellite system dynamics

NASA Technical Reports Server (NTRS)

Lorenzini, E.

1984-01-01

Tethered satellite system (TSS) dynamics were studied. The dynamic response of the TSS during the entire stationkeeping phase for the first electrodynamic mission was investigated. An out of plane swing amplitude and the tether's bowing were observed. The dynamics of the slack tether was studied and computer code, SLACK2, was improved both in capabilities and computational speed. Speed hazard related to tether breakage or plasma contactor failure was examined. Preliminary values of the potential difference after the failure and of the drop of the electric field along the tether axis have been computed. The update of the satellite rotational dynamics model is initiated.

One kilometer (1 km) electric solar wind sail tether produced automatically.

PubMed

Seppänen, Henri; Rauhala, Timo; Kiprich, Sergiy; Ukkonen, Jukka; Simonsson, Martin; Kurppa, Risto; Janhunen, Pekka; Hæggström, Edward

2013-09-01

We produced a 1 km continuous piece of multifilament electric solar wind sail tether of μm-diameter aluminum wires using a custom made automatic tether factory. The tether comprising 90,704 bonds between 25 and 50 μm diameter wires is reeled onto a metal reel. The total mass of 1 km tether is 10 g. We reached a production rate of 70 m/24 h and a quality level of 1‰ loose bonds and 2‰ rebonded ones. We thus demonstrated that production of long electric solar wind sail tethers is possible and practical.

Characteristics and anti-proliferative activity of azelaic acid and its derivatives entrapped in bilayer vesicles in cancer cell lines.

PubMed

Manosroi, Aranya; Panyosak, Atchara; Rojanasakul, Yon; Manosroi, Jiradej

2007-06-01

The hydrophilicity and lipophilicity of azelaic acid (AA) were modified to diethyl azelate (DA) which was synthesized by Fisher esterification reaction and identified by IR, MS and (1)H NMR and to azelaic acid-beta-cyclodextrin complex (AACD) which was prepared by inclusion complexation and identified by IR, DSC and XRD respectively. AA, DA and AACD were entrapped in liposomes and niosomes comprising of L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/cholesterol at 7:3 molar ratio and Tween61/cholesterol at 1:1 molar ratio, respectively, using a thin-film hydration method with sonication. The size and morphology of these bilayer vesicles were determined by optical and transmission electron microscopy. The particle size was found to be in the range of 90-190 nm. The entrapment efficiency of AA, DA and AACD in all vesicular formulations was more than 80%, as analyzed by HPLC for AA and AACD, and GC for DA. Anti-proliferative activity of AA and its derivatives (DA and AACD) both entrapped and not entrapped in bilayer vesicles, using MTT assay in three cancer cell lines (HeLa, KB and B(16)F(10)) comparing with vincristine, were investigated. AACD showed the highest potency comparing to AA in HeLa, KB and B(16)F(10) of 1.48, 1.6 and 1.5 times, respectively. AA entrapped in liposomes was about 90 times more potent than the free AA, and about 1.5 times less potent than vincristine. When entrapped in bilayer vesicles, DA and AACD were more effective than AA in killing cancer cells. AACD entrapped in liposomes gave the highest anti-proliferation activity in HeLa cell lines with the IC(50) of 2.3 and 327 times more potent than vincristine and AA, respectively. DA in liposomes demonstrated the IC(50) of 0.03 times less potent than vincristine in KB cell lines, while in B(16)F(10) AACD in niosomes showed the IC(50) of 0.05 times less potent than vincristine. This study has suggested that the modification of AA by derivatization and complexation as well as the entrapment in bilayer vesicles can enhance its therapeutic efficacy.

Thermomechanical Properties and Glass Dynamics of Polymer-Tethered Colloidal Particles and Films

PubMed Central

2017-01-01

Polymer-tethered colloidal particles (aka “particle brush materials”) have attracted interest as a platform for innovative material technologies and as a model system to elucidate glass formation in complex structured media. In this contribution, Brillouin light scattering is used to sequentially evaluate the role of brush architecture on the dynamical properties of brush particles in both the individual and assembled (film) state. In the former state, the analysis reveals that brush–brush interactions as well as global chain relaxation sensitively depend on grafting density; i.e., more polymer-like behavior is observed in sparse brush systems. This is interpreted to be a consequence of more extensive chain entanglement. In contrast, the local relaxation of films does not depend on grafting density. The results highlight that relaxation processes in particle brush-based materials span a wider range of time and length scales as compared to linear chain polymers. Differentiation between relaxation on local and global scale is necessary to reveal the influence of molecular structure and connectivity on the aging behavior of these complex systems. PMID:29755139

Thermomechanical Properties and Glass Dynamics of Polymer-Tethered Colloidal Particles and Films.

PubMed

Cang, Yu; Reuss, Anna N; Lee, Jaejun; Yan, Jiajun; Zhang, Jianan; Alonso-Redondo, Elena; Sainidou, Rebecca; Rembert, Pascal; Matyjaszewski, Krzysztof; Bockstaller, Michael R; Fytas, George

2017-11-14

Polymer-tethered colloidal particles (aka "particle brush materials") have attracted interest as a platform for innovative material technologies and as a model system to elucidate glass formation in complex structured media. In this contribution, Brillouin light scattering is used to sequentially evaluate the role of brush architecture on the dynamical properties of brush particles in both the individual and assembled (film) state. In the former state, the analysis reveals that brush-brush interactions as well as global chain relaxation sensitively depend on grafting density; i.e., more polymer-like behavior is observed in sparse brush systems. This is interpreted to be a consequence of more extensive chain entanglement. In contrast, the local relaxation of films does not depend on grafting density. The results highlight that relaxation processes in particle brush-based materials span a wider range of time and length scales as compared to linear chain polymers. Differentiation between relaxation on local and global scale is necessary to reveal the influence of molecular structure and connectivity on the aging behavior of these complex systems.

Effects of topology on the adsorption of singly tethered ring polymers to attractive surfaces.

PubMed

Li, Bing; Sun, Zhao-Yan; An, Li-Jia

2015-07-14

We investigate the effect of topology on the equilibrium behavior of singly tethered ring polymers adsorbed on an attractive surface. We focus on the change of square radius of gyration Rg(2), the perpendicular component Rg⊥(2) and the parallel component Rg‖(2) to the adsorbing surface, the mean contacting number of monomers with the surface , and the monomer distribution along z-direction during transition from desorption to adsorption. We find that both of the critical point of adsorption εc and the crossover exponent ϕ depend on the knot type when the chain length of ring ranges from 48 to 400. The behaviors of Rg(2), Rg⊥(2), and Rg‖(2) are found to be dependent on the topology and the monomer-surface attractive strength. At weak adsorption, the polymer chains with more complex topology are more adsorbable than those with simple topology. However, at strong adsorption, the polymer chains with complex topology are less adsorbable. By analyzing the distribution of monomer along z-direction, we give a possible mechanism for the effect of topology on the adsorption behavior.

Sub-Scale Orion Parachute Test Results from the National Full-Scale Aerodynamics Complex 80- By 120-ft Wind Tunnel

NASA Technical Reports Server (NTRS)

Anderson, Brian P.; Greathouse, James S.; Powell, Jessica M.; Ross, James C.; Schairer, Edward T.; Kushner, Laura; Porter, Barry J.; Goulding, Patrick W., II; Zwicker, Matthew L.; Mollmann, Catherine

2017-01-01

A two-week test campaign was conducted in the National Full-Scale Aerodynamics Complex 80 x 120-ft Wind Tunnel in support of Orion parachute pendulum mitigation activities. The test gathered static aerodynamic data using an instrumented, 3-tether system attached to the parachute vent in combination with an instrumented parachute riser. Dynamic data was also gathered by releasing the tether system and measuring canopy performance using photogrammetry. Several canopy configurations were tested and compared against the current Orion parachute design to understand changes in drag performance and aerodynamic stability. These configurations included canopies with varying levels and locations of geometric porosity as well as sails with increased levels of fullness. In total, 37 runs were completed for a total of 392 data points. Immediately after the end of the testing campaign a down-select decision was made based on preliminary data to support follow-on sub-scale air drop testing. A summary of a more rigorous analysis of the test data is also presented.

Dynamics of model blood cells in shear flow

NASA Astrophysics Data System (ADS)

Podgorski, Thomas; Callens, Natacha; Minetti, Christophe; Coupier, Gwennou; Dubois, Frank; Misbah, Chaouqi

The dynamics of a vesicle suspension in shear flow was investigated by digital holographic microscopy [1] in parabolic flights and in the MASER 11 sounding rocket. Vesicles are lipid membranes which mimic the mechanical behaviour of cells, such as red blood cells in flow. In a simple shear flow between parallel walls, a lift force of purely viscous origin pushes vesicles away from walls. Our parabolic flight experiments [2] reveal that the lift velocity in a dilute suspen-sion is well described by theoretical predictions by Olla. As vesicles gather near the center of the flow chamber due to lift forces from both walls, one expects hydrodynamic interactions of pairs of vesicles to result in shear induced diffusion in the suspension. The BIOMICS experi-ment in the MASER 11 sounding rocket revealed a complex spatial structure of a polydisperse vesicle suspension due to the interplay between lift forces from the walls and hydrodynamic interactions. These phenomena have a strong impact on the structure and rheology of blood in small vessels, and a precise knowledge of the dynamics of migration and diffusion of soft particles in flow can lead to alternative ways to separate and sort blood cells. 1. Dubois, F., Schockaert, C., Callens, N., Yourrassowsky, C., "Focus plane detection criteria in digital holography microscopy by amplitude analysis", Opt. Express, Vol. 14, pp 5895-5908, 2006 2. Callens, N., Minetti, C., Coupier, G., Mader, M.-A., Dubois, F., Misbah, C., Podgorski, T., "Hydrodynamics lift of vesicles under shear flow in microgravity", Europhys. Lett., Vol. 83, p. 24002, 2008

Analysis of ProSEDS Test of Bare-Tether Collection

NASA Technical Reports Server (NTRS)

Sanmartin, J. R.; Lorenzini, E. C.; Estes, R. D.; Charro, M.; Cosmo, M. L.

2003-01-01

NASA's tether experiment ProSEDS will be placed in orbit on board a Delta-II rocket to test bare-tether electron collection, deorbiting of the rocket second stage, and the system dynamic stability. ProSEDS performance will vary because ambient conditions change along the orbit and tether-circuit bulk elements at the cathodic end follow the step-by-step sequence for the current cycles of operating modes (open-circuit, shunt and resistor modes for primary cycles; shunt and battery modes for secondary cycles). In this work we discuss expected ProSEDS values of the ratio L,/L*, which jointly with cathodic bulk elements determines bias and current tether profiles; L, is tether length, and L* (changing with tether temperature and ionospheric plasma density and magnetic field) is a characteristic length gauging ohmic versus baretether collection impedances. We discuss how to test bare-tether electron collection during primary cycles, using probe measurements of plasma density, measurements of cathodic current in resistor and shunt modes, and an estimate of tether temperature based on ProSEDS orbital position at the particular cycle concerned. We discuss how a temperature misestimate might occasionally affect the test of bare-tether collection, and how introducing the battery mode in some primary cycles, for an additional current measurement, could obviate the need of a temperature estimate. We also show how to test bare-tether collection by estimating orbit-decay rate from measurements of cathodic current for the shunt and battery modes of secondary cycles.

Efficacy of chordal cutting in alleviating ischemic mitral regurgitation: insights from 3-dimensional echocardiography

PubMed Central

Sai-Sudhakar, Chittoor B; Vandse, Rashmi; Armen, Todd A; Bickle, Katherine M; Nathan, Nadia S

2007-01-01

Background Ischemic mitral regurgitation often complicates severe ischemic heart disease and adversely affects the prognosis in these patients. There is wide variation in the clinical spectrum of ischemic mitral regurgitation due to varying location and chronicity of ischemia and anomalies in annular and ventricular remodeling. As a result, there is lack of consensus in treating these patients. Treatment has to be individualized for each patient. Most of the available surgical options do not consistently correct this condition in all the patients. Chordal cutting is one of the newer surgical approaches in which cutting a limited number of critically positioned basal chordae have found success by relieving the leaflet tethering and thereby improving the coaptation of leaflets. Three-dimensional echocardiography is a potentially valuable tool in identifying the specific pattern of tethering and thus the suitability of this procedure in a given clinical scenario. Case Presentation A 66-year-old man with cardiomyopathy and ischemic mitral regurgitation presented to us with the features of congestive heart failure. The three-dimensional echocardiography revealed severe mitral regurgitation associated with the tethering of the lateral (P1) and medial (P3) scallops of the posterior leaflet of the mitral valve due to secondary chordal attachments. The ejection fraction was only 15% with severe global systolic and diastolic dysfunction. Mitral regurgitation was successfully corrected with mitral annuloplasty and resection of the secondary chordae tethering the medial and lateral scallops of the posterior leaflet of the mitral valve. Conclusion Cutting the second order chordae along with mitral annuloplasty could be a novel method to remedy Ischemic mitral regurgitation by relieving the tethering of the valve leaflets. The preoperative three-dimensional echocardiography should be considered in all patients with Ischemic mitral regurgitation to assess the complex three-dimensional interactions between the mitral valve apparatus and the left ventricle. This aids in timely surgical planning. PMID:17894872

Efficacy of chordal cutting in alleviating ischemic mitral regurgitation: insights from 3-dimensional echocardiography.

PubMed

Sai-Sudhakar, Chittoor B; Vandse, Rashmi; Armen, Todd A; Bickle, Katherine M; Nathan, Nadia S

2007-09-25

Ischemic mitral regurgitation often complicates severe ischemic heart disease and adversely affects the prognosis in these patients. There is wide variation in the clinical spectrum of ischemic mitral regurgitation due to varying location and chronicity of ischemia and anomalies in annular and ventricular remodeling. As a result, there is lack of consensus in treating these patients. Treatment has to be individualized for each patient. Most of the available surgical options do not consistently correct this condition in all the patients. Chordal cutting is one of the newer surgical approaches in which cutting a limited number of critically positioned basal chordae have found success by relieving the leaflet tethering and thereby improving the coaptation of leaflets. Three-dimensional echocardiography is a potentially valuable tool in identifying the specific pattern of tethering and thus the suitability of this procedure in a given clinical scenario. A 66-year-old man with cardiomyopathy and ischemic mitral regurgitation presented to us with the features of congestive heart failure. The three-dimensional echocardiography revealed severe mitral regurgitation associated with the tethering of the lateral (P1) and medial (P3) scallops of the posterior leaflet of the mitral valve due to secondary chordal attachments. The ejection fraction was only 15% with severe global systolic and diastolic dysfunction. Mitral regurgitation was successfully corrected with mitral annuloplasty and resection of the secondary chordae tethering the medial and lateral scallops of the posterior leaflet of the mitral valve. Cutting the second order chordae along with mitral annuloplasty could be a novel method to remedy Ischemic mitral regurgitation by relieving the tethering of the valve leaflets. The preoperative three-dimensional echocardiography should be considered in all patients with Ischemic mitral regurgitation to assess the complex three-dimensional interactions between the mitral valve apparatus and the left ventricle. This aids in timely surgical planning.

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.

Small expendable deployer system measurement analysis

NASA Technical Reports Server (NTRS)

Carrington, Connie K.

1988-01-01

The first on-orbit experiment of the Small Expendable Deployer System (SEDS) for tethered satellites will collect telemetry data for tether length, rate of deployment, and tether tension. The post-flight analysis will use this data to reconstruct the deployment history and determine dynamic characteristics such as tether shape and payload position. Linearized observability analysis has determined that these measurements are adequate to define states for a two-mass tether model, and two state estimators were written.

Energy transduction inside vesicles, photocatalysis by titanium dioxide and formation of NADH

NASA Astrophysics Data System (ADS)

Summers, David; Noveron, Juan; Rodoni, David; Basa, Ranor

A number of theories on the origin and early evolution of life have focused on the role of lipid bilayer membrane structures (vesicles). These vesicles are similar to modern cellular membranes , and have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth to provide compartments for early cellular life. They can contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy to drive metabolism (ie. energy transduction) is also one of the central issues in our attempts to understand the origin and evolution of life. When did energy transduction and photosynthesis begin? What was the original system for capturing photochemical energy? How simple can such a system be? It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has been shown that pH gradients can be photo-chemically created, but it has been found difficult to couple these to drive chemical reactions. Minerals can introduce a number of properties to a vesicle system. The incorporation of clay particles into vesicles can provide catalytic activity that mediates both vesicle assembly and RNA oligomerization. It is known that colloidal semiconducting mineral particles can act as photocatalysts and drive redox chemistry. We show that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry and represent a model system for early photosynthesis. TiO2 particles can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to be able concentrate species inside a vesicle. It is shown that these can be used to produce biochemical species such as enzymatically active NADH in such structures. This system demonstrates a simple energy source inside vesicles/protocells suitable either for simple prebiotic systems and/or for more complex "protobiochemical" systems. It could act as a precursor to metabolic systems and provide a model of how metabolism could have developed prebiotically in a vesicle based "protocell origin of life". It can provide a source of prebiotic compounds inside vesicles, an environment considered to be of great importance for the origin of life. An energy transduction system that is simple enough to have formed at an early stage of the origin of life (even before the formation of enzymatic or ribozymal activity) makes an autotrophic origin of life easier to envision.

Input profile for satellite orbit transfer by tether mechanism; Part I: Tether length profile using feed-forward procedure

NASA Astrophysics Data System (ADS)

Hokamoto, Shinji

This study deals with orbital transfer of a satellite using a tether extension / retrieval mechanism. Instead of using propellant for the orbital transfer, the present concept uses electrical energy. By controlling the pitch motion of the tether system, we can achieve a prescribed velocity of the satellite at a prescribed position. By cutting the tether at that instant, we can inject the satellite into a designed new orbit. This paper considers co-planar motion and proposes a technique to achieve the desired tether length, pitch angle, and pitch angular rate at a designated position in orbit by using only tether length control. These three state variables are adjusted to their target values in three consecutive sections in the orbit; 1) control for the angular momentum of the pitching motion, which implies to adjust the tether length, 2) control for the pitch angle, and 3) control for the pitch angular rate. In each section, a pitch acceleration profile can be formed by using Fourier series as an alternative input for tether length profile. Their coefficients can be obtained without numerical iterations by using the simple initial / final relationships for the pitch angle and pitch angular rate. Therefore, this proposed procedure requires less computational cost than a numerical search, is easily applicable for different models and orbits, and can cope with physical restrictions of the system, such as tether tension or maximum tether length. Furthermore, the resulting final states precisely coincide with the target values. To demonstrate that the proposed procedure can successfully generate proper input profiles, this paper presents an orbital transfer problem as an example, and verifies its effectiveness. The simulation results show that the maximum tether length is less than 5km, and that the tether tension is kept positive during the mission.

Applications of Tethers in Space, Volume 1

NASA Technical Reports Server (NTRS)

Cron, A. C. (Compiler)

1985-01-01

The tethered satellite system is described including tether fundamentals. Applications of very long tethers in space to a broad spectrum of future space missions are explored. Topics covered include: science, transportation, constellations, artificial gravity, technology and test, and electrodynamic interactions. Recommendations to NASA are included.

Tethers in space: Birth and growth of a new avenue to space utilization

NASA Technical Reports Server (NTRS)

Vontiesenhausen, G.

1984-01-01

The evolution of the ideas of tether applications in space are traced from its origin in the last century past a dormant period of sixty-five years to the mid-seventies. At that time as a consequence of major revival efforts, NASA entered into serious investigations of the theoretical and practical feasibility of a large number of tethered concepts in space. These efforts culminated in the establishment of the Tethered Satellite System Project now at NASA in the advanced development phase. Extensive planning efforts are described, first, through a Tether Applications in Space Workshop which generated additional concepts and provided overall assessments and recommendations to NASA, and then through a NASA inter-center Tether Applications in Space Task Group which generated a four year program plan in the areas of further studies, technology, work and science and applications of tethers in space. An outlook into the future of tether applications that approaches some of the goals of the early visionaries is offered.

Summary presentation of the technology and test panel

NASA Technical Reports Server (NTRS)

Siemers, P.

1985-01-01

Tether related technology issues were investigated along with potential applications. Several of the applications do not derive necessarily from nor are they related to a technology issue. Tether designs must concern itself with length requirements (whether the tether is to be flexible or stiff) and what the environmental impact is on the particular material that is proposed for the tether. As far as tether manufacturing techniques, a lot of technology related work is required to develop cost effective manfacturing capabilities for the future tether. There are techniques that are used on the ground now. However, after some of the proposed applications are determined to be feasible, it may be that the best way to manufacture the tether is to pretend the satellite is a spider and allow it to spin its own web in space. The technology required to developed tapered tethers was considered. Definition of the taper, where the center of that taper should be, and the taper's relation to the end masses are all of concern.

3D imaging of vesicles in hyaloclastic fragments - clues to syn-eruptive shear conditions

NASA Astrophysics Data System (ADS)

Helo, C.; Flaws, A.; Hess, K.; Franz, A.; Clague, D. A.; Dingwell, D. B.

2011-12-01

3D imaging of stretched vesicles in hyaloclastic fragments has been used to investigate the shear environment of mild pyroclastic eruptions at mid-ocean ridges. X-ray computed tomography offers an attractive non-invasive method to investigate geomaterials at a high resolution for the geometry of the different phases. In this study, we have imaged vesicles within two types of basaltic glass fragments. Stretched, ellipsoid-shaped vesicles in thin limu o Pele and tubular vesicles in a pumiceous fragment. Both types originate from pyroclastic activity on Axial Seamount, Juan de Fuca ridge. Rapid quenching of the glass has prevented extensive bubble relaxation and information about syn-eruptive shear and differential stress conditions is stored, as the dimensions of a stretched bubble directly relates to the extent and mode of shearing. The X-ray tomography data was processed using a set of codes based on edge detection and ellipsoid fitting to acquire quantitative information on the shape of the stretched vesicles. Preliminary results demonstrate, that the geometry of the stretched vesicles, e.g., the elongation of the vesicle with respect to the calculated undeformed radius, is in accordance with simple shear scenarios. Stored differential stress ranges from 5 kPa to 90 kPa with shear rates between 3.2x102 s-1 and 5.7x3 s-1 within a single limu o Pele fragment. This range may be explained by either variable time available for relaxation as the cooling front proceeds through the fragment, complex interplay in space and time between fragmentation and quenching, bubble clusters mutually inhibiting each others extend of deformation, or any combination of these. Bubble relaxation time scales are less then 0.005 s providing constraints on the timeframe for cooling to the glass transition. Qualitative analyses of the tube pumice indicates that the tubular structures grow in length by coalescence of vertically aligned ellipsoid-shaped vesicles, and in width by coalescence of horizontally contiguous vesicles. Pyroclastic fragments produced in deep-sea environments thus offer the opportunity to explore in-situ shear and stress conditions prevailing during eruption, even in very low viscous systems.

[Regulation of immune responses by exosomes derived from antigen presenting cells].

PubMed

Maravillas-Montero, José Luis; Martínez-Cortés, Ismael

2017-01-01

Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.

Assessing tether anchor labeling and usability in pickup trucks.

PubMed

Klinich, Kathleen D; Manary, Miriam A; Malik, Laura A; Flannagan, Carol A; Jermakian, Jessica S

2018-04-03

The objective of this study was to investigate vehicle factors associated with child restraint tether use and misuse in pickup trucks and evaluate 4 labeling interventions designed to educate consumers on proper tether use. Volunteer testing was performed with 24 subjects and 4 different pickup trucks. Each subject performed 8 child restraint installations among the 4 pickups using 2 forward-facing restraints: a Britax Marathon G4.1 and an Evenflo Triumph. Vehicles were selected to represent 4 different implementations of tether anchors among pickups: plastic loop routers (Chevrolet Silverado), webbing routers (Ram), back wall anchors (Nissan Frontier), and webbing routers plus metal anchors (Toyota Tundra). Interventions included a diagram label, Quick Response (QR) Code linked to video instruction, coordinating text label, and contrasting text tag. Subjects used the child restraint tether in 93% of trials. However, tether use was completely correct in only 9% of trials. An installation was considered functional if the subject attached the tether to a tether anchor and had a tight installation (ignoring routing and head restraint position); 28% of subjects achieved a functional installation. The most common installation error was attaching the tether hook to the anchor/router directly behind the child restraint (near the top of the seatback) rather than placing the tether through the router and attaching it to the anchor in the adjacent seating position. The Nissan Frontier, with the anchor located on the back wall of the cab, had the highest rate of correct installations but also had the highest rate of attaching the tether to components other than the tether anchor (seat adjustor, child restraint storage hook, around head restraint). None of the labeling interventions had a significant effect on correct installation; not a single subject scanned the QR Code to access the video instruction. Subjects with the most successful installations spent extensive time reviewing the vehicle manuals. Current implementations of tether anchors among pickup trucks are not intuitive for child restraint installations, and alternate designs should be explored. Several different labeling interventions were ineffective at achieving correct tether use in pickup trucks.

Electrodynamic Bare Tether Systems as a Thruster for the Momentum-Exchange/Electrodynamic Reboost(MXER)Project

NASA Technical Reports Server (NTRS)

Khazanov, G. V.; Krivorutsky, E. N.; Gallagher, D. L.

2006-01-01

The concept of electrodynamic tether propulsion has a number of attractive features and has been widely discussed for different applications. Different system designs have been proposed and compared during the last 10 years. In spite of this, the choice of proper design for any particular mission is a unique problem. Such characteristics of tether performance as system acceleration, efficiency, etc., should be calculated and compared on the basis of the known capability of a tether to collect electrical current. We discuss the choice of parameters for circular and tape tethers with regard to the Momentum-Exchange/Electrodynamic Reboost (MXER) tether project.

The role of tethers on space station

NASA Technical Reports Server (NTRS)

Vontiesenhausen, G. (Editor)

1985-01-01

The results of research and development that addressed the usefulness of tether applications in space, particularly for space station are described. A well organized and structured effort of considerable magnitude involving NASA, industry and academia have defined the engineering and technological requirements of space tethers and their broad range of economic and operational benefits. The work directed by seven NASA Field Centers is consolidated and structured to cover the general and specific roles of tethers in space as they apply to NASA's planned space station. This is followed by a description of tether systems and operations. A summary of NASA's plans for tether applications in space for years to come is given.

Modeling and Simulation of a Tethered Harpoon for Comet Sampling

NASA Technical Reports Server (NTRS)

Quadrelli, Marco B.

2014-01-01

This paper describes the development of a dynamic model and simulation results of a tethered harpoon for comet sampling. This model and simulation was done in order to carry out an initial sensitivity analysis for key design parameters of the tethered system. The harpoon would contain a canister which would collect a sample of soil from a cometary surface. Both a spring ejected canister and a tethered canister are considered. To arrive in close proximity of the spacecraft at the end of its trajectory so it could be captured, the free-flying canister would need to be ejected at the right time and with the proper impulse, while the tethered canister must be recovered by properly retrieving the tether at a rate that would avoid an excessive amplitude of oscillatory behavior during the retrieval. The paper describes the model of the tether dynamics and harpoon penetration physics. The simulations indicate that, without the tether, the canister would still reach the spacecraft for collection, that the tether retrieval of the canister would be achievable with reasonable fuel consumption, and that the canister amplitude upon retrieval would be insensitive to variations in vertical velocity dispersion.

Space Station Reboost with Electrodynamic Tethers

NASA Technical Reports Server (NTRS)

Vas, Irwin E.; Kelly, Thomas J.; Scarl, Ethan A.

1999-01-01

This paper presents the results of a study of an electrodynamic tether system to reboost the International Space Station (ISS). One recommendation is to use a partially bare tether for electron collection. Locations are suggested as to where the tether system is to be attached at the space station. The effects of the tether system on the microgravity environment may actually be beneficial, because the system can neutralize aerodrag during quiescent periods and, if deployed from a movable boom, can permit optimization of laboratory positioning with respect to acceleration contours. Alternative approaches to tether deployment and retrieval are discussed. It is shown that a relatively short tether system, 7 km long, operating at a power level of 5 kW could provide cumulative savings or over a billion dollars during a 10-year period ending in 2012. This savings is the direct result of a reduction in the number or nights that would otherwise be required to deliver propellant for reboost, with larger cost savings for higher tether usage. In addition to economic considerations, an electrodynamic tether promises a practical backup system that could ensure ISS survival in the event of an (otherwise) catastrophic delay in propellant delivery.

Analysis of the effect of attachment point bias during large space debris removal using a tethered space tug

NASA Astrophysics Data System (ADS)

Chu, Zhongyi; Di, Jingnan; Cui, Jing

2017-10-01

Space debris occupies a valuable orbital resource and is an inevitable and urgent problem, especially for large space debris because of its high risk and the possible crippling effects of a collision. Space debris has attracted much attention in recent years. A tethered system used in an active debris removal scenario is a promising method to de-orbit large debris in a safe manner. In a tethered system, the flexibility of the tether used in debris removal can possibly induce tangling, which is dangerous and should be avoided. In particular, attachment point bias due to capture error can significantly affect the motion of debris relative to the tether and increase the tangling risk. Hence, in this paper, the effect of attachment point bias on the tethered system is studied based on a dynamic model established based on a Newtonian approach. Next, a safety metric of avoiding a tangle when a tether is tensioned with attachment point bias is designed to analyse the tangling risk of the tethered system. Finally, several numerical cases are established and simulated to validate the effects of attachment point bias on a space tethered system.

Practicality of Using a Tether for Electrodynamic Reboost of the International Space Station

NASA Technical Reports Server (NTRS)

Blumer, J. H.; Donahue, Benjamin B.; Bangham, Michal E.; Roth, A. (Technical Monitor)

2001-01-01

ElectroDynamic (ED) Tethers can generate continuous low thrust in a low Earth orbit. An induced current running through the length of the tether reacts with the geomagnetic field to produce thrust. The amount of thrust scales with tether lens!th and current. The International Space Station (ISS) requires periodic reboost to maintain an approximately circular orbit t above the Earth. The baseline reboost method is a traditional bi-propellant rocket thruster and tankage system which must to be refueled via Soyuz / Progress or other launch vehicle. The estimated propellant costs associated with keeping ISS in the designated orbit over a 10-year life have been extremely high. The ED Tether would draw energy from the renewable ISS Solar Array electrical power system. Propulsion requirements for ISS vary depending on solar wind and other conditions. It is projected that a ED Tether could provide the majority of the required reboost thrust for ISS for a nominal solar year. For above nominal solar wind years the ISS would have to use the rocket reboost system, but at a greatly reduced level. Thus resulting in substantial cost savings, via the reduction in the number of Earth-to-orbit launch vehicle flights to the ISS that must bring reboost propellant. However, the purposes of this paper is to further Previous research on an ISS ED Tether and examine the operational and technical issues working against using a ED Tether on ISS. Issues such as Shuttle rendezvous and flight path concerns raise serious safety concerns and restrictions on tether use. Tether issues such as tether librations and off angle thrust raise concerns about impacts to microgravity payloads and the long-term effect on ISS orbital path and inclination. Operational issues such as peak power available to an ED Tether and allowable duty cycle may impose severe restrictions on tether design and ultimately limit the practicality of an ED Tether on ISS. Thus while at first glance the cost numbers appear to be strongly in favor of an ED Tether the limitations imposed by safety, operations and technical concerns may severely undermine the economic model. Possible Solutions to these problems have been investigated and proposed, however some items like off angle thrust are still being actively investigated for an adequate solution.

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.

PubMed

Vasiljev, Andreja; Ahting, Uwe; Nargang, Frank E; Go, Nancy E; Habib, Shukry J; Kozany, Christian; Panneels, Valérie; Sinning, Irmgard; Prokisch, Holger; Neupert, Walter; Nussberger, Stephan; Rapaport, Doron

2004-03-01

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

Effects of predators on fish and crayfish survival in intermittent streams

USGS Publications Warehouse

Dekar, Matthew P.; Magoulick, Daniel D.

2013-01-01

Predation from aquatic and terrestrial predators arc important factors structuring the size and depth distribution of aquatic prey. We conducted mesocosm and tethering experiments on Little Mulberry Creek in northwest Arkansas during low flows to examine the effects of predators on fish and crayfish survival in intermittent streams Using shallow artificial pools (10 cm deep) and predator exclusions, we tested the hypothesis that large-bodied fish are at greater risk from terrestrial predators in shallow habitats compared to small-bodied individuals. Twenty-four circular pools (12 open top. 12 closed top) were stocked with two size classes of Campostoma anomalum (Central Stonerller) and deployed systematically in a single stream pool. In addition, we used a crayfish tethering experiment to test the hypothesis that the survival of small and large crayfish is greater in shallow and deep habitats, respectively. We tethered two size classes of Orconectes meeki meeki (Meek's Crayfish) along shallow and deep transects in two adjacent stream pools and measured survival for 15 days. During both experiments, we monitored the presence or absence of predators by visual observation and from scat surveys. We demonstrated a negative effect of terrestrial predators on Central Stonerller survival in the artificial pools, and larger individuals were more susceptible to predation. In contrast, small crayfish experienced low survival at all depths and large crayfish were preyed upon much less intensively during the tethering study, particularly in the pool with larger substrate. More studies are needed to understand how stream drying and environmental heterogeneity influence the complex interactions between predator and prey populations in intermittent streams.

An Overview of Electrodynamic Tether Performance in the Jovian System

NASA Technical Reports Server (NTRS)

Gallagher, Dennis; Johnson, Les; Bagenal, Fran; Moore, James

1998-01-01

The Jovian magnetosphere with its strong magnetic field and rapid planetary rotation present new opportunities and challenges for the use of electrodynamic tethers. An overview of the basic plasma physics properties of an electrodynamic tether moving through the Jovian magnetosphere is examined. Tether use for both propulsion and power generation are considered. Close to the planet, tether propulsive forces are found to be as high as 50 Newtons and power levels as high as 1 million Watts.

The tethered galaxy problem: a possible window to explore cosmological models

NASA Astrophysics Data System (ADS)

Tangmatitham, Matipon; Nemiroff, Robert J.

2017-01-01

In the tethered galaxy problem, a hypothetical galaxy is being held at a fixed proper distance. Contrary to Newtonian intuition, it has been shown that this tethered galaxy can have a nonzero redshift. However, constant proper distance has been suggested as unphysical in a cosmological setting and therefore other definitions have been suggested. The tethered galaxy problem is therefore reviewed in Friedmann cosmology. In this work, different tethers are considered as possible local cosmological discriminators.

Orbital transfer and release of tethered payloads. Continuation of investigation of electrodynamic stabilization and control of long orbiting tethers Martinez-Sanchez, Manuel

NASA Technical Reports Server (NTRS)

Colombo, G.; Grossi, M. D.; Arnold, D.

1983-01-01

The effect of reeling operations on the orbital altitude of the tether system and the development of control laws to minimize tether rebound upon payload release were studied. The use of the tether for LEO/GEO payload orbital transfer was also investigated. It was concluded that (1) reeling operations can contribute a significant amount of energy to the orbit of the system and should be considered in orbit calculations and predictions, (2) deployment of payloads, even very large payloads, using tethers is a practical and fully stable operation, (3) tether augmented LEO/GEO transfer operations yield useful payload gains under the practical constraint of fixed size OTV's, and (4) orbit to orbit satellite retrieval is limited by useful revisit times to orbital inclinations of less than forty-five degrees.

Review of deployment technology for tethered satellite systems

NASA Astrophysics Data System (ADS)

Yu, B. S.; Wen, H.; Jin, D. P.

2018-03-01

Tethered satellite systems (TSSs) have attracted significant attention due to their potential and valuable applications for scientific research. With the development of various launched on-orbit missions, the deployment of tethers is considered a crucial technology for operation of a TSS. Both past orbiting experiments and numerical results have shown that oscillations of the deployed tether due to the Coriolis force and environmental perturbations are inevitable and that the impact between the space tether and end-body at the end of the deployment process leads to complicated nonlinear phenomena. Hence, a set of suitable control methods plays a fundamental role in tether deployment. This review article summarizes previous work on aspects of the dynamics, control, and ground-based experiments of tether deployment. The relevant basic principles, analytical expressions, simulation cases, and experimental results are presented as well.

Tethered Satellite System (TSS-1R)-Post Flight (STS-75) Engineering Performance Report

NASA Technical Reports Server (NTRS)

Lavoie, Anthony R.

1996-01-01

The first mission of the Tethered Satellite deployer was flown onboard Atlantis in 1992 during the Space Transportation System (STS) flight STS-46. Due to a mechanical interference with the level wind mechanism the satellite was only Deployed to 256 m rather than the planned 20,000 m. Other problems were also experienced during the STS-46 flight and several modifications were made to the Deployer and Satellite. STS-75 was a reflight of the Tethered Satellite System 1 (TSS-1) designated as Tethered Satellite System 1 Reflight (TSS-1 R) onboard Columbia. As on STS-46, the TSS payload consisted of the Deployer, the Satellite, 3 cargo bay mounted experiments: Shuttle Electrodynamic Tether System (SETS), Shuttle Potential and Return Electron Experiment (SPREE), Deployer Core Equipment (DCORE) 4 Satellite mounted experiments: Research on Electrodynamics Tether Effects (RETE), Research on Orbital Plasma Electrodynamics (ROPE), Satellite Core Instruments (SCORE), Tether Magnetic Field Experiment (TEMAG) and an aft flight deck camera: Tether Optical Phenomena Experiment (TOP). Following successful pre-launch, launch and pre-deployment orbital operations, the Deployer deployed the Tethered Satellite to 19,695 m at which point the tether broke within the Satellite Deployment Boom (SDB). The planned length for On-Station I (OST1) was 20,700 m The Satellite flew away from the Orbiter with the tether attached. The satellite was "safed" and placed in a limited power mode via the RF link. The Satellite was contacted periodically during overflights of ground stations. Cargo bay science activities continued for the period of time allocated to TSS-1 R operations.

Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity.

PubMed

Arntzen, Magnus Ø; Várnai, Anikó; Mackie, Roderick I; Eijsink, Vincent G H; Pope, Phillip B

2017-07-01

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

PubMed Central

Mollenhauer, Hilton H.; Zebrun, William

1960-01-01

Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

The first mission of the Tethered Satellite System

NASA Technical Reports Server (NTRS)

Powers, C. Blake (Editor); Shea, Charlotte; Mcmahan, Tracy

1992-01-01

The era of space-age tethered operations moves toward reality with the launch of Tethered Satellite System-1 (TSS-1). The primary objective of this mission is to demonstrate the technology of long tethered systems in space and to demonstrate, through scientific investigations, that such systems are useful for research.

Tethered acoustic doppler current profiler platforms for measuring streamflow

USGS Publications Warehouse

Rehmel, Michael S.; Stewart, James A.; Morlock, Scott E.

2003-01-01

A tethered-platform design with a trimaran hull and 900-megahertz radio modems is now commercially available. Continued field use has resulted in U.S. Geological Survey procedures for making tethered-platform discharge measurements, including methods for tethered-boat deployment, moving-bed tests, and measurement of edge distances.

Tethered gravity laboratories study

NASA Technical Reports Server (NTRS)

Lucchetti, F.

1989-01-01

The use is studied of tether systems to improve the lowest possible steady gravity level on the Space Station. Particular emphasis is placed by the microgravity community on the achievement of high quality microgravity conditions. The tether capability is explored for active control of the center of gravity and the analysis of possible tethered configurations.

Electrodynamic Tether

NASA Technical Reports Server (NTRS)

Johnson, Charles L. (Inventor); Ballance, Judy L. (Inventor); Welzyn, Kenneth J. (Inventor); Vaughn, Jason A. (Inventor); Lorenzini, Enrico (Inventor); Schuler, Peter S. (Inventor)

2006-01-01

A tether system for providing thrust to or power subsystems of an artificial satellite in a low earth orbit. The tether has three main sections, an insulated section connected to the satellite, a conducting section connected to the insulating section for drawing in and releasing electrons from the space plasma and a non-conducting section for providing a tension to the other sections of the tether. An oxygen resistant coating is applied to the bare wire of the conducting section as well as the insulated wires of the insulated section that prevents breakdown during tether operations in the space plasma. The insulated and bare wire sections also surround a high tensile flexible polymer core to prevent any debris from breaking the tether during use.

The tether inspection and repair experiment (TIRE)

NASA Technical Reports Server (NTRS)

Wood, George M.; Loria, Alberto; Harrison, James K.

1988-01-01

The successful development and deployment of reusable tethers for space applications will require methods for detecting, locating, and repairing damage to the tether. This requirement becomes especially important whenever the safety of the STS or the Space Station may be diminished or when critical supplies or systems would be lost in the event of a tether failure. A joint NASA/PSN study endeavor has recently been initiated to evaluate and address the problems to be solved for such an undertaking. The objectives of the Tether Inspection and Repair Experiment (TIRE) are to develop instrumentation and repair technology for specific classes of tethers defined as standards, and to demonstrate the technologies in ground-based and in-flight testing on the STS.

Space Environmental Effects on Coated Tether Materials

NASA Technical Reports Server (NTRS)

Gittemeier, Keith A.; Hawk, Clark W.; Finckenor, Miria M.; Watts, Ed

2005-01-01

The University of Alabama in Huntsville s Propulsion Research Center has teamed with NASA's Marshall Space Flight Center (MSFC) to research the effects of atomic oxygen (AO) bombardment on coated tether materials. Tethers Unlimited Inc. has provided several candidate tether materials with various coatings for AO exposure in MSFC s Atomic Oxygen Beam Facility. Additional samples were exposed to ultraviolet (UV) radiation at MSFC. AO erodes most organic materials, and ultraviolet radiation embrittles polymers. This test series was performed to determine the effect of AO and UV on the mechanical integrity of tether materials that were treated with AO-protective coatings, such as polyhedral oligomeric silsesquioxane (POSS) or metallization. Both TUI's Multi-Application Survivable Tether (MAST) Experiment and Marshall Space Flight Center s Momentum Exchange Electrodynamic Reboost (MXER) programs will benefit from this research by helping to determine tether materials and coatings that give the longest life with the lowest mass penalty.

Rigorous approaches to tether dynamics in deployment and retrieval

NASA Technical Reports Server (NTRS)

Antona, Ettore

1987-01-01

Dynamics of tethers in a linearized analysis can be considered as the superposition of propagating waves. This approach permits a new way for the analysis of tether behavior during deployment and retrieval, where a tether is composed by a part at rest and a part subjected to propagation phenomena, with the separating section depending on time. The dependence on time of the separating section requires the analysis of the reflection of the waves travelling toward the part at rest. Such a reflection generates a reflected wave, whose characteristics are determined. The propagation phenomena of major interest in a tether are transverse waves and longitudinal waves, all mathematically modelled by the vibrating chord equations, if the tension is considered constant along the tether. An interesting problem also considered is concerned with the dependence of the tether tension from the longitudinal position, due to microgravity, and the influence of this dependence on the propagation waves.

Atomic Oxygen Effects on Coated Tether Materials

NASA Technical Reports Server (NTRS)

Gittemeier, Keith A.; Hawk, Clark W.; Finckenor, Miria M.; Watts, Ed

2005-01-01

The University of Alabama in Huntsville s Propulsion Research Center has teamed with NASA's Marshall Space Flight Center (MSFC) to research the effects of atomic oxygen (AO) bombardment on coated tether materials. Tethers Unlimited Inc. has provided several candidate tether materials with various coatings for (AO) exposure in MSFC's Atomic Oxygen Beam Facility. Additional samples were exposed to ultraviolet (UV) radiation at MSFC. AO erodes most organic materials, and ultraviolet radiation embrittles polymers. This test series was performed to determine the effect of AO and UV on the mechanical integrity of tether materials that were treated with AO-protective coatings, such as Photosil or metallization. Both TUI's Multi-Application Survivable Tether (MAST) Experiment and Marshall Space Flight Center's Momentum Exchange Electrodynamic Reboost (MXER) programs will benefit from this research by helping to determine tether materials and coatings that give the longest life with the lowest mass penalty.

The development of optimal control laws for orbiting tethered platform systems

NASA Technical Reports Server (NTRS)

Bainum, P. M.; Woodard, S.; Juang, J.-N.

1986-01-01

A mathematical model of the open and closed loop in-orbit plane dynamics of a space platform-tethered-subsatellite system is developed. The system consists of a rigid platform from which an (assumed massless) tether is deploying (retrieving) a subsatellite from an attachment point which is, in general, offset from the platform's mass center. A Lagrangian formulation yields equations describing platform pitch, subsatellite tether-line swing, and varying tether length motions. These equations are linearized about the nominal station keeping motion. Control can be provided by both modulation of the tether tension level and by a momentum type platform-mounted device; system controllability depends on the presence of both control inputs. Stability criteria are developed in terms of the control law gains, the platform inertia ratio, and tether offset parameter. Control law gains are obtained based on linear quadratic regulator techniques. Typical transient responses of both the state and required control effort are presented.

Models of dynamic extraction of lipid tethers from cell membranes.

PubMed

Nowak, Sarah A; Chou, Tom

2010-05-07

When a ligand that is bound to an integral membrane receptor is pulled, the membrane and the underlying cytoskeleton can deform before either the membrane delaminates from the cytoskeleton or the ligand detaches from the receptor. If the membrane delaminates from the cytoskeleton, it may be further extruded and form a membrane tether. We develop a phenomenological model for this process by assuming that deformations obey Hooke's law up to a critical force at which the cell membrane locally detaches from the cytoskeleton and a membrane tether forms. We compute the probability of tether formation and show that tethers can be extruded only within an intermediate range of force loading rates and pulling velocities. The mean tether length that arises at the moment of ligand detachment is computed as are the force loading rates and pulling velocities that yield the longest tethers.

Ultrastructure of identified fast excitatory, slow excitatory and inhibitory neuromuscular junctions in the locust.

PubMed

Titmus, M J

1981-06-01

The specialized jumping muscle of the locust, the metathoracic extensor tibiae (ETi), is innervated by four physiologically different motoneurons, including FETi, a phasic excitor, SETi, a tonic excitor, and CI, a tonic common inhibitor. FETi neuromuscular junctions were examined in three phasic ETi bundles innervated by FETi. FETi terminals were characterized by patchy contacts on to granular sarcoplasm. The ETi accessory extensor, innervated by both SETi and CI, contains two morphologically different types of axon ending. When this muscle was soaked in horseradish peroxidase, stimulation of SETi led to selective uptake in vesicles in terminals similar to those of FETi axons but containing smaller vesicles, while stimulation by CI caused increased uptake into terminals with more extensive contact directly on to fibrillar sarcoplasm. As has been observed in excitatory and inhibitory synapses in some crustacean and vertebrate nervous systems, the synaptic vesicles in the locust excitatory endings are round and electron-lucent while those in the inhibitory endings are more irregular in shape. The tonic neuromuscular junctions, SETi and CI, are more densely packed with vesicles, larger in cross-sectional area and appear to be of more complex shape than the smaller, vesicle-sparse, phasic FETi terminals. Following long duration stimulation at 10 Hz, the tonic neuromuscular junctions showed little morphological change. FETi endings, which fatigue within minutes at the same stimulation frequency, showed a 20% decrease in synaptic vesicle density and an increase in irregularly shaped membrane inclusions.

Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study.

PubMed

Mathias, Jordan D; Ran, Yong; Carter, Jeffery D; Fanucci, Gail E

2009-09-02

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms.

PubMed

Couto, Narciso; Schooling, Sarah R; Dutcher, John R; Barber, Jill

2015-10-02

In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations.

Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.

PubMed

DeRocco, Vanessa; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A; Weninger, Keith

2010-11-01

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.

Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445

Oncoplastic Breast Reconstruction: Should All Patients be Considered?

PubMed

Habibi, Mehran; Broderick, Kristen P; Sebai, Mohamad E; Jacobs, Lisa K

2018-01-01

Oncoplastic surgery of the tissue defect from partial mastectomy should be considered for all patients. It can result in in significant asymmetries from scar contraction, skin tethering, and alterations in the nipple areolar complex location. Indications, risks, and benefits are discussed. Optimal procedures are described, considering resected specimen volume, primary tumor location, tumor to breast size ratio, and the impact on the nipple areolar complex. Indications for plastic surgery consultation and joint surgery are discussed. Surgical management includes incision planning, preservation of the nipple areolar complex pedicle and position, patient positioning, incision location, and recovery. Copyright © 2017 Elsevier Inc. All rights reserved.

Applications of Tethers in Space, Volume 2

NASA Technical Reports Server (NTRS)

Cron, A. C. (Compiler)

1985-01-01

Topics discussed include tethered satellites, tether deployment, satellite systems, science applications, electrodynamic interactions, transportation applications, artificial gravity, constellations, and technology and testing.

Coiled-coil protein composition of 22 proteomes--differences and common themes in subcellular infrastructure and traffic control.

PubMed

Rose, Annkatrin; Schraegle, Shannon J; Stahlberg, Eric A; Meier, Iris

2005-11-16

Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

PubMed

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

Surface electrostatics of lipid bilayers by EPR of a pH-sensitive spin-labeled lipid.

PubMed

Voinov, Maxim A; Rivera-Rivera, Izarys; Smirnov, Alex I

2013-01-08

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids' polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

PubMed Central

Voinov, Maxim A.; Rivera-Rivera, Izarys; Smirnov, Alex I.

2013-01-01

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. PMID:23332063

Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

PubMed Central

Rose, Annkatrin; Schraegle, Shannon J; Stahlberg, Eric A; Meier, Iris

2005-01-01

Background Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. Results Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. Conclusion MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. PMID:16288662

Tether System for Exchanging Payloads Between the International Space Station and the Lunar Surface

NASA Technical Reports Server (NTRS)

Hoyt, Robert P.

1998-01-01

Systems composed of several rotating and/or hanging tethers may provide a means of exchanging supplies between low Earth orbit facilities and lunar bases without requiring the use of propellant. This work develops methods for designing a tether system capable of repeatedly exchanging payloads between a LEO facility such as the International Space Station or a Space Business Park and a base on the lunar surface. In this system, a hanging tether extended upwards from the LEO facility, places a payload into a slightly elliptical orbit, where it is caught by a rotating tether in a higher elliptical orbit. This rotating tether then tosses the payload to the moon. At the moon, a long rotating "Lunavator" tether catches the payload and deposits it on the surface of the moon. By transporting an equal mass of lunar materials such as oxygen back down to the LEO facility through the tether transport system, the momentum and energy of the system is conserved, allowing frequent traffic between LEO and the lunar surface with minimal propellant requirements.

Comparative analysis of plant genomes allows the definition of the "Phytolongins": a novel non-SNARE longin domain protein family

PubMed Central

2009-01-01

Background Subcellular trafficking is a hallmark of eukaryotic cells. Because of their pivotal role in the process, a great deal of attention has been paid to the SNARE proteins. Most R-SNAREs, or "longins", however, also possess a highly conserved, N-terminal fold. This "longin domain" is known to play multiple roles in regulating SNARE activity and targeting via interaction with other trafficking proteins. However, the diversity and complement of longins in eukaryotes is poorly understood. Results Our comparative genome survey identified a novel family of longin-related proteins, dubbed the "Phytolongins" because they are specific to land plants. Phytolongins share with longins the N-terminal longin domain and the C-terminal transmembrane domain; however, in the central region, the SNARE motif is replaced by a novel region. Phylogenetic analysis pinpoints the Phytolongins as a derivative of the plant specific VAMP72 longin sub-family and allows elucidation of Phytolongin evolution. Conclusion "Longins" have been defined as R-SNAREs composed of both a longin domain and a SNARE motif. However, expressed gene isoforms and splice variants of longins are examples of non-SNARE motif containing longins. The discovery of Phytolongins, a family of non-SNARE longin domain proteins, together with recent evidence on the conservation of the longin-like fold in proteins involved in both vesicle fusion (e.g. the Trs20 tether) and vesicle formation (e.g. σ and μ adaptin) highlight the importance of the longin-like domain in protein trafficking and suggest that it was one of the primordial building blocks of the eukaryotic membrane-trafficking machinery. PMID:19889231

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

PubMed Central

Nikolaus, Joerg; Karatekin, Erdem

2016-01-01

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. PMID:27585113

Chromatin boundaries in budding yeast: the nuclear pore connection.

PubMed

Ishii, Kojiro; Arib, Ghislaine; Lin, Clayton; Van Houwe, Griet; Laemmli, Ulrich K

2002-05-31

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Dynamic modeling and Super-Twisting Sliding Mode Control for Tethered Space Robot

NASA Astrophysics Data System (ADS)

Zhao, Yakun; Huang, Panfeng; Zhang, Fan

2018-02-01

Recent years, tethered space capturing systems have been considered as one of the most promising solutions for active space debris removal due to the increasing threat of space debris to spacecraft and astronauts. In this paper, one of the tethered space capturing systems, Tethered Space Robot (TSR), is investigated. TSR includes a space platform, a space tether, and a gripper as the terminal device. Based on the assumptions that the platform and the gripper are point masses and the tether is rigid, inextensible and remaining straight, the dynamic model of TSR is presented, in which the disturbances from space environment is considered. According to the previous study, the in-plane and out-of-plane angles of the tether oscillate periodically although the tether is released to the desired length. A super-twisting adaptive sliding mode control scheme is designed for TSR to eliminate the vibration of the tether to assure a successful capture in station-keeping phase. Both uncontrolled and controlled situations are simulated. The simulation results show that the proposed controller is effective. Additionally, after comparing with normal sliding mode control algorithm, it is verified that the proposed control scheme can avoid the chattering of normal sliding mode control and is robust for unknown boundary perturbations.

Pathfinder

NASA Image and Video Library

2001-07-01

This photograph shows two Marshall Space Flight Center (MSFC) engineers, Mark Vaccaro (left) and Ken Welzyn, testing electrodynamic tethers in the MSFC Tether Winding and Spark Testing Facility. For 4 years, MSFC and industry partners have been developing the Propulsive Small Expendable Deployer System experiment, called ProSEDS. ProSEDS will test electrodynamic tether propulsion technology. Electrodynamic tethers are long, thin wires that collect electrical current when passing through a magnetic field. The tether works as a thruster as a magnetic field exerts a force on a current-carrying wire. Since electrodynamic tethers require no propellant, they could substantially reduce the weight of the spacecraft and provide a cost-effective method of reboosting spacecraft. The initial flight of ProSEDS is scheduled to fly aboard an Air Force Delta II rocket in the summer of 2002. In orbit, ProSEDS will deploy from a Delta II second stage. It will be a 3.1-mile (5 kilometer) long, ultrathin base-wire tether cornected with a 6.2-mile (10 kilometer) long non-conducting tether. This photograph shows Less Johnson, a scientist at MSFC, inspecting the nonconducting part of a tether as it exits a deployer similar to the one to be used in the ProSEDS experiment. The ProSEDS experiment is managed by the Space Transportation Directorate at MSFC.

Analytical investigation of the dynamics of tethered constellations in earth orbit

NASA Technical Reports Server (NTRS)

Lorenzini, Enrico C.; Gullahorn, Gordon E.; Estes, Robert D.

1988-01-01

This Quarterly Report on Tethering in Earth Orbit deals with three topics: (1) Investigation of the propagation of longitudinal and transverse waves along the upper tether. Specifically, the upper tether is modeled as three massive platforms connected by two perfectly elastic continua (tether segments). The tether attachment point to the station is assumed to vibrate both longitudinally and transversely at a given frequency. Longitudinal and transverse waves propagate along the tethers affecting the acceleration levels at the elevator and at the upper platform. The displacement and acceleration frequency-response functions at the elevator and at the upper platform are computed for both longitudinal and transverse waves. An analysis to optimize the damping time of the longitudinal dampers is also carried out in order to select optimal parameters. The analytical evaluation of the performance of tuned vs. detuned longitudinal dampers is also part of this analysis. (2) The use of the Shuttle primary Reaction Control System (RCS) thrusters for blowing away a recoiling broken tether is discussed. A microcomputer system was set up to support this operation. (3) Most of the effort in the tether plasma physics study was devoted to software development. A particle simulation code has been integrated into the Macintosh II computer system and will be utilized for studying the physics of hollow cathodes.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Evolution of insect proteomes: insights into synapse organization and synaptic vesicle life cycle

PubMed Central

Yanay, Chava; Morpurgo, Noa; Linial, Michal

2008-01-01

Background The molecular components in synapses that are essential to the life cycle of synaptic vesicles are well characterized. Nonetheless, many aspects of synaptic processes, in particular how they relate to complex behaviour, remain elusive. The genomes of flies, mosquitoes, the honeybee and the beetle are now fully sequenced and span an evolutionary breadth of about 350 million years; this provides a unique opportunity to conduct a comparative genomics study of the synapse. Results We compiled a list of 120 gene prototypes that comprise the core of presynaptic structures in insects. Insects lack several scaffolding proteins in the active zone, such as bassoon and piccollo, and the most abundant protein in the mammalian synaptic vesicle, namely synaptophysin. The pattern of evolution of synaptic protein complexes is analyzed. According to this analysis, the components of presynaptic complexes as well as proteins that take part in organelle biogenesis are tightly coordinated. Most synaptic proteins are involved in rich protein interaction networks. Overall, the number of interacting proteins and the degrees of sequence conservation between human and insects are closely correlated. Such a correlation holds for exocytotic but not for endocytotic proteins. Conclusion This comparative study of human with insects sheds light on the composition and assembly of protein complexes in the synapse. Specifically, the nature of the protein interaction graphs differentiate exocytotic from endocytotic proteins and suggest unique evolutionary constraints for each set. General principles in the design of proteins of the presynaptic site can be inferred from a comparative study of human and insect genomes. PMID:18257909

THE IN VITRO DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES

PubMed Central

Cohn, Zanvil A.; Fedorko, Martha E.; Hirsch, James G.

1966-01-01

A combined morphological, autoradiographic, and cytochemical study at the electron microscope level has been directed towards the formation of electron-opaque granules of cultured macrophages. Labeling of the membrane-bound vesicular structures of pinocytic origin was accomplished with colloidal gold. The initial uptake of gold occurred within micropinocytic vesicles. These electron-lucent vesicles subsequently fused with and discharged their contents into larger pinocytic vacuoles. Colloidal gold was homogeneously distributed in the large pinosomes. In contrast, gold was initially deposited in the periphery of preformed dense granules indicating that these structures were also in constant interaction with the external environment. Colloidal gold was not observed within the cisternae of the endoplasmic reticulum nor within the saccules or vesicles of the Golgi apparatus. There were, however, many small, gold-free vesicles, indistinguishable from Golgi vesicles, which were preferentially aligned about and appeared to fuse with the large pinosomes. The intracellular flow of leucine-H3-labeled protein was followed by electron microscopic autoradiography. After a 15 min pulse of labeled amino acid there was initial labeling of the rough endoplasmic reticulum. Subsequently, much of the label appeared in the Golgi complex. At still later time periods the cytoplasmic dense granules contained the majority of the isotope. Acid phosphatase activity was localized to the dense granules and in the majority of cells to the Golgi apparatus. It is suggested that hydrolytic enzymes are initially synthesized in the endoplasmic reticulum and are then transferred to the Golgi apparatus. Here they are packaged into small Golgi vesicles which represent the primary lysosome of macrophages. The Golgi vesicles subsequently fuse with pinosomes, thereby discharging their hydrolases and forming digestive granules or secondary lysosomes. PMID:5931922

Investigating Degassing in Felsic and Mafic Magmas by 3-D Imaging of Vesicle Pathways

NASA Astrophysics Data System (ADS)

Polacci, M.; Baker, D. R.; Piochi, M.; Mancini, L.

2009-12-01

Volatiles are the motor of volcanic eruptions. Studies of vesiculation in erupted products can provide information on how volatiles exsolve, grow and are lost from magmas as lava and tephra fragments bear the fingerprints of such processes in vesicle and crystal textures. We summarize here the results of a series of X-ray computed microtomographic experiments that were performed on about 70 volcanic specimens of mainly basaltic and trachytic compositions. A first sample suite comprises samples collected from explosive activity at persistently degassing basaltic volcanoes, namely Stromboli (Aeolian Islands), Etna (Eastern Sicily) and Ambrym (Vanuatu Islands); a second suite consists of pumice and scoria clasts from Plinian to Subplinian to Vulcanian eruptions that occurred in the Campi Flegrei caldera (Southern Italy). The tomographic images provide us with a complete 3-D view of our sampled material through which it is possible to reconstruct the geometry of the vesicle network and explore how gas was transported in the investigated magmas. We find that basaltic scoriae exhibit two types of vesicles: large (~ mm^3), coalescing vesicles with complex, convoluted shapes and small-to-intermediate sized (<~1x10^-3 mm^3), spherical to sub-spherical, poorly connected or isolated vesicles. The former vesicles were interpreted as percolation pathways for gas to flow non-explosively to the volcano crater and thought to sustain the persistent passive gas release that characterizes these volcanoes. The fact that such vesicles were found in products erupted from active basaltic volcanoes located in different tectonic settings and characterized by different explosivity strongly suggests that basaltic systems appear to follow a common degassing pathway. However, not all explosive basaltic rocks contain large, coalescing vesicles. Pumice clasts from the much more violent, dangerous and less frequent paroxysmal explosions at Stromboli do not have this type of vesicles, demonstrating that basaltic volcanoes develop different vesicle textures and therefore degassing dynamics with increasing explosive activity. Trachytic pumices from highly explosive eruptions display a much finer structure in comparison to scoriae having sub-spherical to slightly deformed large vesicles and a large population of small spherical vesicles (1x10^-3 - <1x10^-5 mm^3). These two vesicle textures were mainly ascribed to the rapid ascent of a supersaturated magma under closed-system degassing, in comparison to the open-system conditions of basaltic magmas. Large interconnected vesicles that form micro-cracks are, however, found in some denser pyroclasts from Campi Flegrei. This suggests that gas was percolating in the conduit system before the eruption and that open-system degassing may be an effective way through which gas is lost in a moderately violent manner at the crater surface in some explosive felsic eruptions. Ultimately this study reveals that 3-D imaging of volcanic rocks is an essential tool for investigating degassing conditions in erupted magmas.

The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome

PubMed Central

Xie, Shuwei; Bahl, Kriti; Reinecke, James B.; Hammond, Gerald R. V.; Naslavsky, Naava; Caplan, Steve

2016-01-01

The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous “membrane bridges.” Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC. PMID:26510502

Dynamics and stability of spinning flexible space tether systems

NASA Astrophysics Data System (ADS)

Tyc, George

This dissertation focuses on a detailed dynamical investigation of a previously unexplored tether configuration that involves a spinning two-body tethered system with flexible appendages on each end-body where the spin axis is nominally aligned along the tether. The original motivation for this work came after the flight of the first Canadian sub-orbital tether mission OEDIPUS-A in 1989 which employed this spinning tethered configuration. To everyone's surprise, one of the end-bodies was observed to exhibit a rapid divergence of its nutation angle. It was clear after this flight that there were some fundamental mechanisms associated with the interaction between the tether and the end-body that were not fully understood at that time. Hence, a Tether Dynamics Experiment (TDE) was formed and became a formal part of the scientific agenda for the follow-on mission OEDIPUS-C which flew in 1995. This dissertation describes the work that was conducted as part of the TDE and involves: theoretical investigations into the dynamics of this spinning tethered flexible body system; ground testing to validate the models and establish the tether properties; application of the models to develop a stabilization approach for OEDIPUS-C, and comparisons between theory and flight data from both OEDIPUS-A and OEDIPUS-C. Nonlinear equations of motion are developed for a spinning tethered system where the tether could be either spinning with the end-bodies or attached to small de-spun platforms on the end-bodies. Since the tether used for the OEDIPUS missions is not a string, as is often assumed, but rather a wire that has some bending stiffness, albeit small, the tether bending was also taken into account in the formulation. Two sets of ground tests are described that were used to validate the stability conditions and gain confidence in the mathematical models. One set involved hanging a body by a tether and spinning at different speeds to investigate the end-body stability. The other set used a tethered spinning end-body suspended on a set of gimbals and had a means to measure the end-body attitude in real-time. The mathematical models were then applied to investigate suitable stabilization approaches for OEDIPUS-C. In general, very good agreement was found between the theory and both the ground experiments and flight data. One of the surprising results from this work is the significance of the tether root bending effects. It is shown that it is this subtle effect that caused the rapid divergence in one of the end-bodies in the OEDIPUS-A mission which was unstable. For OEDIPUS-C, the situation was rectified by adding the booms to ensure "short term" stability and also by not spinning as rapidly. The OEDIPUS-C was very successful as all systems worked as planned and hence a superb set of flight dynamics data was collected. (Abstract shortened by UMI.)

Imaging Vesicular Traffic at the Immune Synapse.

PubMed

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Alcover, Andrés

2017-01-01

Immunological synapse formation is the result of a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, as well as intracellular vesicle traffic. Endosomal vesicle traffic ensures the targeting of the T cell receptor (TCR) and various signaling molecules to the synapse, being necessary for the generation of signaling complexes downstream of the TCR. Here we describe the microscopy imaging methods that we currently use to unveil how TCR and signaling molecules are associated with endosomal compartments and deliver their cargo to the immunological synapse.

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

PubMed Central

Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

2017-01-01

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly. DOI: http://dx.doi.org/10.7554/eLife.26691.001 PMID:28621666

Investigation of dynamic noise affecting geodynamics information in a tethered subsatellite

NASA Technical Reports Server (NTRS)

Gullahorn, G. E.

1985-01-01

Work performed as part of an investigation of noise affecting instrumentation in a tethered subsatellite, was studied. The following specific topics were addressed during the reporting period: a method for stabilizing the subsatellite against the rotational effects of atmospheric perturbation was developed; a variety of analytic studies of tether dynamics aimed at elucidating dynamic noise processes were performed; a novel mechanism for coupling longitudinal and latitudinal oscillations of the tether was discovered, and random vibration analysis for modeling the tethered subsatellite under atmospheric perturbation were studied.

Effects of tether attachments on the Shuttle/Tethered Satellite System dynamics

NASA Technical Reports Server (NTRS)

Gresham, L. L.; Rupp, C. C.

1979-01-01

The dynamics of the Shuttle Tethered Satellite System are influenced by attaching the tether at some point other than the center-of-masses of the Shuttle and the subsatellite. At the Shuttle, the tether attachment is made at the end of a boom deployed out of the payload bay. This attachment noticeably affects retrieval dynamics of the satellite pendulous motion. At the satellite, the tether attachment is assumed to be made on the circumference of the satellite. This attachment greatly affects the attitude motion of the satellite about its own center-of-mass. Computer simulation results are presented showing the effects of the Shuttle boom in a three-dimensional model and the effects of satellite attachment in a planar model.

Space Station tethered elevator system

NASA Technical Reports Server (NTRS)

Haddock, Michael H.; Anderson, Loren A.; Hosterman, K.; Decresie, E.; Miranda, P.; Hamilton, R.

1989-01-01

The optimized conceptual engineering design of a space station tethered elevator is presented. The tethered elevator is an unmanned, mobile structure which operates on a ten-kilometer tether spanning the distance between Space Station Freedom and a platform. Its capabilities include providing access to residual gravity levels, remote servicing, and transportation to any point along a tether. The report discusses the potential uses, parameters, and evolution of the spacecraft design. Emphasis is placed on the elevator's structural configuration and three major subsystem designs. First, the design of elevator robotics used to aid in elevator operations and tethered experimentation is presented. Second, the design of drive mechanisms used to propel the vehicle is discussed. Third, the design of an onboard self-sufficient power generation and transmission system is addressed.

Lipid deposits and lipo-mucosomes in human cholecystitis and epithelial metaplasia in chronic cholecystitis.

PubMed

Gilloteaux, Jacques; Tomasello, Lisa M; Elgison, Deborah A

2003-01-01

Among the inflammatory changes seen in cholecystitis, the ultrastructural alterations of the human gallbladder epithelium include lipid and lipofuscin deposits, fusions of lipid deposits and mucus-containing vesicles forming complex substructural formations called lipo-mucosomes, and microvillar changes of sparse microvilli and basal bodies. Small, lipid-laden structures, such as VLDL-like vesicles, also are fused with the mucus vesicles. Epithelial cell sloughing could liberate and add lipo-mucosomes to the biliary sludge and participate in gallstone formation. With chronic cholelithiasis, fatty degeneration of scattered epithelial cells appears to alter the epithelial lining and favors metaplastic change that could lead to other pathologic changes, including carcinoma in situ-like lesions. In addition to lipid deposition in macrophages, lipid is also incorporated in other cells and tissues of the gallbladder wall (endothelium of capillaries, smooth muscles and fibrocytes).

Stepwise Synthesis of Giant Unilamellar Vesicles on a Microfluidic Assembly Line

PubMed Central

2011-01-01

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore. PMID:21309555

Proceedings of a Workshop on the Applications of Tethers in Space, Volume 1

NASA Technical Reports Server (NTRS)

1983-01-01

Project overview; tether deployment; satellite system description; tether fundamentals; science applications; electrodynamic interactions; transportation; artificial gravity; and constellations; were described.

Isocyanide or nitrosyl complexation to hemes with varying tethered axial base ligand donors: synthesis and characterization.

PubMed

Sharma, Savita K; Kim, Hyun; Rogler, Patrick J; A Siegler, Maxime; Karlin, Kenneth D

2016-09-01

A series of ferrous-heme 2,6-dimethylphenyl isocyanide (DIMPI) and ferrous-heme mononitrosyl complexes have been synthesized and characterized. The heme portion of the complexes studied is varied with respect to the nature of the axial ligand, including complexes, where it is covalently tethered to the porphyrinate periphery. Reduced heme complexes, [(F8)Fe(II)], [(P(Py))Fe(II)], [(P(Im))Fe(II)], and [(P(ImH))Fe(II)], where F8 = tetrakis(2,6-difluorophenyl)-porphyrinate and P(Py), P(Im), and P(ImH) are partially fluorinated tetraaryl porphyrinates with covalently appended axial base pyridyl/imidazolyl or histamine moieties, were employed; P(ImH) is a new construct. Room temperature addition of DIMPI to these iron(II) complexes affords the bis-isocyanide species [(F8)Fe(II)-(DIMPI)2] in the case of [(F8)Fe(II)], while for the other hemes, mono-DIMPI compounds are obtained, [(P(Py))Fe(II)-(DIMPI)] [(2)-DIMPI], [(P(Im))Fe(II)-(DIMPI)] [(3)-DIMPI], and [(P(ImH))Fe(II)-(DIMPI)] [(4)-DIMPI]. The structures of complexes (3)-DIMPI and (4)-DIMPI have been determined by single crystal X-ray crystallography, where interesting H…F(porphryinate aryl group) interactions are observed. (19)F-NMR spectra determined for these complexes suggest that H…F(porphyrinate aryl groups) attractions also occur in solution, the H atom coming either from the DIMPI methyl groups or from a porphyinate axial base imidazole or porphyrinate pyrrole. Similarly, we have used nitrogen monoxide to generate ferrous-nitrosyl complexes, a five-coordinate species for F8, [(F8)Fe(II)-(NO)], or low-spin six-coordinate compounds [(P(Py))Fe(II)-(NO)], [(P(Im))Fe(II)-(NO)], and [(P(ImH))Fe(II)-(NO)]. The DIMPI and mononitrosyl complexes have also been characterized using UV-Vis, IR, (1)H-NMR, and EPR spectroscopies.

Mechanical properties of complex biological systems using AFM-based force spectroscopy

NASA Astrophysics Data System (ADS)

Graham, John Stephen

An atomic force microscope (AFM) was designed and built to study the mechanical properties of small collagen fibrils and the plasma membrane of living cells. Collagen is a major component of bone, skin and connective tissues, and is abundant in the extracellular matrix (ECM). Because of its abundance, an understanding of how disease affects collagen mechanics is crucial in disease prevention efforts. Two levels of type I collagen structure were investigated, subfibrils (on the order of 1 mum in length) and longer fibrils. Comparisons were made between measurements of wild-type (wt) collagen and collagen from the mouse model of osteogenesis imperfecta (OI). Significant differences between OI and wt collagen were observed, primarily that intermolecular bonds in OI collagen fibrils are weaker than in wt, or not ruptured, as in the case of OI subfibrils. As cells interact with collagen in the ECM, the mechanical properties of the plasma membrane are also of great interest. Membrane tethers were extracted from living cells under varied conditions in order to assess the contributions of membrane-associated macromolecules such as the actin cytoskeleton and the glycocalyx, and intracellular signaling. Tether extraction force was found to be sensitive to all of these altered conditions, suggesting that tether extraction may be used to monitor various cellular processes.

Assessing the Impact of Electrostatic Drag on Processive Molecular Motor Transport.

PubMed

Smith, J Darby; McKinley, Scott A

2018-06-04

The bidirectional movement of intracellular cargo is usually described as a tug-of-war among opposite-directed families of molecular motors. While tug-of-war models have enjoyed some success, recent evidence suggests underlying motor interactions are more complex than previously understood. For example, these tug-of-war models fail to predict the counterintuitive phenomenon that inhibiting one family of motors can decrease the functionality of opposite-directed transport. In this paper, we use a stochastic differential equations modeling framework to explore one proposed physical mechanism, called microtubule tethering, that could play a role in this "co-dependence" among antagonistic motors. This hypothesis includes the possibility of a trade-off: weakly bound trailing molecular motors can serve as tethers for cargoes and processing motors, thereby enhancing motor-cargo run lengths along microtubules; however, this introduces a cost of processing at a lower mean velocity. By computing the small- and large-time mean-squared displacement of our theoretical model and comparing our results to experimental observations of dynein and its "helper protein" dynactin, we find some supporting evidence for microtubule tethering interactions. We extrapolate these findings to predict how dynein-dynactin might interact with the opposite-directed kinesin motors and introduce a criterion for when the trade-off is beneficial in simple systems.

Orbital Winch for High-Strength, Space-Survivable Tethers

NASA Technical Reports Server (NTRS)

Hoyt, Robert; Barnes, Ian; Slostad, Jeffrey; Frank, Scott

2010-01-01

An Orbital Winch mechanism enables high-load, multi-line tethers to be deployed and retracted without rotating the spool on which the tether is wound. To minimize damage to the tether and the wound package during retraction or deployment under load, it can incorporate a Tension Management Module that reduces the infeed tension by a factor of 15 through the use of a powered capstan with guide rollers. This design eliminates the need for rotating high-voltage electrical connections in tether systems that use propellantless electro-dynamic propulsion. It can also eliminate the need for rotating optical connections in applications where the tether contains optical fibers. This winch design was developed to deploy a 15-km-long, 15-kg high-strength Hoytether structure incorporating conductive wires as part of the MXER-1 demonstration mission concept. Two slewing rings that orbit around the tether spool, combined with translation of one of the slewing rings back and forth along the spool axis to traverse the wind point, enables the winch to wind the tether. Variations of the traverse motion of the slewing ring can accomplish level winds and conical pirn winds. By removing the non-traversing slewing ring, and adding an actuated guide arm, the winch can manage rapid, low-drag deployment of a tether off the end of a pirn-wound spool, followed by controlled retraction and rewinding, in a manner very similar to a spin-casting reel. The winch requires at least two motor driver controller units to coordinate the action of two stepper motors to accomplish tether deployment or retraction.

Self-assembly of core-polyethylene glycol-lipid shell (CPLS) nanoparticles and their potential as drug delivery vehicles

NASA Astrophysics Data System (ADS)

Shen, Zhiqiang; Loe, David T.; Awino, Joseph K.; Kröger, Martin; Rouge, Jessica L.; Li, Ying

2016-08-01

Herein a new multifunctional formulation, referred to as a core-polyethylene glycol-lipid shell (CPLS) nanoparticle, has been proposed and studied in silico via large scale coarse-grained molecular dynamics simulations. A PEGylated core with surface tethered polyethylene glycol (PEG) chains is used as the starting configuration, where the free ends of the PEG chains are covalently bonded with lipid molecules (lipid heads). A complete lipid bilayer is formed at the surface of the PEGylated particle core upon addition of free lipids, driven by the hydrophobic properties of the lipid tails, leading to the formation of a CPLS nanoparticle. The self-assembly process is found to be sensitive to the grafting density and molecular weight of the tethered PEG chains, as well as the amount of free lipids added. At low grafting densities the assembly of CPLS nanoparticles cannot be accomplished. As demonstrated by simulations, a lipid bud/vesicle can be formed on the surface when an excess amount of free lipids is added at high grafting density. Therefore, the CPLS nanoparticles can only be formed under appropriate conditions of both PEG and free lipids. The CPLS nanoparticle has been recognized to be able to store a large quantity of water molecules, particularly with high molecular weight of PEG chains, indicating its capacity for carrying hydrophilic molecules such as therapeutic biomolecules or imaging agents. Under identical size and surface chemistry conditions of a liposome, it has been observed that the CPLS particle can be more efficiently wrapped by the lipid membrane, indicating its potential for a greater efficiency in delivering its hydrophilic cargo. As a proof-of-concept, the experimental realization of CPLS nanoparticles is explicitly demonstrated in this study. To test the capacity of the CPLS to store small molecule cargo a hydrophilic dye was successfully encapsulated in the particles' water soluble layer. The results of this study show the power and potential of simulation-driven approaches for guiding the design of more efficient nanomaterial delivery platforms.Herein a new multifunctional formulation, referred to as a core-polyethylene glycol-lipid shell (CPLS) nanoparticle, has been proposed and studied in silico via large scale coarse-grained molecular dynamics simulations. A PEGylated core with surface tethered polyethylene glycol (PEG) chains is used as the starting configuration, where the free ends of the PEG chains are covalently bonded with lipid molecules (lipid heads). A complete lipid bilayer is formed at the surface of the PEGylated particle core upon addition of free lipids, driven by the hydrophobic properties of the lipid tails, leading to the formation of a CPLS nanoparticle. The self-assembly process is found to be sensitive to the grafting density and molecular weight of the tethered PEG chains, as well as the amount of free lipids added. At low grafting densities the assembly of CPLS nanoparticles cannot be accomplished. As demonstrated by simulations, a lipid bud/vesicle can be formed on the surface when an excess amount of free lipids is added at high grafting density. Therefore, the CPLS nanoparticles can only be formed under appropriate conditions of both PEG and free lipids. The CPLS nanoparticle has been recognized to be able to store a large quantity of water molecules, particularly with high molecular weight of PEG chains, indicating its capacity for carrying hydrophilic molecules such as therapeutic biomolecules or imaging agents. Under identical size and surface chemistry conditions of a liposome, it has been observed that the CPLS particle can be more efficiently wrapped by the lipid membrane, indicating its potential for a greater efficiency in delivering its hydrophilic cargo. As a proof-of-concept, the experimental realization of CPLS nanoparticles is explicitly demonstrated in this study. To test the capacity of the CPLS to store small molecule cargo a hydrophilic dye was successfully encapsulated in the particles' water soluble layer. The results of this study show the power and potential of simulation-driven approaches for guiding the design of more efficient nanomaterial delivery platforms. Electronic supplementary information (ESI) available: Simulation protocol, simulation results for the self-assembly of CPLS nanoparticles, membrane wrapping and free energy change of grafted PEG polymers. See DOI: 10.1039/C6NR04134E

Attitude control analysis of tethered de-orbiting

NASA Astrophysics Data System (ADS)

Peters, T. V.; Briz Valero, José Francisco; Escorial Olmos, Diego; Lappas, V.; Jakowski, P.; Gray, I.; Tsourdos, A.; Schaub, H.; Biesbroek, R.

2018-05-01

The increase of satellites and rocket upper stages in low earth orbit (LEO) has also increased substantially the danger of collisions in space. Studies have shown that the problem will continue to grow unless a number of debris are removed every year. A typical active debris removal (ADR) mission scenario includes launching an active spacecraft (chaser) which will rendezvous with the inactive target (debris), capture the debris and eventually deorbit both satellites. Many concepts for the capture of the debris while keeping a connection via a tether, between the target and chaser have been investigated, including harpoons, nets, grapples and robotic arms. The paper provides an analysis on the attitude control behaviour for a tethered de-orbiting mission based on the ESA e.Deorbit reference mission, where Envisat is the debris target to be captured by a chaser using a net which is connected to the chaser with a tether. The paper provides novel insight on the feasibility of tethered de-orbiting for the various mission phases such as stabilization after capture, de-orbit burn (plus stabilization), stabilization during atmospheric pass, highlighting the importance of various critical mission parameters such as the tether material. It is shown that the selection of the appropriate tether material while using simple controllers can reduce the effort needed for tethered deorbiting and can safely control the attitude of the debris/chaser connected with a tether, without the danger of a collision.

Tethered gravity laboratories study

NASA Technical Reports Server (NTRS)

Lucchetti, F.

1990-01-01

The scope of the study is to investigate ways of controlling the microgravity environment of the International Space Station by means of a tethered system. Four main study tasks were performed. First, researchers analyzed the utilization of the tether systems to improve the lowest possible steady gravity level on the Space Station and the tether capability to actively control the center of gravity position in order to compensate for activities that would upset the mass distribution of the Station. The purpose of the second task was to evaluate the whole of the experiments performable in a variable gravity environment and the related beneficial residual accelerations, both for pure and applied research in the fields of fluid, materials, and life science, so as to assess the relevance of a variable g-level laboratory. The third task involves the Tethered Variable Gravity Laboratory. The use of the facility that would crawl along a deployed tether and expose experiments to varying intensities of reduced gravity is discussed. Last, a study performed on the Attitude Tether Stabilizer concept is discussed. The stabilization effect of ballast masses tethered to the Space Station was investigated as a means of assisting the attitude control system of the Station.

Uniform and Janus-like nanoparticles in contact with vesicles: energy landscapes and curvature-induced forces.

PubMed

Agudo-Canalejo, Jaime; Lipowsky, Reinhard

2017-03-15

Biological membranes and lipid vesicles often display complex shapes with non-uniform membrane curvature. When adhesive nanoparticles with chemically uniform surfaces come into contact with such membranes, they exhibit four different engulfment regimes as recently shown by a systematic stability analysis. Depending on the local curvature of the membrane, the particles either remain free, become partially or completely engulfed by the membrane, or display bistability between free and completely engulfed states. Here, we go beyond stability analysis and develop an analytical theory to leading order in the ratio of particle-to-vesicle size. This theory allows us to determine the local and global energy landscapes of uniform nanoparticles that are attracted towards membranes and vesicles. While the local energy landscape depends only on the local curvature of the vesicle membrane and not on the overall membrane shape, the global energy landscape describes the variation of the equilibrium state of the particle as it probes different points along the membrane surface. In particular, we find that the binding energy of a partially engulfed particle depends on the 'unperturbed' local curvature of the membrane in the absence of the particle. This curvature dependence leads to local forces that pull the partially engulfed particles towards membrane segments with lower and higher mean curvature if the particles originate from the exterior and interior solution, respectively, corresponding to endo- and exocytosis. Thus, for partial engulfment, endocytic particles undergo biased diffusion towards the membrane segments with the lowest membrane curvature, whereas exocytic particles move towards segments with the highest curvature. The curvature-induced forces are also effective for Janus particles with one adhesive and one non-adhesive surface domain. In fact, Janus particles with a strongly adhesive surface domain are always partially engulfed which implies that they provide convenient probes for experimental studies of the curvature-induced forces that arise for complex-shaped membranes.

Flowing Plasma Interaction with an Electric Sail Tether Element

NASA Technical Reports Server (NTRS)

Schneider, Todd; Vaughn, Jason; Wright, Kenneth; Anderson, Allen; Stone, Nobie

2017-01-01

Harnessing the power of the solar wind, an Electric Sail, or E-sail, is a relatively new concept that promises to deliver high speed propellant-less propulsion. The electric sail is an invention made in 2006 at the Kumpula Space Centre in Finland by Pekka Janhunen [Janhunen and Sandroos, 2007]. At its core, an electric sail utilizes multiple positively biased tethers which exchange momentum with solar wind protons via the repelling electric field established around each tether, in other words, by reflecting the solar wind protons. Recognizing the solar wind is a plasma, the effective repelling area of each tether is increased significantly by the formation a plasma sheath around each tether. Fig. 1 shows schematically a spacecraft employing an electric sail. The positive voltage bias (greater than10kV) applied to each tether naturally results in electron collection. Therefore, the electric sail concept necessarily includes an electron source (electron gun) to return collected electrons to space and maintain the positive bias of the tether system.

Tethered Satellite System Project Overview

NASA Technical Reports Server (NTRS)

Laue, J. H.

1985-01-01

The Skyhook concept is reviewed and the use of a tethered satellite system (TSS) to enable scientific investigations from the shuttle using a closed loop control system is examined. The tethered satellite system has capabilities for deployment toward or away from Earth, for multiple round trip missions, and for deployment at distances up to 100 KN from the orbiter. The deployer, which consists of an entendable boom, a reel for the tether, and the tether itself, permits deployment and retrieval at a safe distance, allows alignment of the force vector of the tether through the center of gravity of the shuttle, and gives some initial gravity gradient separation to aid in deployment and ultimate retrieval of the tethered satellite. Charts show TSS activities in terms of systems studies, key guidelines, Italian and U.S. responsibilities, user activities, and major science and applications accommodation features. Scientific objectives for TSS-1 and TSS-2 verification missions and the current status of the project are also given.

SPHERES tethered formation flight testbed: application to NASA's SPECS mission

NASA Astrophysics Data System (ADS)

Chung, Soon-Jo; Kong, Edmund M.; Miller, David W.

2005-08-01

This paper elaborates on theory and experiment of the formation flight control for the future space-borne tethered interferometers. The nonlinear equations of multi-vehicle tethered spacecraft system are derived by Lagrange equations and decoupling method. The preliminary analysis predicts unstable dynamics depending on the direction of the tether motor. The controllability analysis indicates that both array resizing and spin-up are fully controllable only by the reaction wheels and the tether motor, thereby eliminating the need for thrusters. Linear and nonlinear decentralized control techniques have been implemented into the tethered SPHERES testbed, and tested at the NASA MSFC's flat floor facility using two and three SPHERES configurations. The nonlinear control using feedback linearization technique performed successfully in both two SPHERES in-line configuration and three triangular configuration while varying the tether length. The relative metrology system, using the ultra sound metrology system and the inertial sensors as well as the decentralized nonlinear estimator, is developed to provide necessary state information.

Dynamic analysis of the tether transportation system using absolute nodal coordinate formulation

NASA Astrophysics Data System (ADS)

Sun, Xin; Xu, Ming; Zhong, Rui

2017-10-01

Long space tethers are becoming a rising concern as an alternate way for transportation in space. It benefits from fuel economizing. This paper focuses on the dynamics of the tether transportation system, which consists of two end satellites connected by a flexible tether, and a movable vehicle driven by the actuator carried by itself. The Absolute Nodal Coordinate Formulation is applied to the establishment of the equation of motion, so that the influence caused by the distributed mass and elasticity of the tether is introduced. Moreover, an approximated method for accelerating the calculation of the generalized gravitational forces on the tether is proposed by substituting the volume integral every step into summation of finite terms. Afterwards, dynamic evolutions of such a system in different configurations are illustrated using numerical simulations. The deflection of the tether and the trajectory of the crawler during the transportation is investigated. Finally, the effect on the orbit of the system due to the crawler is revealed.

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bienz, K.; Egger, D.; Troxler, M.

1990-03-01

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but didmore » not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.« less

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation

PubMed Central

Neufeld, Thomas P.

2017-01-01

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration. PMID:28100687

Liposomes entrapping β-cyclodextrin/ibuprofen inclusion complex: Role of the host and the guest on the bilayer integrity and microviscosity.

PubMed

Angelini, Guido; Campestre, Cristina; Boncompagni, Simona; Gasbarri, Carla

2017-12-01

Multilamellar vesicles (MLVs) from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared by using the dehydration-rehydration method. The β-cyclodextrin/Ibuprofen inclusion complex (β-CD/Ibu) was formed and solubilised into the aqueous compartments of the investigated vesicles. The resulting POPC MLVs entrapping β-CD/Ibu complex were essentially homogeneous in shape as demonstrated by Transmission Electron Microscopy (TEM). The liposomal stability was determined at 37.0±0.1°C by following the outflux rate of 5(6)-carboxyfluorescein (CF) at pH 7.40, while the membrane microviscosity was estimated by the ratio of the fluorescence intensities of pyrene in excimer and monomer state. The results presented herein confirm that interactions between POPC and β-CD occur and suggest that associations between POPC and Ibuprofen are also involved in the properties of the investigated liposomes. Copyright © 2017 Elsevier B.V. All rights reserved.

The investigation of tethered satellite system dynamics

NASA Technical Reports Server (NTRS)

Lorenzini, E.

1985-01-01

Progress in tethered satellite system dynamics research is reported. A retrieval rate control law with no angular feedback to investigate the system's dynamic response was studied. The initial conditions for the computer code which simulates the satellite's rotational dynamics were extended to a generic orbit. The model of the satellite thrusters was modified to simulate a pulsed thrust, by making the SKYHOOK integrator suitable for dealing with delta functions without loosing computational efficiency. Tether breaks were simulated with the high resolution computer code SLACK3. Shuttle's maneuvers were tested. The electric potential around a severed conductive tether with insulator, in the case of a tether breakage at 20 km from the Shuttle, was computed. The electrodynamic hazards due to the breakage of the TSS electrodynamic tether in a plasma are evaluated.

Design of a reusable kinetic energy absorber for an astronaut safety tether to be used during extravehicular activities on the Space Station

NASA Technical Reports Server (NTRS)

Borthwick, Dawn E.; Cronch, Daniel F.; Nixon, Glen R.

1991-01-01

The goal of this project is to design a reusable safety device for a waist tether which will absorb the kinetic energy of an astronaut drifting away from the Space Station. The safety device must limit the tension of the tether line in order to prevent damage to the astronaut's space suit or to the structure of the spacecraft. The tether currently used on shuttle missions must be replaced after the safety feature has been developed. A reusable tether for the Space Station would eliminate the need for replacement tethers, conserving space and mass. This report presents background information, scope and limitations, methods of research and development, alternative designs, a final design solution and its evaluation, and recommendations for further work.

The radiation impedance of an electrodynamic tether with end connectors

NASA Technical Reports Server (NTRS)

Hastings, Daniel E.; Wang, J.

1987-01-01

Electrodynamic tethers are wires deployed across the earth's geomagnetic field through which a current is flowing. The radiation impedance of a tether with end connectors carrying an ac current is computed from classical antenna theory. This simulates the use of a tether on a space structure. It is shown that the current flow pattern at the tether connector is critical to determining the overall radiation impedance. If the tether makes direct electrical contact with the ionosphere then radiation impedances of the order of several thousand Ohms can be expected. If the only electrical contact is through the end connectors then the impedance is only a few Ohms for a dc current rising to several tens of Ohms for an ac current with frequencies in the whistler range.

Method and apparatus for advancing tethers

DOEpatents

Zollinger, W. Thor

1998-01-01

A tether puller for advancing a tether through a channel may include a bellows assembly having a leading end fixedly attached to the tether at a first position and a trailing end fixedly attached to the tether at a second position so that the leading and trailing ends of the bellows assembly are located a substantially fixed distance apart. The bellows assembly includes a plurality of independently inflatable elements each of which may be separately inflated to an extended position and deflated to a retracted position. Each of the independently inflatable elements expands radially and axially upon inflation. An inflation system connected to the independently inflatable elements inflates and deflates selected ones of the independently inflatable elements to cause the bellows assembly to apply a tractive force to the tether and advance it in the channel.

Investigation of dynamic noise affecting geodynamics information in a tethered subsatellite

NASA Technical Reports Server (NTRS)

Gullahorn, G. E.

1984-01-01

The effects of a tethered satellite system's internal dynamics on the subsatellite were calculated including both overall motions (libration and attitude oscillations) and internal tether oscillations. The SKYHOOK tether simulation program was modified to operate with atmospheric density variations and to output quantities of interest. Techniques and software for analyzing the results were developed including noise spectral analysis. A program was begun for computing a stable configuration of a tether system subject to air drag. These configurations will be of use as initial conditions for SKYHOOK and, through linearized analysis, directly for stability and dynamical studies. A case study in which the subsatellite traverses an atmospheric density enhancement confirmed some theoretical calculations, and pointed out some aspects of the interaction with the tether system dynamics.

Development of the follicle complex and oocyte staging in red drum, Sciaenops ocellatus Linnaeus, 1776 (Perciformes, Sciaenidae).

PubMed

Grier, Harry J

2012-08-01

Pelagic egg development in red drum, Sciaenops ocellatus, is described using tiered staging. Based on mitosis and meiosis, there are five periods: Mitosis of Oogonia, Active Meiosis I, Arrested Meiosis I, Active Meiosis II, and Arrested Meiosis II. The Periods are divided into six stages: Mitotic Division of Oogonia, Chromatin Nucleolus, Primary Growth, Secondary Growth, Oocyte Maturation and Ovulation. The Chromatin Nucleolus Stage is divided into four steps: Leptotene, Zygotene, Pachytene, and Early Diplotene. Oocytes in the last step possess one nucleolus, dispersed chromatin with forming lampbrush chromosomes and lack basophilic ooplasm. The Primary Growth Stage, characterized by basophilic ooplasm and absence of yolk in oocytes, is divided into five steps: One-Nucleolus, Multiple Nucleoli, Perinucleolar, Oil Droplets, and Cortical Alveolar. During primary growth, the Balbiani body develops from nuage, enlarges and disperses throughout the ooplasm as both endoplasmic reticulum and Golgi develop within it. Secondary growth or vitellogenesis has three steps: Early Secondary Growth, Late Secondary Growth and Full-Grown. The Oocyte Maturation Stage, including ooplasmic and germinal vesicle maturation, has four steps: Eccentric Germinal Vesicle, Germinal Vesicle Migration, Germinal Vesicle Breakdown and Resumption of Meiosis when complete yolk hydration occurs. The period is Arrested Meiosis II. When folliculogenesis is completed, the ovarian follicle, an oocyte and encompassing follicle cells, is surrounded by a basement membrane and developing theca, all forming a follicle complex. After ovulation, a newly defined postovulatory follicle complex remains attached to the germinal epithelium. It is composed of a basement membrane that separates the postovulatory follicle from the postovulatory theca. Arrested Meiosis I encompasses primary and secondary growth (vitellogenesis) and includes most of oocyte maturation until the resumption of meiosis (Active Meiosis II). The last stage, Ovulation, is the emergence of the oocyte from the follicle when it becomes an egg or ovum. Copyright © 2012 Wiley Periodicals, Inc.

Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling

PubMed Central

Dobson, Katharine L.; Jackson, Claire; Balakrishnan, Saju; Bellamy, Tomas C.

2015-01-01

Background Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites—a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. Methods Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. Key Results Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine—intracellular calcium release, and cAMP signalling—had no impact on these effects. Conclusions We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections. PMID:25933382

β-COP as a Component of Transport Vesicles for HDL Apolipoprotein-Mediated Cholesterol Exocytosis

PubMed Central

Ma, Weilie; Lin, Margarita; Ding, Hang; Lin, Guorong; Zhang, Zhizhen

2016-01-01

Objective HDL and its apolipoproteins protect against atherosclerotic disease partly by removing excess cholesterol from macrophage foam cells. But the underlying mechanisms of cholesterol clearance are still not well defined. We investigated roles of vesicle trafficking of coatomer β-COP in delivering cholesterol to the cell surface during apoA-1 and apoE-mediated lipid efflux from fibroblasts and THP-1 macrophages. Methods shRNA knockout, confocal and electron microscopy and biochemical analysis were used to investigate the roles of β-COP in apolipoprotein-mediated cholesterol efflux in fibroblasts and THP-1 macrophages. Results We showed that β-COP knockdown by lentiviral shRNA resulted in reduced apoA-1-mediated cholesterol efflux, while increased cholesterol accumulation and formation of larger vesicles were observed in THP-1 macrophages by laser scanning confocal microscopy. Immunogold electron microscopy showed that β-COP appeared on the membrane protrusion complexes and colocalized with apoA-1 or apoE during cholesterol efflux. This was associated with releasing heterogeneous sizes of small particles into the culture media of THP-1 macrophage. Western blotting also showed that apoA-1 promotes β-COP translocation to the cell membrane and secretion into culture media, in which a total of 17 proteins were identified by proteomics. Moreover, β-COP exclusively associated with human plasma HDL fractions. Conclusion ApoA-1 and apoE promoted transport vesicles consisting of β-COP and other candidate proteins to exocytose cholesterol, forming the protrusion complexes on cell surface, which were then released from the cell membrane as small particles to media. PMID:26986486

Energy dissipation/transfer and stable attitude of spatial on-orbit tethered system

NASA Astrophysics Data System (ADS)

Hu, Weipeng; Song, Mingzhe; Deng, Zichen

2018-01-01

For the Tethered Satellite System, the coupling between the platform system and the solar panel is a challenge in the dynamic analysis. In this paper, the coupling dynamic behaviors of the Tethered Satellite System that is idealized as a planar flexible damping beam-spring-mass composite system are investigated via a structure-preserving method. Considering the coupling between the plane motion of the system, the oscillation of the spring and the transverse vibration of the beam, the dynamic model of the composite system is established based on the Hamiltonian variational principle. A symplectic dimensionality reduction method is proposed to decouple the dynamic system into two subsystems approximately. Employing the complex structure-preserving approach presented in our previous work, numerical iterations are performed between the two subsystems with weak damping to study the energy dissipation/transfer in the composite system, the effect of the spring stiffness on the energy distribution and the effect of the particle mass on the stability of the composite system. The numerical results show that: the energy transfer approach is uniquely determined by the initial attitude angle, while the energy dissipation speed is mainly depending on the initial attitude angle and the spring stiffness besides the weak damping. In addition, the mass ratio between the platform system and the solar panel determines the stable state as well as the time needed to reach the stable state of the composite system. The numerical approach presented in this paper provides a new way to deal with the coupling dynamic system and the conclusions obtained give some useful advices on the overall design of the Tethered Satellite System.

Space webs based on rotating tethered formations

NASA Astrophysics Data System (ADS)

Palmerini, Giovanni B.; Sgubini, Silvano; Sabatini, Marco

2009-07-01

Several on-going studies indicate the interest for large, light orbiting structures, shaped as fish nets or webs: along the ropes of the web small spacecraft can move like spiders to position and re-locate, at will, pieces of hardware devoted to specific missions. The concept could be considered as an intermediate solution between the large monolithic structure, heavy and expensive to realize, but easy to control, and the formations of satellites, where all system members are completely free and should manoeuvre in order to acquire a desired configuration. Instead, the advantage of having a "hard-but-light" link among the different grids lays in the partition of the tasks among system components and in a possible overall reduction of the control system complexity and cost. Unfortunately, there is no stable configuration for an orbiting, two-dimensional web made by light, flexible tethers which cannot support compression forces. A possible solution is to make use of centrifugal forces to pull the net, with a reduced number of simple thrusters located at the tips of the tethers to initially acquire the required spin. In this paper a dynamic analysis of a simplified rotating web is performed, in order to evaluate the spinning velocity able to satisfy the requirement for the stability of the system. The model adopted overlaps simpler elements, each of them given by a tether (made up of a number of linear finite elements) connecting two extreme bodies accommodating the spinning thrusters. The combination of these "diameter-like" elements provides the web, shaped according to the specific requirements. The net is primarily considered as subjected to Keplerian attraction and J2 and drag perturbations only, but its behaviour under thermal inputs is also investigated.

Tethering sockets and wrenches

NASA Technical Reports Server (NTRS)

Johnson, E. P.

1990-01-01

The tethering of sockets and wrenches was accomplished to improve the safety of working over motor segments. To accomplish the tethering of the sockets to the ratchets, a special design was implemented in which a groove was machined into each socket. Each socket was then fitted with a snap ring that can spin around the machined groove. The snap ring is tethered to the handle of the ratchet. All open end wrenches are also tethered to the ratchet or to the operator, depending upon the type. Tests were run to ensure that the modified tools meet torque requirements. The design was subsequently approved by Space Safety.

Selected tether applications in space: Phase 2

NASA Technical Reports Server (NTRS)

Thorsen, M. H.; Lippy, L. J.

1985-01-01

System characteristics and design requirements are assessed for tether deployment. Criteria are established for comparing alternate concepts for: (1) deployment of 220 klb space shuttle from the space station; (2) tether assisted launch of a 20,000 lb payload to geosynchronous orbit; (3) placement of the 20,000 lb AXAF into 320 nmi orbit via orbiter; (4) retrieval of 20,000 lb AXAF from 205 nmi circular orbit for maintenance and reboost to 320 nmi; and (5) tethered OMV rendezvous and retrieval of OTV returning from a geosynchronous mission. Tether deployment systems and technical issues are discussed.

Lyapunov Orbits in the Jupiter System Using Electrodynamic Tethers

NASA Technical Reports Server (NTRS)

Bokelmann, Kevin; Russell, Ryan P.; Lantoine, Gregory

2013-01-01

Various researchers have proposed the use of electrodynamic tethers for power generation and capture from interplanetary transfers. The effect of tether forces on periodic orbits in Jupiter-satellite systems are investigated. A perturbation force is added to the restricted three-body problem model and a series of simplifications allows development of a conservative system that retains the Jacobi integral. Expressions are developed to find modified locations of equilibrium positions. Modified families of Lyapunov orbits are generated as functions of tether size and Jacobi integral. Zero velocity curves and stability analyses are used to evaluate the dynamical properties of tether-modified orbits.

Protection of children in forward-facing child restraint systems during oblique side impact sled tests: Intrusion and tether effects.

PubMed

Hauschild, Hans W; Humm, John R; Pintar, Frank A; Yoganandan, Narayan; Kaufman, Bruce; Kim, Jinyong; Maltese, Matthew R; Arbogast, Kristy B

2016-09-01

Testing was conducted to quantify the kinematics, potential for head impact, and influence on head injury metrics for a center-seated Q3s in a forward-facing child restraint system (FFCRS) in oblique impacts. The influences of a tether and intruded door on these measures were explored. Nine lateral oblique sled tests were conducted on a convertible forward-facing child restraint seat (FFCRS). The FFCRSs were secured to a bench seat from a popular production small SUV at the center seating position utilizing the lower anchor and tether for children (LATCH). The vehicle seat was fixed on the sled carriage at 60° and 80° from full frontal (30° and 10° forward rotation from pure lateral) providing an oblique lateral acceleration to the Q3s and FFCRS. A structure simulating an intruded door was mounted to the near (left) side of vehicle seat. The sled input acceleration was the proposed FMVSS 213 lateral pulse scaled to a 35 km/h delta-V. Tests were conducted with and without the tether attached to the FFCRS. Results indicate the influence of the tether on kinematics and injury measures in oblique side impact crashes for a center- or far-side-seated child occupant. All tests without a tether resulted in head contact with the simulated door, and 2 tests at the less oblique angle (80°) with a tether also resulted in head contact. No head-to-door contact was observed in 2 tests utilizing a tether. High-speed video analysis showed that the head moved beyond the CRS head side wings and made contact with the simulated intruded door. Head injury criterion (HIC) 15 median values were 589 without the tether vs. 332 with the tether attached. Tests utilizing a tether had less lateral head excursion than tests without a tether (median 400 vs. 442 mm). These tests demonstrate the important role of the tether in controlling head excursion for center- or far-side-seated child occupants in oblique side impact crashes and limiting the head injury potential with an intruded door. The tether may not influence the kinematics of a near-side-seated occupant as strongly where the vehicle door or side structure interacts with the CRS and influences its motion. The results indicate that there may be an opportunity to improve child head kinematics and head protection in oblique side impacts through different CRS attachment methods and/or alternative vehicle side structure protection or padding.

Tethered subsatellite study

NASA Technical Reports Server (NTRS)

Baker, W. P.; Dunkin, J. A.; Galaboff, Z. J.; Johnston, K. D.; Kissel, R. R.; Rheinfurth, M. H.; Siebel, M. P. L.

1976-01-01

The results are presented of studies performed relating to the feasibility of deploying a subsatellite from the shuttle by means of a tether. The dynamics, the control laws, the aerodynamics, the heating, and some communication considerations of the tethered subsatellite system are considered. Nothing was found that prohibits the use of a subsatellite joined to the shuttle by a long (100 km) tether. More detailed studies directed at specific applications are recommended.

Tethers in space handbook, second edition

NASA Technical Reports Server (NTRS)

Penzo, Paul A. (Editor); Ammann, Paul W. (Editor)

1989-01-01

The Tethers in Space Handbook, Second Edition represents an update to the initial volume issued in September 1986. As originally intended, this handbook is designed to serve as a reference manual for policy makers, program managers, educators, engineers, and scientists alike. It contains information for the uninitiated, providing insight into the fundamental behavior of tethers in space. For those familiar with space tethers, it includes a summary of past and ongoing studies and programs, a complete bibliography of tether publications, and names, addresses, and phone numbers of workers in the field. Perhaps its most valuable asset is the brief description of nearly 50 tether applications which have been proposed and analyzed over the past 10 years. The great variety of these applications, from energy generation to boosting satellites to gravity wave detection is an indication that tethers will play a significant part in the future of space development. This edition of the handbook preserves the major characteristics of the original; however, some significant rearrangements and additions have been made. The first section on Tether Programs has been brought up to date, and now includes a description of TSS-2, the aerodynamic NASA/Italian Space Agency (ASI) mission. Tether Applications follows, and this section has been substantially rearranged. First, the index and cross-reference for the applications have been simplified. Also, the categories have changed slightly, with Technology and Test changed to Aerodynamics, and the Constellations category removed. In reality, tether constellations may be applicable to many of the other categories, since it is simply a different way of using tethers. Finally, to separate out those applications which are obviously in the future, a Concepts category has been added. A new section included here on Conference Summaries recognizes the fact that the tether community is growing internationally, and that meetings provide a means of rapid communication and interaction. Finally, the Bibliography section has been considerably updated to include all known references. These are listed by author and by subject and include the papers to be presented at the Third International Conference in May 1989.

Top tether effectiveness during side impacts.

PubMed

Majstorovic, Jordan; Bing, Julie; Dahle, Eric; Bolte, John; Kang, Yun-Seok

2018-02-28

Few studies have looked at the effectiveness of the top tether during side impacts. In these studies, limited anthropomorphic test device (ATD) data were collected and/or few side impact scenarios were observed. The goal of this study was to further understand the effects of the top tether on ATD responses and child restraint system (CRS) kinematics during various side impact conditions. A series of high-speed near-side and far-side sled tests were performed using the FMVSS213 side impact sled buck and Q3s ATD. Tests were performed at both 10° and 30° impacts with respect to the pure lateral direction. Two child restraints, CRS A and CRS B, were attached to the bench using flexible lower anchors. Each test scenario was performed with the presence and absence of a top tether. Instrumentation recorded Q3s responses and CRS kinematics, and the identical test scenarios with and without a top tether attachment were compared. For the far-side lateral (10°) and oblique (30°) impacts, top tether attachment increased resultant head accelerations by 8-38% and head injury criterion (HIC 15 ) values by 20-140%. However, the top tether was effective in reducing lateral head excursion by 5-25%. For near-side impacts, the top tether resulted in less than 10% increases in both resultant head acceleration and HIC 15 in the lateral impact direction. For near-side oblique impacts, the top tether increased HIC 15 by 17.3% for CRS A and decreased it by 19.5% for CRS B. However, the injury values determined from both impact conditions were below current injury assessment reference values (IARVs). Additionally, the top tether proved beneficial in preventing forward and lateral CRS rotations. The results show that the effects of the top tether on Q3s responses were dependent on impact type, impact angle, and CRS. Tether attachments that increased head accelerations and HIC 15 values were generally counterbalanced by a reduction in head excursion and CRS rotation compared to nontethered scenarios.

The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis.

PubMed

Coonrod, Emily M; Stevens, Tom H

2010-12-01

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

Biomechanical simulations of costo-vertebral and anterior vertebral body tethers for the fusionless treatment of pediatric scoliosis.

PubMed

Aubin, Carl-Éric; Clin, Julien; Rawlinson, Jeremy

2018-01-01

Compression-based fusionless tethers are an alternative to conventional surgical treatments of pediatric scoliosis. Anterior approaches place an anterior (ANT) tether on the anterolateral convexity of the deformed spine to modify growth. Posterior, or costo-vertebral (CV), approaches have not been assessed for biomechanical and corrective effectiveness. The objective was to biomechanically assess CV and ANT tethers using six patient-specific, finite element models of adolescent scoliotic patients (11.9 ± 0.7 years, Cobb 34° ± 10°). A validated algorithm simulated the growth and Hueter-Volkmann growth modulation over a period of 2 years with the CV and ANT tethers at two initial tensions (100, 200 N). The models without tethering also simulated deformity progression with Cobb angle increasing from 34° to 56°, axial rotation 11° to 13°, and kyphosis 28° to 32° (mean values). With the CV tether, the Cobb angle was reduced to 27° and 20° for tensions of 100 and 200 N, respectively, kyphosis to 21° and 19°, and no change in axial rotation. With the ANT tether, Cobb was reduced to 32° and 9° for 100 and 200 N, respectively, kyphosis unchanged, and axial rotation to 3° and 0°. While the CV tether mildly corrected the coronal curve over a 2-year growth period, it had sagittal lordosing effect, particularly with increasing initial axial rotation (>15°). The ANT tether achieved coronal correction, maintained kyphosis, and reduced the axial rotation, but over-correction was simulated at higher initial tensions. This biomechanical study captured the differences between a CV and ANT tether and indicated the variability arising from the patient-specific characteristics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:254-264, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Why Not Space Tethers?

NASA Technical Reports Server (NTRS)

Stone, Noble H.

2007-01-01

The Tethered Satellite System Space Shuttle missions, TSS-1 in 1993 and TSS-1R in 1996, were the height of space tether technology development. Since NASA's investment of some $200M and two Shuttle missions in those two pioneering missions, there have been several smaller tether flight experiments, but interest in this promising technology has waned within NASA as well as the DOD agencies. This is curious in view of the unique capabilities of space tether systems and the fact that they have been flight validated and shown to perform as, or better than, expected in earth orbit. While it is true that the TSS-1, TSS-1R and SEDS-2 missions experienced technical difficulties, the causes of these early developmental problems are now known to be design or materials flaws that are (1) unrelated to the basic viability of space tether technology, and (2) they are readily corrected. The purpose of this paper is to review the dynamic and electrodynamic fundamentals of space tethers and the unique capabilities they afford (that are enabling to certain types of space missions); to elucidate the nature, cause, and solution of the early developmental problems; and to provide an update on progress made in development of the technology. Finally, it is shown that (1) all problems experienced during early development of the technology now have solutions; and (2) the technology has been matured by advances made in strength and robustness of tether materials, high voltage engineering in the space environment, tether health and status monitoring, and the elimination of the broken tether hazard. In view of this, it is inexplicable why this flight-validated technology has not been utilized in the past decade, considering the powerful and unique capabilities that space tethers can afford that are, not only required to carryout, otherwise, unobtainable missions, but can also greatly reduce the cost of certain on-going space operations.

The kinetics of the phospholipase A2-catalyzed hydrolysis of Egg phosphatidylcholine in unilamellar vesicles. Product inhibition and its relief by serum albumin.

PubMed

Kupferberg, J P; Yokoyama, S; Kézdy, F J

1981-06-25

Only the lecithin in the outer leaflet (representing 70% of the total) of egg lecithin unilamellar vesicles is hydrolyzed by Crotalus atrox phospholipase A2. Hydrolyzed vesicles remain intact and impermeable to ionic solutes. The fatty acids produced in the hydrolysis remain on the vesicle and are only partially ionized at neutral pH due to electrostatic repulsions. About 40% of the lysolecithin product is desorbed from the vesicle. In the presence of a large excess of bovine serum albumin, the reaction is first order with respect to both the enzyme and the substrate. At 21 degrees C, pH 7.2, I = 0.16 M, and [Ca2+] = 7 mM, the second order rate constant is kex(2) = 1.5 X 10(6) M-1 s-1. In the absence of albumin, the reaction is inhibited competitively by both the monomeric (KIm = 4.5 X 10(-8) M) and micellar (nKIa = 3.7 X 10(-7) M) forms of lysolecithin ([critical micelle concentration] = 4.3 X 10(-6) M). Bovine serum albumin complexes two molecules of lysolecithin with a dissociation constant, Kb = 5 X 10(-8) M. With substoichiometric albumin, the reaction is biphasic, and, when the albumin is saturated with lysolecithin, the kinetics become similar to those observed in the absence of albumin. The action of phospholipase A2 shows that in unilamellar vesicles there is only one major lecithin conformation in the outer leaflet, or that all conformations are rapidly interconvertible.

Experiments on and Numerical Modeling of the Capture and Concentration of Transcription-Translation Machinery inside Vesicles.

PubMed

Mavelli, Fabio; Stano, Pasquale

2015-01-01

Synthetic or semi-synthetic minimal cells are those cell-like artificial compartments that are based on the encapsulation of molecules inside lipid vesicles (liposomes). Synthetic cells are currently used as primitive cell models and are very promising tools for future biotechnology. Despite the recent experimental advancements and sophistication reached in this field, the complete elucidation of many fundamental physical aspects still poses experimental and theoretical challenges. The interplay between solute capture and vesicle formation is one of the most intriguing ones. In a series of studies, we have reported that when vesicles spontaneously form in a dilute solution of proteins, ribosomes, or ribo-peptidic complexes, then, contrary to statistical predictions, it is possible to find a small fraction of liposomes (<1%) that contain a very large number of solutes, so that their local (intravesicular) concentrations largely exceed the expected value. More recently, we have demonstrated that this effect (spontaneous crowding) operates also on multimolecular mixtures, and can drive the synthesis of proteins inside vesicles, whereas the same reaction does not proceed at a measurable rate in the external bulk phase. Here we firstly introduce and discuss these already published observations. Then, we present a computational investigation of the encapsulation of transcription-translation (TX-TL) machinery inside vesicles, based on a minimal protein synthesis model and on different solute partition functions. Results show that experimental data are compatible with an entrapment model that follows a power law rather than a Gaussian distribution. The results are discussed from the viewpoint of origin of life, highlighting open questions and possible future research directions.

The space station tethered elevator system

NASA Technical Reports Server (NTRS)

Anderson, Loren A.

1989-01-01

The optimized conceptual engineering design of a space station tethered elevator is presented. The elevator is an unmanned mobile structure which operates on a ten kilometer tether spanning the distance between the Space Station and a tethered platform. Elevator capabilities include providing access to residual gravity levels, remote servicing, and transportation to any point along a tether. The potential uses, parameters, and evolution of the spacecraft design are discussed. Engineering development of the tethered elevator is the result of work conducted in the following areas: structural configurations; robotics, drive mechanisms; and power generation and transmission systems. The structural configuration of the elevator is presented. The structure supports, houses, and protects all systems on board the elevator. The implementation of robotics on board the elevator is discussed. Elevator robotics allow for the deployment, retrieval, and manipulation of tethered objects. Robotic manipulators also aid in hooking the elevator on a tether. Critical to the operation of the tethered elevator is the design of its drive mechanisms, which are discussed. Two drivers, located internal to the elevator, propel the vehicle along a tether. These modular components consist of endless toothed belts, shunt-wound motors, regenerative power braking, and computer controlled linear actuators. The designs of self-sufficient power generation and transmission systems are reviewed. Thorough research indicates all components of the elevator will operate under power provided by fuel cells. The fuel cell systems will power the vehicle at seven kilowatts continuously and twelve kilowatts maximally. A set of secondary fuel cells provides redundancy in the unlikely event of a primary system failure. Power storage exists in the form of Nickel-Hydrogen batteries capable of powering the elevator under maximum loads.

Swarms: Optimum aggregations of spacecraft

NASA Technical Reports Server (NTRS)

Mayer, H. L.

1980-01-01

Swarms are aggregations of spacecraft or elements of a space system which are cooperative in function, but physically isolated or only loosely connected. For some missions the swarm configuration may be optimum compared to a group of completely independent spacecraft or a complex rigidly integrated spacecraft or space platform. General features of swarms are induced by considering an ensemble of 26 swarms, examples ranging from Earth centered swarms for commercial application to swarms for exploring minor planets. A concept for a low altitude swarm as a substitute for a space platform is proposed and a preliminary design studied. The salient design feature is the web of tethers holding the 30 km swarm in a rigid two dimensional array in the orbital plane. A mathematical discussion and tutorial in tether technology and in some aspects of the distribution of services (mass, energy, and information to swarm elements) are included.

Harnessing bistability for directional propulsion of soft, untethered robots.

PubMed

Chen, Tian; Bilal, Osama R; Shea, Kristina; Daraio, Chiara

2018-05-29

In most macroscale robotic systems, propulsion and controls are enabled through a physical tether or complex onboard electronics and batteries. A tether simplifies the design process but limits the range of motion of the robot, while onboard controls and power supplies are heavy and complicate the design process. Here, we present a simple design principle for an untethered, soft swimming robot with preprogrammed, directional propulsion without a battery or onboard electronics. Locomotion is achieved by using actuators that harness the large displacements of bistable elements triggered by surrounding temperature changes. Powered by shape memory polymer (SMP) muscles, the bistable elements in turn actuate the robot's fins. Our robots are fabricated using a commercially available 3D printer in a single print. As a proof of concept, we show the ability to program a vessel, which can autonomously deliver a cargo and navigate back to the deployment point.

Multifunctional-layered materials for creating membrane-restricted nanodomains and nanoscale imaging

NASA Astrophysics Data System (ADS)

Srinivasan, P.

2016-01-01

Experimental platform that allows precise spatial positioning of biomolecules with an exquisite control at nanometer length scales is a valuable tool to study the molecular mechanisms of membrane bound signaling. Using micromachined thin film gold (Au) in layered architecture, it is possible to add both optical and biochemical functionalities in in vitro. Towards this goal, here, I show that docking of complementary DNA tethered giant phospholiposomes on Au surface can create membrane-restricted nanodomains. These nanodomains are critical features to dissect molecular choreography of membrane signaling complexes. The excited surface plasmon resonance modes of Au allow label-free imaging at diffraction-limited resolution of stably docked DNA tethered phospholiposomes, and lipid-detergent bicelle structures. Such multifunctional building block enables realizing rigorously controlled in vitro set-up to model membrane anchored biological signaling, besides serving as an optical tool for nanoscale imaging.

Quantifying parenchymal tethering in a finite element simulation of a human lung slice under bronchoconstriction.

PubMed

Breen, Barbara J; Donovan, Graham M; Sneyd, James; Tawhai, Merryn H

2012-08-15

Airway hyper-responsiveness (AHR), a hallmark of asthma, is a highly complex phenomenon characterised by multiple processes manifesting over a large range of length and time scales. Multiscale computational models have been derived to embody the experimental understanding of AHR. While current models differ in their derivation, a common assumption is that the increase in parenchymal tethering pressure P(teth) during airway constriction can be described using the model proposed by Lai-Fook (1979), which is based on intact lung experimental data for elastic moduli over a range of inflation pressures. Here we reexamine this relationship for consistency with a nonlinear elastic material law that has been parameterised to the pressure-volume behaviour of the intact lung. We show that the nonlinear law and Lai-Fook's relationship are consistent for small constrictions, but diverge when the constriction becomes large. Copyright © 2012 Elsevier B.V. All rights reserved.

A Stimulation Function of Synaptotagmin-1 in Ternary SNARE Complex Formation Dependent on Munc18 and Munc13

PubMed Central

Li, Yun; Wang, Shen; Li, Tianzhi; Zhu, Le; Xu, Yuanyuan; Ma, Cong

2017-01-01

The Ca2+ sensor synaptotagmin-1 (Syt1) plays an essential function in synaptic exocytosis. Recently, Syt1 has been implicated in synaptic vesicle priming, a maturation step prior to Ca2+-triggered membrane fusion that is believed to involve formation of the ternary SNARE complex and require priming proteins Munc18-1 and Munc13-1. However, the mechanisms of Syt1 in synaptic vesicle priming are still unclear. In this study, we found that Syt1 stimulates the transition from the Munc18-1/syntaxin-1 complex to the ternary SNARE complex catalyzed by Munc13-1. This stimulation can be further enhanced in a membrane-containing environment. Further, we showed that Syt1, together with Munc18-1 and Munc13-1, stimulates trans ternary SNARE complex formation on membranes in a manner resistant to disassembly factors NSF and α-SNAP. Disruption of a proposed Syt1/SNARE binding interface strongly abrogated the stimulation function of Syt1. Our results suggest that binding of Syt1 to an intermediate SNARE assembly with Munc18-1 and Munc13-1 is critical for the stimulation function of Syt1 in ternary SNARE complex formation, and this stimulation may underlie the priming function of Syt1 in synaptic exocytosis. PMID:28860966

Electric Sail (E-Sail) Tether Team

NASA Image and Video Library

2017-08-03

Electric Sail (E-Sail) Tether Team Discusses 6U CubeSat Test Article and Tether Deployment System (Right to left: Tom Bryan, Davis Hunter (student intern), Jonathan MacArthur (student intern), Charles Cowen, Mike Tinker)

Dynamics and stability of a tethered centrifuge in low earth orbit

NASA Technical Reports Server (NTRS)

Quadrelli, B. M.; Lorenzini, E. C.

1992-01-01

The three-dimensional attitude dynamics of a spaceborne tethered centrifuge for artificial gravity experiments in low earth orbit is analyzed using two different methods. First, the tethered centrifuge is modeled as a dumbbell with a straight viscoelastic tether, point tip-masses, and sophisticated environmental models such as nonspherical gravity, thermal perturbations, and a dynamic atmospheric model. The motion of the centrifuge during spin-up, de-spin, and steady-rotation is then simulated. Second, a continuum model of the tether is developed for analyzing the stability of lateral tether oscillations. Results indicate that the maximum fluctuation about the 1-g radial acceleration level is less than 0.001 g; the time required for spin-up and de-spin is less than one orbit; and lateral oscillations are stable for any practical values of the system parameters.

Method and apparatus for advancing tethers

DOEpatents

Zollinger, W.T.

1998-06-02

A tether puller for advancing a tether through a channel may include a bellows assembly having a leading end fixedly attached to the tether at a first position and a trailing end fixedly attached to the tether at a second position so that the leading and trailing ends of the bellows assembly are located a substantially fixed distance apart. The bellows assembly includes a plurality of independently inflatable elements each of which may be separately inflated to an extended position and deflated to a retracted position. Each of the independently inflatable elements expands radially and axially upon inflation. An inflation system connected to the independently inflatable elements inflates and deflates selected ones of the independently inflatable elements to cause the bellows assembly to apply a tractive force to the tether and advance it in the channel. 9 figs.

Flowing Plasma Interaction with an Electric Sail Tether Element

NASA Technical Reports Server (NTRS)

Schneider, Todd; Vaughn, Jason; Wright, Kenneth; Andersen, Allen; Stone, Nobie

2017-01-01

Electric sails are a relatively new concept for providing high speed propellant-less propulsion. Employing multiple tethers biased to high positive voltage levels (kV), electric sails are designed to gain momentum from the solar wind by repelling solar wind protons. To maximize the area of the sail that interacts with the solar wind, electric sails rely on the formation of a large plasma sheath around each small diameter tether. Motivated by interest in advancing the development of electric sails, a set of laboratory tests has been conducted to study the interaction of a drifting plasma with a sheath formed around a small diameter tether element biased at positive voltages. The laboratory test setup was created with Debye length scaling in mind to offer a path to extrapolate (via modeling) to full scale electric sail missions. Using an instrument known as a Differential Ion Flux Probe (DIFP) the interaction between a positively biased tether element and a drifting plasma has been measured for several scenarios. Clear evidence of the tether element sheath deflecting ions has been obtained. Maps of the flow angle downstream from the tether element have been made and they show the influence of the plasma sheath. Finally, electron current collection measurements have been made for a wide range of plasma conditions and tether element bias voltages. The electron collection data will have an impact on electric sail power requirements, as high voltage power supplies and electron guns will have to be sized to accommodate the electron currents collected by each tether.

The stochastic dynamics of tethered microcantilevers in a viscous fluid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robbins, Brian A.; Paul, Mark R.; Radiom, Milad

2014-10-28

We explore and quantify the coupled dynamics of a pair of micron scale cantilevers immersed in a viscous fluid that are also directly tethered to one another at their tips by a spring force. The spring force, for example, could represent the molecular stiffness or elasticity of a biomolecule or material tethered between the cantilevers. We use deterministic numerical simulations with the fluctuation-dissipation theorem to compute the stochastic dynamics of the cantilever pair for the conditions of experiment when driven only by Brownian motion. We validate our approach by comparing directly with experimental measurements in the absence of the tethermore » which shows excellent agreement. Using numerical simulations, we quantify the correlated dynamics of the cantilever pair over a range of tether stiffness. Our results quantify the sensitivity of the auto- and cross-correlations of equilibrium fluctuations in cantilever displacement to the stiffness of the tether. We show that the tether affects the magnitude of the correlations which can be used in a measurement to probe the properties of an attached tethering substance. For the configurations of current interest using micron scale cantilevers in water, we show that the magnitude of the fluid coupling between the cantilevers is sufficiently small such that the influence of the tether can be significant. Our results show that the cross-correlation is more sensitive to tether stiffness than the auto-correlation indicating that a two-cantilever measurement has improved sensitivity when compared with a measurement using a single cantilever.« less

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

PubMed

Remis, Jonathan P; Wei, Dongguang; Gorur, Amita; Zemla, Marcin; Haraga, Jessica; Allen, Simon; Witkowska, H Ewa; Costerton, J William; Berleman, James E; Auer, Manfred

2014-02-01

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

The vesicle-to-micelle transition of phosphatidylcholine vesicles induced by nonionic detergents: effects of sodium chloride, sucrose and urea.

PubMed

Walter, A; Kuehl, G; Barnes, K; VanderWaerdt, G

2000-11-23

The vesicle-to-micelle transition of egg phosphatidylcholine LUVs induced by octylglucoside was studied in buffers with 0-4 M sodium chloride, sucrose or urea. We used both light scattering and fluorescent probes to follow the lipid-detergent complexes in these buffers. The vesicle-to-micelle transition process was fundamentally the same in each solute. However, the detergent-to-lipid ratio required for micelle formation shifted in ways that depended on the aqueous solute. The partitioning of octylglucoside between the vesicles and the aqueous phase was primarily determined by the change in its critical micelle concentration (cmc) induced by each solute. Specifically, the cmc decreased in high salt and sucrose buffers but increased in high concentrations of urea. Cmc for two additional nonionic detergents, decyl- and dodecyl-maltoside, and three zwittergents (3-12, 3-14 and 3-16) were determined as a function of concentration for each of the solutes. In all cases NaCl and sucrose decreased the solubility of the detergents, whereas urea increased their solubilities. The effects clearly depended on acyl chain length in urea-containing solutions, but this dependence was less clear with increasing NaCl and sucrose concentrations. The contributions of these solutes to solubility and to interfacial interactions in the bilayers, pure and mixed micelles are considered.

Model of reversible vesicular transport with exclusion

NASA Astrophysics Data System (ADS)

Bressloff, Paul C.; Karamched, Bhargav R.

2016-08-01

A major question in neurobiology concerns the mechanics behind the motor-driven transport and delivery of vesicles to synaptic targets along the axon of a neuron. Experimental evidence suggests that the distribution of vesicles along the axon is relatively uniform and that vesicular delivery to synapses is reversible. A recent modeling study has made explicit the crucial role that reversibility in vesicular delivery to synapses plays in achieving uniformity in vesicle distribution, so called synaptic democracy (Bressloff et al 2015 Phys. Rev. Lett. 114 168101). In this paper we generalize the previous model by accounting for exclusion effects (hard-core repulsion) that may occur between molecular motor-cargo complexes (particles) moving along the same microtubule track. The resulting model takes the form of an exclusion process with four internal states, which distinguish between motile and stationary particles, and whether or not a particle is carrying vesicles. By applying a mean field approximation and an adiabatic approximation we reduce the system of ODEs describing the evolution of occupation numbers of the sites on a 1D lattice to a system of hydrodynamic equations in the continuum limit. We find that reversibility in vesicular delivery allows for synaptic democracy even in the presence of exclusion effects, although exclusion does exacerbate nonuniform distributions of vesicles in an axon when compared with a model without exclusion. We also uncover the relationship between our model and other models of exclusion processes with internal states.

Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations

PubMed Central

Stein, Hannah; Spindler, Susann; Bonakdar, Navid; Wang, Chun; Sandoghdar, Vahid

2017-01-01

The cell membrane forms a dynamic and complex barrier between the living cell and its environment. However, its in vivo studies are difficult because it consists of a high variety of lipids and proteins and is continuously reorganized by the cell. Therefore, membrane model systems with precisely controlled composition are used to investigate fundamental interactions of membrane components under well-defined conditions. Giant unilamellar vesicles (GUVs) offer a powerful model system for the cell membrane, but many previous studies have been performed in unphysiologically low ionic strength solutions which might lead to altered membrane properties, protein stability and lipid-protein interaction. In the present work, we give an overview of the existing methods for GUV production and present our efforts on forming single, free floating vesicles up to several tens of μm in diameter and at high yield in various buffer solutions with physiological ionic strength and pH. PMID:28243205

GEMINI-TITAN (GT)-11 - MISC. - EXPERIMENT D-16 - KNEE TETHER - MSC

NASA Image and Video Library

1966-01-28

S66-00933 (28 Jan. 1966) --- Gemini-11 Experiment D-16 Knee Tether, sponsored by the Department of Defense and the United States Air Force. The astronaut tightens and loosens bolts in a prescribed pattern during his extravehicular activity, once with his body held to the spacecraft by a nine-inch tether looped around his knee and through the handrail, and once without the tether. Photo credit: NASA

Space tether dynamics: an introduction

NASA Astrophysics Data System (ADS)

Denny, Mark

2018-05-01

The dynamics of orbiting tethers (space elevators and skyhooks) is developed from an unusual direction: Lagrangian rather than Newtonian mechanics. These basic results are derived among others: space elevator required length with and without counterweight, location and magnitude of maximum tether tension, skyhook orbital parameters and tether tension. These conceptual devices are being increasingly discussed as technologically feasible; here they make an interesting pedagogical application of Lagrangian mechanics suitable for undergraduate physics students.

Design and fabrication of the 20 km/10 kV electromechanical tether for TSS-1 using high impact conductor (Hiwire)

NASA Technical Reports Server (NTRS)

Scala, E.; Bentley, D. P.; Marshall, L. S.

1986-01-01

The development of a 20-km electromechanical tether for the tethered satellite system (TSS) is described. The basic design requirements for electromagnetic cables and for conductors in cables subject to stresses and cyclic loading are discussed. The tether fabricatioon procedures involve: (1) conductor twisting around the core, (2) insulation extrusion, (3) strength member braiding, and (4) protective jacket braiding.