Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

PubMed Central

Fox, Philip D.; Haberkorn, Christopher J.; Weigel, Aubrey V.; Higgins, Jenny L.; Akin, Elizabeth J.; Kennedy, Matthew J.; Krapf, Diego; Tamkun, Michael M.

2013-01-01

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking. PMID:23864710

Mitochondrial cardiolipin/phospholipid trafficking: the role of membrane contact site complexes and lipid transfer proteins.

PubMed

Schlattner, Uwe; Tokarska-Schlattner, Malgorzata; Rousseau, Denis; Boissan, Mathieu; Mannella, Carmen; Epand, Richard; Lacombe, Marie-Lise

2014-04-01

Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2016-02-12

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell biology symposium: Membrane trafficking and signal transduction

USDA-ARS?s Scientific Manuscript database

In general, membrane trafficking is a broad group of processes where proteins and other large molecules are distributed throughout the cell as well as adjacent extracellular spaces. Whereas signal transduction is a process where signals are transmitted through a series of chemical or molecular event...

Trafficking Ion Transporters to the Apical Membrane of Polarized Intestinal Enterocytes.

PubMed

Engevik, Amy Christine; Goldenring, James R

2018-01-02

Epithelial cells lining the gastrointestinal tract require distinct apical and basolateral domains to function properly. Trafficking and insertion of enzymes and transporters into the apical brush border of intestinal epithelial cells is essential for effective digestion and absorption of nutrients. Specific critical ion transporters are delivered to the apical brush border to facilitate fluid and electrolyte uptake. Maintenance of these apical transporters requires both targeted delivery and regulated membrane recycling. Examination of altered apical trafficking in patients with Microvillus Inclusion disease caused by inactivating mutations in MYO5B has led to insights into the regulation of apical trafficking by elements of the apical recycling system. Modeling of MYO5B loss in cell culture and animal models has led to recognition of Rab11a and Rab8a as critical regulators of apical brush border function. All of these studies show the importance of apical membrane trafficking dynamics in maintenance of polarized epithelial cell function. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

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.

Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading

PubMed Central

Wurtzel, Jeremy G.T.; Kumar, Puneet; Goldfinger, Lawrence E.

2012-01-01

In this study we investigated the dynamics of R-Ras intracellular trafficking and its contributions to the unique roles of R-Ras in membrane ruffling and cell spreading. Wild type and constitutively active R-Ras localized to membranes of both Rab11- and transferrin-positive and -negative vesicles, which trafficked anterograde to the leading edge in migrating cells. H-Ras also co-localized with R-Ras in many of these vesicles in the vicinity of the Golgi, but R-Ras and H-Ras vesicles segregated proximal to the leading edge, in a manner dictated by the C-terminal membrane-targeting sequences. These segregated vesicle trafficking patterns corresponded to distinct modes of targeting to membrane ruffles at the leading edge. Geranylgeranylation was required for membrane anchorage of R-Ras, whereas palmitoylation was required for exit from the Golgi in post-Golgi vesicle membranes and trafficking to the plasma membrane. R-Ras vesicle membranes did not contain phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), whereas R-Ras co-localized with PtdIns(3,4,5)P3 in membrane ruffles. Finally, palmitoylation-deficient R-Ras blocked membrane ruffling, R-Ras/PI3-kinase interaction, enrichment of PtdIns(3,4,5)P3 at the plasma membrane, and R-Ras-dependent cell spreading. Thus, lipid modification of R-Ras dictates its vesicle trafficking, targeting to membrane ruffles, and its unique roles in localizing PtdIns(3,4,5)P3 to ruffles and promoting cell spreading. PMID:22751447

Insights into plant plasma membrane aquaporin trafficking.

PubMed

Hachez, Charles; Besserer, Arnaud; Chevalier, Adrien S; Chaumont, François

2013-06-01

Plasma membrane intrinsic proteins (PIPs) are plant aquaporins that facilitate the diffusion of water and small uncharged solutes through the cell membrane. Deciphering the network of interacting proteins that modulate PIP trafficking to and activity in the plasma membrane is essential to improve our knowledge about PIP regulation and function. This review highlights the most recent advances related to PIP subcellular routing and dynamic redistribution, identifies some key molecular interacting proteins, and indicates exciting directions for future research in this field. A better understanding of the mechanisms by which plants optimize water movement might help in identifying new molecular players of agronomical relevance involved in the control of cellular water uptake and drought tolerance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

PubMed

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Common α2A and α2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking.

PubMed

Hurt, Carl M; Sorensen, Matt W; Angelotti, Timothy

2014-06-01

Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.

Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells.

PubMed

Offringa, Remko; Huang, Fang

2013-09-01

In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples. © 2013 Institute of Botany, Chinese Academy of Sciences.

Multiple roles for membrane-associated protein trafficking and signaling in gravitropism

PubMed Central

Strohm, Allison K.; Baldwin, Katherine L.; Masson, Patrick H.

2012-01-01

Gravitropism is a process that allows plant organs to guide their growth relative to the gravity vector. It requires them to sense changes in their orientation and generate a biochemical signal that they transmit to the tissues that drive organ curvature. Trafficking between the plasma membrane and endosomal compartments is important for all of these phases of the gravitropic response. The sedimentation of starch-filled organelles called amyloplasts plays a key role in sensing reorientation, and vacuolar integrity is required for amyloplast sedimentation in shoots. Other proteins associated with the vesicle trafficking pathway contribute to early gravity signal transduction independently of amyloplast sedimentation in both roots and hypocotyls. Phosphatidylinositol signaling, which starts at the plasma membrane and later affects the localization of auxin efflux facilitators, is a likely second messenger in the signal transduction phase of gravitropism. Finally, membrane-localized auxin influx and efflux facilitators contribute to a differential auxin gradient across the gravistimulated organs, which directs root curvature. PMID:23248632

Multiple roles for membrane-associated protein trafficking and signaling in gravitropism.

PubMed

Strohm, Allison K; Baldwin, Katherine L; Masson, Patrick H

2012-01-01

Gravitropism is a process that allows plant organs to guide their growth relative to the gravity vector. It requires them to sense changes in their orientation and generate a biochemical signal that they transmit to the tissues that drive organ curvature. Trafficking between the plasma membrane and endosomal compartments is important for all of these phases of the gravitropic response. The sedimentation of starch-filled organelles called amyloplasts plays a key role in sensing reorientation, and vacuolar integrity is required for amyloplast sedimentation in shoots. Other proteins associated with the vesicle trafficking pathway contribute to early gravity signal transduction independently of amyloplast sedimentation in both roots and hypocotyls. Phosphatidylinositol signaling, which starts at the plasma membrane and later affects the localization of auxin efflux facilitators, is a likely second messenger in the signal transduction phase of gravitropism. Finally, membrane-localized auxin influx and efflux facilitators contribute to a differential auxin gradient across the gravistimulated organs, which directs root curvature.

Inhibition of phosphatidylinositol 3,5-bisphosphate production has pleiotropic effects on various membrane trafficking routes in Arabidopsis.

PubMed

Hirano, Tomoko; Munnik, Teun; Sato, Masa H

2017-01-01

Phosphoinositides play an important role in various membrane trafficking events in eukaryotes. One of them, however, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], has not been studied widely in plants. Using a combination of fluorescent reporter proteins and the PI(3,5)P2-specific inhibitor YM202636, here we demonstrated that in Arabidopsis thaliana, PI(3,5)P2 affects various membrane trafficking events, mostly in the post-Golgi routes. We found that YM201636 treatment effectively reduced PI(3,5)P2 concentration not only in the wild type but also in FAB1A-overexpressing Arabidopsis plants. In particular, reduced PI(3,5)P2 levels caused abnormal membrane dynamics of plasma membrane proteins, AUX1 and BOR1, with different trafficking patterns. Secretion and morphological characteristics of late endosomes and vacuoles were also affected by the decreased PI(3,5)P2 production. These pleiotropic defects in the post-Golgi trafficking events were caused by the inhibition of PI(3,5)P2 production. This effect is probably mediated by the inhibition of maturation of FAB1-positive late endosomes, thereby impairing late endosome function. In conclusion, our results imply that in Arabidopsis, late endosomes are involved in multiple post-Golgi membrane trafficking routes including not only vacuolar trafficking and endocytosis but also secretion. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway.

PubMed

Hein, Zeynep; Hooper, Nigel M; Naim, Hassan Y

2009-01-15

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

PubMed Central

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles. PMID:28462015

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum.

PubMed

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona; Patankar, Swati

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPx Gl ) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF 4 - and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPx Gl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPx Gl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPx Gl ; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.

A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane.

PubMed

Bryant, Kirsten L; Baird, Barbara; Holowka, David

2015-02-27

Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. To investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking. Taken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.

Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

PubMed

Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

2014-01-01

The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

PubMed Central

Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

2010-01-01

β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans.

PubMed

Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V; Nechipurenko, Inna V; Blacque, Oliver E; Sengupta, Piali

2013-04-01

The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs.

From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

PubMed

Farinha, Carlos M; Canato, Sara

2017-01-01

CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

PubMed

Chevalier, Adrien S; Chaumont, François

2015-05-01

Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Trafficking to the apical and basolateral membranes in polarized epithelial cells.

PubMed

Stoops, Emily H; Caplan, Michael J

2014-07-01

Renal epithelial cells must maintain distinct protein compositions in their apical and basolateral membranes in order to perform their transport functions. The creation of these polarized protein distributions depends on sorting signals that designate the trafficking route and site of ultimate functional residence for each protein. Segregation of newly synthesized apical and basolateral proteins into distinct carrier vesicles can occur at the trans-Golgi network, recycling endosomes, or a growing assortment of stations along the cellular trafficking pathway. The nature of the specific sorting signal and the mechanism through which it is interpreted can influence the route a protein takes through the cell. Cell type-specific variations in the targeting motifs of a protein, as are evident for Na,K-ATPase, demonstrate a remarkable capacity to adapt sorting pathways to different developmental states or physiologic requirements. This review summarizes our current understanding of apical and basolateral trafficking routes in polarized epithelial cells. Copyright © 2014 by the American Society of Nephrology.

Trafficking to the Apical and Basolateral Membranes in Polarized Epithelial Cells

PubMed Central

Stoops, Emily H.

2014-01-01

Renal epithelial cells must maintain distinct protein compositions in their apical and basolateral membranes in order to perform their transport functions. The creation of these polarized protein distributions depends on sorting signals that designate the trafficking route and site of ultimate functional residence for each protein. Segregation of newly synthesized apical and basolateral proteins into distinct carrier vesicles can occur at the trans-Golgi network, recycling endosomes, or a growing assortment of stations along the cellular trafficking pathway. The nature of the specific sorting signal and the mechanism through which it is interpreted can influence the route a protein takes through the cell. Cell type–specific variations in the targeting motifs of a protein, as are evident for Na,K-ATPase, demonstrate a remarkable capacity to adapt sorting pathways to different developmental states or physiologic requirements. This review summarizes our current understanding of apical and basolateral trafficking routes in polarized epithelial cells. PMID:24652803

One-way membrane trafficking of SOS in receptor-triggered Ras activation.

PubMed

Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

2016-09-01

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

One-way membrane trafficking of SOS in receptor-triggered Ras activation

PubMed Central

Christensen, Sune M.; Tu, Hsiung-Lin; Jun, Jesse E.; Alvarez, Steven; Triplet, Meredith G.; Iwig, Jeffrey S.; Yadav, Kamlesh K.; Bar-Sagi, Dafna; Roose, Jeroen P.; Groves, Jay T.

2016-01-01

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane-recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2:SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted membrane experiments, these Grb2-independent interactions are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis. PMID:27501536

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane.

PubMed

Zhang, Liang; Trushin, Sergey; Christensen, Trace A; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E; Howell, Kyle G; Poduslo, Joseph F; Trushina, Eugenia

2018-06-01

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. Copyright © 2018 The Authors. Published by

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane

PubMed Central

Zhang, Liang; Trushin, Sergey; Christensen, Trace A.; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E.; Howell, Kyle G.; Poduslo, Joseph F.; Trushina, Eugenia

2018-01-01

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer’s Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. PMID:29477640

Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability.

PubMed

Hachez, Charles; Laloux, Timothée; Reinhardt, Hagen; Cavez, Damien; Degand, Hervé; Grefen, Christopher; De Rycke, Riet; Inzé, Dirk; Blatt, Michael R; Russinova, Eugenia; Chaumont, François

2014-07-01

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane. © 2014 American Society of Plant Biologists. All rights reserved.

Palaeontological evidence of membrane relationship in step-by-step membrane fusion

PubMed Central

WANG, XIN; LIU, WENZHE; DU, KAIHE

2011-01-01

Studies on membrane fusion in living cells indicate that initiation of membrane fusion is a transient and hard to capture process. Despite previous research, membrane behaviour at this point is still poorly understood. Recent palaeobotanical research has revealed snapshots of membrane fusion in a 15-million-year-old fossil pinaceous cone. To reveal the membrane behaviour during the fusion, we conducted more observations on the same fossil material. Several discernible steps of membrane fusion have been fixed naturally and observed in the fossil material. This observation provides transmission electron microscope (TEM) images of the transient intermediate stage and clearly shows the relationship between membranes. Observing such a transient phenomenon in fossil material implies that the fixing was most likely accomplished quickly by a natural process. The mechanism behind this phenomenon is clearly worthy of further enquiry. PMID:21190428

Role of Passive Diffusion, Transporters, and Membrane Trafficking-Mediated Processes in Cellular Drug Transport.

PubMed

Cocucci, E; Kim, J Y; Bai, Y; Pabla, N

2017-01-01

Intracellular drug accumulation is thought to be dictated by two major processes, passive diffusion through the lipid membrane or membrane transporters. The relative role played by these distinct processes remains actively debated. Moreover, the role of membrane-trafficking in drug transport remains underappreciated and unexplored. Here we discuss the distinct processes involved in cellular drug distribution and propose that better experimental models are required to elucidate the differential contributions of various processes in intracellular drug accumulation. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Fluvastatin delays propagation of viral infection in isolated rat FDB myofibers but does not affect exocytic membrane trafficking.

PubMed

Nevalainen, Mika; Metsikkö, Kalervo

2015-11-01

We have utilized the enveloped viral model to study the effect of fluvastatin on membrane trafficking in isolated rat myofibers. Our immunofluorescence studies constantly showed that infections in myofibers, which were treated with fluvastatin prior and during the infection with either vesicular stomatitis virus (VSV) or influenza A virus, propagated more slowly than in control myofibers without drug treatment. Experiments with a virus expressing Dad1 tagged with green fluorescent protein (GFP-Dad1) showed that fluvastatin did not affect its distribution within the ER/SR network and immunofluorescence staining for GM130 did not show any marked effect on the structure of the Golgi components. Furthermore, fluvastatin did not inhibit trafficking of the chimeric transport marker VSV temperature sensitive G protein (tsG-GFP) from the ER to the Golgi. We next subjected VSV infected myofibers for pulse-chase labeling experiments and found that fluvastatin did not slow down the ER-to-Golgi trafficking or Golgi to plasma membrane trafficking of the viral glycoprotein. These studies show that fluvastatin inhibited the propagation of viral infection in skeletal myofibers but no adverse effect on the exocytic trafficking could be demonstrated. These results suggest that other effects of statins rather than inhibition of ER-to-Golgi trafficking might be behind the myotoxic effects of the statins. © 2015 International Federation for Cell Biology.

Lysosomal trafficking regulator Lyst links membrane trafficking to toll-like receptor–mediated inflammatory responses

PubMed Central

Krautkrämer, Martina

2017-01-01

Subcellular compartmentalization of receptor signaling is an emerging principle in innate immunity. However, the functional integration of receptor signaling pathways into membrane trafficking routes and its physiological relevance for immune responses is still largely unclear. In this study, using Lyst-mutant beige mice, we show that lysosomal trafficking regulator Lyst links endolysosomal organization to the selective control of toll-like receptor 3 (TLR3)– and TLR4-mediated proinflammatory responses. Consequently, Lyst-mutant mice showed increased susceptibility to bacterial infection and were largely resistant to endotoxin-induced septic shock. Mechanistic analysis revealed that Lyst specifically controls TLR3- and TLR4-induced endosomal TRIF (TIR domain–containing adapter-inducing interferon β) signaling pathways. Loss of functional Lyst leads to dysregulated phagosomal maturation, resulting in a failure to form an activation-induced Rab7+ endosomal/phagosomal compartment. This specific Rab7+ compartment was further demonstrated to serve as a major site for active TRIF signaling events, thus linking phagosomal maturation to specific TLR signaling pathways. The immunoregulatory role of Lyst on TLR signaling pathways was confirmed in human cells by CRISPR/Cas9-mediated gene inactivation. As mutations in LYST cause human Chédiak-Higashi syndrome, a severe immunodeficiency, our findings also contribute to a better understanding of human disease mechanisms. PMID:27881733

EMBO workshop al fin del mundo: a meeting on membrane trafficking and its implication for polarity and diseases.

PubMed

Marzolo, María-Paz; Faundez, Victor; Galli, Thierry

2015-07-01

The EMBO worskhop at the "end of the world'" (al fin del mundo), a meeting on membrane trafficking and its implication for polarity and diseases, took place in the Chilean Patagonia surrounded by the landscapes once witnessed by Charles Darwin. The meeting showcased some of the best membrane trafficking science with an emphasis in neuroscience and disease models. Speakers from Europe, USA, South America and the graduate students behind it; embarked on an enthusiastic and eclectic dialog where a wide range of cell types, model genetic systems, and diseases where discussed. This meeting demonstrated the power of trafficking concepts to integrate diverse biology and to formulate mechanisms of normal and disease cells. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-β2.

PubMed

Roussa, Eleni; Speer, Jan Manuel; Chudotvorova, Ilona; Khakipoor, Shokoufeh; Smirnov, Sergei; Rivera, Claudio; Krieglstein, Kerstin

2016-09-15

Functional activation of the neuronal K(+)-Cl(-) co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor β2 (TGF-β2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-β2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl(-) extrusion. We also identify the signaling pathway from TGF-β2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-β2-mediated KCC2 trafficking and functional activation. TGF-β2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-β2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-β2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission. © 2016. Published by The Company of Biologists Ltd.

The RING finger E3 ligase STRF1 is involved in membrane trafficking and modulates salt-stress response in Arabidopsis thaliana.

PubMed

Tian, Miaomiao; Lou, Lijuan; Liu, Lijing; Yu, Feifei; Zhao, Qingzhen; Zhang, Huawei; Wu, Yaorong; Tang, Sanyuan; Xia, Ran; Zhu, Baoge; Serino, Giovanna; Xie, Qi

2015-04-01

Salt stress is a detrimental factor for plant growth and development. The response to salt stress has been shown to involve components in the intracellular trafficking system, as well as components of the ubiquitin-proteasome system (UPS). In this article, we have identified in Arabidopsis thaliana a little reported ubiquitin ligase involved in salt-stress response, which we named STRF1 (Salt Tolerance RING Finger 1). STRF1 is a member of RING-H2 finger proteins and we demonstrate that it has ubiquitin ligase activity in vitro. We also show that STRF1 localizes mainly at the plasma membrane and at the intracellular endosomes. strf1-1 loss-of-function mutant seedlings exhibit accelerated endocytosis in roots, and have altered expression of several genes involved in the membrane trafficking system. Moreover, protein trafficking inhibitor, brefeldin A (BFA), treatment has increased BFA bodies in strf1-1 mutant. This mutant also showed increased tolerance to salt, ionic and osmotic stresses, reduced accumulation of reactive oxygen species during salt stress, and increased expression of AtRbohD, which encodes a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase involved in H2 O2 production. We conclude that STRF1 is a membrane trafficking-related ubiquitin ligase, which helps the plant to respond to salt stress by monitoring intracellular membrane trafficking and reactive oxygen species (ROS) production. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

PubMed

Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

2005-03-29

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors

PubMed Central

Gesemann, Matthias; Mateos, José M.; Barmettler, Gery; Forbes, Austin; Ziegler, Urs

2017-01-01

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

PubMed

Ojeda Naharros, Irene; Gesemann, Matthias; Mateos, José M; Barmettler, Gery; Forbes, Austin; Ziegler, Urs; Neuhauss, Stephan C F; Bachmann-Gagescu, Ruxandra

2017-12-01

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

Cdk1-dependent control of membrane-trafficking dynamics

PubMed Central

McCusker, Derek; Royou, Anne; Velours, Christophe; Kellogg, Douglas

2012-01-01

Cyclin-dependent kinase 1 (Cdk1) is required for initiation and maintenance of polarized cell growth in budding yeast. Cdk1 activates Rho-family GTPases, which polarize the actin cytoskeleton for delivery of membrane to growth sites via the secretory pathway. Here we investigate whether Cdk1 plays additional roles in the initiation and maintenance of polarized cell growth. We find that inhibition of Cdk1 causes a cell surface growth defect that is as severe as that caused by actin depolymerization. However, unlike actin depolymerization, Cdk1 inhibition does not result in a massive accumulation of intracellular secretory vesicles or their cargoes. Analysis of post-Golgi vesicle dynamics after Cdk1 inhibition demonstrates that exocytic vesicles are rapidly mistargeted away from the growing bud, possibly to the endomembrane/vacuolar system. Inhibition of Cdk1 also causes defects in the organization of endocytic and exocytic zones at the site of growth. Cdk1 thus modulates membrane-trafficking dynamics, which is likely to play an important role in coordinating cell surface growth with cell cycle progression. PMID:22767578

The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

PubMed

Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

2017-04-20

Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

PubMed

Rudell, Jolene Chang; Borges, Lucia S; Rudell, John B; Beck, Kenneth A; Ferns, Michael J

2014-01-03

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

GOLGI IN COPPER HOMEOSTASIS: A VIEW FROM THE MEMBRANE TRAFFICKING FIELD

PubMed Central

Polishchuk, Roman; Lutsenko, Svetlana

2013-01-01

Copper is essential for a variety of important biological processes as a cofactor and regulator of many enzymes. Incorporation of copper into the secreted and plasma membrane-targeted cuproenzymes takes place in Golgi, a compartment central for normal copper homeostasis. The Golgi complex harbors copper-transporting ATPases, ATP7A and ATP7B, that transfer copper from the cytosol into Golgi lumen for incorporation into copper-dependent enzymes. The Golgi complex also sends these ATPases to appropriate post-Golgi destinations to ensure correct Cu fluxes in the body and to avoid potentially toxic copper accumulation. Mutations in ATP7A or ATP7B or in the proteins that regulate their trafficking affect their exit from Golgi or subsequent retrieval to this organelle. This, in turn, disrupts the homeostatic Cu balance, resulting in copper deficiency (Menkes disease) or copper overload (Wilson disease). Research over the last decade has yielded significant insights into the enzymatic properties and cell biology of the copper-ATPases. However, the mechanisms through which the Golgi regulates trafficking of ATP7A/7B and, therefore, maintain Cu homeostasis remain unclear. This review summarizes current data on the role of the Golgi in Cu metabolism and outlines questions and challenges that should be addressed to understand ATP7A and ATP7B trafficking mechanisms in health and disease. PMID:23846821

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

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

Kita, Ayako; Higa, Mari; Doi, Akira

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis withmore » multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.« less

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.

Desmoglein 3 regulates membrane trafficking of cadherins, an implication in cell-cell adhesion.

PubMed

Moftah, Hanan; Dias, Kasuni; Apu, Ehsanul Hoque; Liu, Li; Uttagomol, Jutamas; Bergmeier, Lesley; Kermorgant, Stephanie; Wan, Hong

2017-05-04

E-cadherin mediated cell-cell adhesion plays a critical role in epithelial cell polarization and morphogenesis. Our recent studies suggest that the desmosomal cadherin, desmoglein 3 (Dsg3) cross talks with E-cadherin and regulates its adhesive function in differentiating keratinocytes. However, the underlying mechanism remains not fully elucidated. Since E-cadherin trafficking has been recognized to be a central determinant in cell-cell adhesion and homeostasis we hypothesize that Dsg3 may play a role in regulating E-cadherin trafficking and hence the cell-cell adhesion. Here we investigated this hypothesis in cells with loss of Dsg3 function through RNAi mediated Dsg3 knockdown or the stable expression of the truncated mutant Dsg3ΔC. Our results showed that loss of Dsg3 resulted in compromised cell-cell adhesion and reduction of adherens junction and desmosome protein expression as well as the cortical F-actin formation. As a consequence, cells failed to polarize but instead displayed aberrant cell flattening. Furthermore, retardation of E-cadherin internalization and recycling was consistently observed in these cells during the process of calcium induced junction assembling. In contrast, enhanced cadherin endocytosis was detected in cells with overexpression of Dsg3 compared to control cells. Importantly, this altered cadherin trafficking was found to be coincided with the reduced expression and activity of Rab proteins, including Rab5, Rab7 and Rab11 which are known to be involved in E-cadherin trafficking. Taken together, our findings suggest that Dsg3 functions as a key in cell-cell adhesion through at least a mechanism of regulating E-cadherin membrane trafficking.

Desmoglein 3 regulates membrane trafficking of cadherins, an implication in cell-cell adhesion

PubMed Central

Moftah, Hanan; Dias, Kasuni; Apu, Ehsanul Hoque; Liu, Li; Uttagomol, Jutamas; Bergmeier, Lesley; Kermorgant, Stephanie; Wan, Hong

2017-01-01

ABSTRACT E-cadherin mediated cell-cell adhesion plays a critical role in epithelial cell polarization and morphogenesis. Our recent studies suggest that the desmosomal cadherin, desmoglein 3 (Dsg3) cross talks with E-cadherin and regulates its adhesive function in differentiating keratinocytes. However, the underlying mechanism remains not fully elucidated. Since E-cadherin trafficking has been recognized to be a central determinant in cell-cell adhesion and homeostasis we hypothesize that Dsg3 may play a role in regulating E-cadherin trafficking and hence the cell-cell adhesion. Here we investigated this hypothesis in cells with loss of Dsg3 function through RNAi mediated Dsg3 knockdown or the stable expression of the truncated mutant Dsg3ΔC. Our results showed that loss of Dsg3 resulted in compromised cell-cell adhesion and reduction of adherens junction and desmosome protein expression as well as the cortical F-actin formation. As a consequence, cells failed to polarize but instead displayed aberrant cell flattening. Furthermore, retardation of E-cadherin internalization and recycling was consistently observed in these cells during the process of calcium induced junction assembling. In contrast, enhanced cadherin endocytosis was detected in cells with overexpression of Dsg3 compared to control cells. Importantly, this altered cadherin trafficking was found to be coincided with the reduced expression and activity of Rab proteins, including Rab5, Rab7 and Rab11 which are known to be involved in E-cadherin trafficking. Taken together, our findings suggest that Dsg3 functions as a key in cell-cell adhesion through at least a mechanism of regulating E-cadherin membrane trafficking. PMID:27254775

Superresolution Imaging Identifies That Conventional Trafficking Pathways Are Not Essential for Endoplasmic Reticulum to Outer Mitochondrial Membrane Protein Transport.

PubMed

Salka, Kyle; Bhuvanendran, Shivaprasad; Wilson, Kassandra; Bozidis, Petros; Mehta, Mansi; Rainey, Kristin; Sesaki, Hiromi; Patterson, George H; Jaiswal, Jyoti K; Colberg-Poley, Anamaris M

2017-02-02

Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA's ability to organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts facilitate proper organization of vMIA on the OMM.

Cellular membrane trafficking of mesoporous silica nanoparticles

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

Fang, I-Ju

This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulfmore » some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to

Regulation of podocalyxin trafficking by Rab small GTPases in epithelial cells

PubMed Central

Mrozowska, Paulina S.; Fukuda, Mitsunori

2016-01-01

ABSTRACT The characteristic feature of polarity establishment in MDCK II cells is transcytosis of apical glycoprotein podocalyxin (PCX) from the outer plasma membrane to the newly formed apical domain. This transcytotic event consists of multiple steps, including internalization from the plasma membrane, transport through early endosomes and Rab11-positive recycling endosomes, and delivery to the apical membrane. These steps are known to be tightly coordinated by Rab small GTPases, which act as molecular switches cycling between active GTP-bound and inactive GDP-bound states. However, our knowledge regarding which sets of Rabs regulate particular steps of PCX trafficking was rather limited. Recently, we have performed a comprehensive analysis of Rab GTPase engagement in the transcytotic pathway of PCX during polarity establishment in 2-dimensional (2D) and 3-dimensional (3D) MDCK II cell cultures. In this Commentary we summarize our findings and set them in the context of previous reports. PMID:27463697

Membrane Diffusion Occurs by Continuous-Time Random Walk Sustained by Vesicular Trafficking.

PubMed

Goiko, Maria; de Bruyn, John R; Heit, Bryan

2018-06-19

Diffusion in cellular membranes is regulated by processes that occur over a range of spatial and temporal scales. These processes include membrane fluidity, interprotein and interlipid interactions, interactions with membrane microdomains, interactions with the underlying cytoskeleton, and cellular processes that result in net membrane movement. The complex, non-Brownian diffusion that results from these processes has been difficult to characterize, and moreover, the impact of factors such as membrane recycling on membrane diffusion remains largely unexplored. We have used a careful statistical analysis of single-particle tracking data of the single-pass plasma membrane protein CD93 to show that the diffusion of this protein is well described by a continuous-time random walk in parallel with an aging process mediated by membrane corrals. The overall result is an evolution in the diffusion of CD93: proteins initially diffuse freely on the cell surface but over time become increasingly trapped within diffusion-limiting membrane corrals. Stable populations of freely diffusing and corralled CD93 are maintained by an endocytic/exocytic process in which corralled CD93 is selectively endocytosed, whereas freely diffusing CD93 is replenished by exocytosis of newly synthesized and recycled CD93. This trafficking not only maintained CD93 diffusivity but also maintained the heterogeneous distribution of CD93 in the plasma membrane. These results provide insight into the nature of the biological and biophysical processes that can lead to significantly non-Brownian diffusion of membrane proteins and demonstrate that ongoing membrane recycling is critical to maintaining steady-state diffusion and distribution of proteins in the plasma membrane. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Early steps in steroidogenesis: intracellular cholesterol trafficking

PubMed Central

Miller, Walter L.; Bose, Himangshu S.

2011-01-01

Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and “free” cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis. PMID:21976778

Ciliopathies: The Trafficking Connection

PubMed Central

Madhivanan, Kayalvizhi; Aguilar, R. Claudio

2014-01-01

The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multi-organ diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies seems to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e., affecting cilia assembly) will lead to more severe symptoms, while those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology. PMID:25040720

Caveolin, sterol carrier protein-2, membrane cholesterol-rich microdomains and intracellular cholesterol trafficking.

PubMed

Schroeder, Friedhelm; Huang, Huan; McIntosh, Avery L; Atshaves, Barbara P; Martin, Gregory G; Kier, Ann B

2010-01-01

While the existence of membrane lateral microdomains has been known for over 30 years, interest in these structures accelerated in the past decade due to the discovery that cholesterol-rich microdomains serve important biological functions. It is increasingly appreciated that cholesterol-rich microdomains in the plasma membranes of eukaryotic cells represent an organizing nexus for multiple cellular proteins involved in transmembrane nutrient uptake (cholesterol, fatty acid, glucose, etc.), cell-signaling, immune recognition, pathogen entry, and many other roles. Despite these advances, however, relatively little is known regarding the organization of cholesterol itself in these plasma membrane microdomains. Although a variety of non-sterol markers indicate the presence of microdomains in the plasma membranes of living cells, none of these studies have demonstrated that cholesterol is enriched in these microdomains in living cells. Further, the role of cholesterol-rich membrane microdomains as targets for intracellular cholesterol trafficking proteins such as sterol carrier protein-2 (SCP-2) that facilitate cholesterol uptake and transcellular transport for targeting storage (cholesterol esters) or efflux is only beginning to be understood. Herein, we summarize the background as well as recent progress in this field that has advanced our understanding of these issues.

The "Polar Light Sign" is a useful tool to detect discrete membranous supravalvular mitral stenosis.

PubMed

Hertwig, Christine; Haas, Nikolaus A; Habash, Sheeraz; Hanslik, Andreas; Kececioglu, Deniz; Sandica, Eugen; Laser, Kai-Thorsten

2015-02-01

Mitral valve stenosis caused by a discrete supravalvular membrane is a rare congenital malformation haemodynamically leading to significant mitral valve stenosis. When the supravalvular mitral stenosis consists of a discrete supravalvular membrane adherent to the mitral valve, it is usually not clearly detectable by routine echocardiography. We report about the typical echocardiographic finding in three young patients with this rare form of a discrete membranous supravalvular stenosis caused by a membrane adherent to the mitral valve. These cases present a typical echocardiographic feature in colour Doppler generated by the pathognomonic supramitral flow acceleration. Whereas typical supravalvular mitral stenosis caused by cor triatriatum or a clearly visible supravalvular ring is easily detectable by echocardiography, a discrete supravalvular membrane adjacent to the mitral valve leaflets resembling valvular mitral stenosis is difficult to differentiate by routine echocardiography. In our opinion, this colour phenomenon does resemble the visual impression of polar lights in the northern hemisphere; owing to its typical appearance, it may therefore be named as "Polar Light Sign". This phenomenon may help to detect this anatomical entity by echocardiography in time and therefore improve the prognosis for repair.

Endocytosis and Endosomal Trafficking in Plants.

PubMed

Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

2016-04-29

Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.

Lysosome trafficking is necessary for EGF-driven invasion and is regulated by p38 MAPK and Na+/H+ exchangers.

PubMed

Dykes, Samantha S; Steffan, Joshua J; Cardelli, James A

2017-10-04

Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins. Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion. EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells. These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking.

Anterograde Trafficking of KCa3.1 in Polarized Epithelia Is Rab1- and Rab8-Dependent and Recycling Endosome-Independent

PubMed Central

Bertuccio, Claudia A.; Lee, Shih-Liang; Wu, Guangyu; Butterworth, Michael B.; Hamilton, Kirk L.; Devor, Daniel C.

2014-01-01

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

PubMed Central

Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří

2018-01-01

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. PMID:29463731

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

PubMed

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei.

PubMed

Gilden, Julia K; Umaer, Khan; Kruzel, Emilia K; Hecht, Oliver; Correa, Renan O; Mansfield, John M; Bangs, James D

2017-06-01

Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P 2 in T. brucei. We find a low steady state level of PI(3,5)P 2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P 2 pool leading to rapid cell death. TbFab1 and PI(3,5)P 2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P 2 metabolism, the PI(3,5)P 2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P 2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology. Copyright © 2017 Elsevier B.V. All rights reserved.

The large discretization step method for time-dependent partial differential equations

NASA Technical Reports Server (NTRS)

Haras, Zigo; Taasan, Shlomo

1995-01-01

A new method for the acceleration of linear and nonlinear time dependent calculations is presented. It is based on the Large Discretization Step (LDS) approximation, defined in this work, which employs an extended system of low accuracy schemes to approximate a high accuracy discrete approximation to a time dependent differential operator. Error bounds on such approximations are derived. These approximations are efficiently implemented in the LDS methods for linear and nonlinear hyperbolic equations, presented here. In these algorithms the high and low accuracy schemes are interpreted as the same discretization of a time dependent operator on fine and coarse grids, respectively. Thus, a system of correction terms and corresponding equations are derived and solved on the coarse grid to yield the fine grid accuracy. These terms are initialized by visiting the fine grid once in many coarse grid time steps. The resulting methods are very general, simple to implement and may be used to accelerate many existing time marching schemes.

Resection of subaortic membrane for discrete subaortic stenosis.

PubMed

Talwar, Sachin; Anand, Abhishek; Gupta, Saurabh Kumar; Ramakrishnan, Sivasubramanian; Kothari, Shyam Sunder; Saxena, Anita; Juneja, Rajnish; Choudhary, Shiv Kumar; Airan, Balram

2017-07-01

We reviewed the long-term results of surgery for discrete subaortic membrane (SubAM) from a single institute. A retrospective review of medical records of all patients (n = 146) who underwent resection of a SubAM for discrete subaortic stenosis between 1990 and 2015 at the All India Institute of Medical Sciences, New Delhi, India was undertaken. Median age at surgery was 9.0 years (9 months-47 years). There was one early death. Preoperative peak left ventricular outflow tract (LVOT) Doppler gradient was 83.4 ± 26.2 mmHg (range: 34-169 mmHg). On preoperative echocardiography, aortic regurgitation (AR) was absent in 69 (47.3%), mild in 35 (24%), moderate in 30 (20.5%), and severe in 12 (8.2%). After surgery, the LVOT gradient was reduced to 15.1 ± 6.2 mmHg (P < 0.001). Fourteen patients (9.6%) who had residual/recurrent significant gradients are currently being followed-up or awaiting surgery. There was improvement in AR for operated patients with freedom from AR of 92.6 ± 0.03% at 15 years. Kaplan-Meier survival at 25 years was 93.0 ± 3.9% (95% confidence interval: 79.6, 97.7). Freedom from re-operation at 25 years was 96.9 ± 1.8%. Long-term results of surgery for discrete SubAM are good. Resection of the membrane along with septal myectomy decreases the risk of recurrence. © 2017 Wiley Periodicals, Inc.

Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

PubMed Central

Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

2014-01-01

To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

Endomembrane Cation Transporters and Membrane Trafficking

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

Sze, Heven

localization and mutant analyses, we conclude that CHXs modulate the ion balance, pH or both in micro-regions of endoplasmic reticulum, endosomes and prevacuolar compartment (PVC), and so influence membrane trafficking and signaling resulting in proper osmoregulation in guard cells and seed formation. We also demonstrated for the first time that AtKEA2 associates with chloroplasts, especially at the two poles of developing plastids. These results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation. The first 3-D structure model of AtCHX was generated, and architecture-directed mutagenesis identified critical residues of the transport core giving insights to the transport mode of this family. Thus we have revealed for the first time crucial roles of an unknown K +/H + transport family on plant growth (KEA), gas exchange, pollen cell wall, and different phases of reproduction (CHXs). The dynamic endomembrane of plant cells is integral to cytokinesis, cell expansion, defense, and cell wall formation, thus these studies are directly relevant to the mission of the Department of Energy and to a better understanding of determinants for enhancing plant biomass and plant tolerance to abiotic stress.« less

Downregulations of TRPM8 expression and membrane trafficking in dorsal root ganglion mediate the attenuation of cold hyperalgesia in CCI rats induced by GFRα3 knockdown.

PubMed

Su, Lin; Shu, Ruichen; Song, Chengcheng; Yu, Yonghao; Wang, Guolin; Li, Yazhuo; Liu, Changxiao

2017-10-01

Cold hyperalgesia is an intractable sensory abnormality commonly seen in peripheral neuropathies. Although glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) is required for the formation of pathological cold pain has been revealed, potential transduction mechanism is poorly elucidated. We have previously demonstrated the contribution of enhanced activity of transient receptor potential melastatin 8 (TRPM8) to cold hyperalgesia in neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve. Recently, the enhancement of TRPM8 activity is attributed to the increased TRPM8 plasma membrane trafficking. In addition, TRPM8 can be sensitized by the activation of GFRα3, leading to increased cold responses in vivo. The aim of this study was to investigate whether GFRα3 could influence cold hyperalgesia of CCI rats via modulating TRPM8 expression and plasma membrane trafficking in dorsal root ganglion (DRG). Mechanical allodynia, cold and heat hyperalgesia were measured on 1day before CCI and the 1st, 4th, 7th, 10th and 14th day after CCI. TRPM8 total expression and membrane trafficking as well as GFRα3 expression in DRG were detected by immunofluorescence and western blot. Furthermore, GFRα3 small interfering RNA (siRNA) was intrathecally administrated to reduce GFRα3 expression in DRG, and the effects of GFRα3 knockdown on CCI-induced behavioral sensitization as well as TRPM8 total expression and membrane trafficking in both mRNA and protein levels were investigated, and the change in coexpression of TRPM8 with GFRα3 was also evaluated. Then, the effect of GFRα3 activation with artemin on pain behavior of CCI rats pretreated with the selective TRPM8 antagonist RQ-00203078 was observed. Here we found that TRPM8 total expression and plasma membrane trafficking as well as GFRα3 expression in DRG were initially increased on the 4th day after CCI, and maintained at the peak level from the 10th to the 14th day

Calcium Signaling Regulates Trafficking of Familial Hypocalciuric Hypercalcemia (FHH) Mutants of the Calcium Sensing Receptor

PubMed Central

Grant, Michael P.; Stepanchick, Ann

2012-01-01

Calcium-sensing receptors (CaSRs) regulate systemic Ca2+ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2+ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2+, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2+ signaling, which were substantially rescued by coexpression with wild-type CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2+ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSRΔ868 robustly rescued trafficking and Ca2+ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2+ response when extracellular Ca2+ is elevated and argues that Ca2+ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane. PMID:23077345

Tunable-Porosity Membranes From Discrete Nanoparticles

PubMed Central

Marchetti, Patrizia; Mechelhoff, Martin; Livingston, Andrew G.

2015-01-01

Thin film composite membranes were prepared through a facile single-step wire-wound rod coating procedure in which internally crosslinked poly(styrene-co-butadiene) polymer nanoparticles self-assembled to form a thin film on a hydrophilic ultrafiltration support. This nanoparticle film provided a defect-free separation layer 130–150 nm thick, which was highly permeable and able to withstand aggressive pH conditions beyond the range of available commercial membranes. The nanoparticles were found to coalesce to form a rubbery film when heated above their glass transition temperature (Tg). The retention properties of the novel membrane were strongly affected by charge repulsion, due to the negative charge of the hydroxyl functionalized nanoparticles. Porosity was tuned by annealing the membranes at different temperatures, below and above the nanoparticle Tg. This enabled fabrication of membranes with varying performance. Nanofiltration properties were achieved with a molecular weight cut-off below 500 g mol−1 and a low fouling tendency. Interestingly, after annealing above Tg, memory of the interstitial spaces between the nanoparticles persisted. This memory led to significant water permeance, in marked contrast to the almost impermeable films cast from a solution of the same polymer. PMID:26626565

CLIC4 regulates cell adhesion and β1 integrin trafficking.

PubMed

Argenzio, Elisabetta; Margadant, Coert; Leyton-Puig, Daniela; Janssen, Hans; Jalink, Kees; Sonnenberg, Arnoud; Moolenaar, Wouter H

2014-12-15

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking. © 2014. Published by The Company of Biologists Ltd.

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

PubMed Central

Rismanchi, Neggy; Puertollano, Rosa; Blackstone, Craig

2009-01-01

Signal transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. Here we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein (VSVG)-GFP trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with COPII proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex. PMID:19054391

A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

PubMed Central

Goldenring, James R.

2014-01-01

Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

Membrane-trafficking sorting hubs: cooperation between PI4P and small GTPases at the trans-Golgi Network

PubMed Central

Santiago-Tirado, Felipe H.; Bretscher, Anthony

2011-01-01

Cell polarity in eukaryotes requires constant sorting, packaging, and transport of membrane-bound cargo within the cell. These processes occur in two sorting hubs: the recycling endosome for incoming material, and the trans-Golgi Network for outgoing. Phosphatidylinositol 3-phosphate and 4–5 phosphate are enriched at the endocytic and exocytic sorting hubs, respectively, where they act together with small GTPases to recruit factors to segregate cargo and regulate carrier formation and transport. In this review, we summarize the current understanding of how these lipids and GTPases directly regulate membrane trafficking, emphasizing the recent discoveries of phosphatidylinositol 4-phosphate functions at the trans-Golgi Network. PMID:21764313

Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux.

PubMed

Leventhal, A R; Chen, W; Tall, A R; Tabas, I

2001-11-30

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

PubMed

Bahouth, Suleiman W; Nooh, Mohammed M

2017-08-01

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction

Step-by-step seeding procedure for preparing HKUST-1 membrane on porous α-alumina support.

PubMed

Nan, Jiangpu; Dong, Xueliang; Wang, Wenjin; Jin, Wanqin; Xu, Nanping

2011-04-19

Metal-organic framework (MOF) membranes have attracted considerable attention because of their striking advantages in small-molecule separation. The preparation of an integrated MOF membrane is still a major challenge. Depositing a uniform seed layer on a support for secondary growth is a main route to obtaining an integrated MOF membrane. A novel seeding method to prepare HKUST-1 (known as Cu(3)(btc)(2)) membranes on porous α-alumina supports is reported. The in situ production of the seed layer was realized in step-by-step fashion via the coordination of H(3)btc and Cu(2+) on an α-alumina support. The formation process of the seed layer was observed by ultraviolet-visible absorption spectroscopy and atomic force microscopy. An integrated HKUST-1 membrane could be synthesized by the secondary hydrothermal growth on the seeded support. The gas permeation performance of the membrane was evaluated. © 2011 American Chemical Society

LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

PubMed

Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

2014-01-01

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

Endocytosis and Trafficking of Natriuretic Peptide Receptor-A: Potential Role of Short Sequence Motifs

PubMed Central

Pandey, Kailash N.

2015-01-01

The targeted endocytosis and redistribution of transmembrane receptors among membrane-bound subcellular organelles are vital for their correct signaling and physiological functions. Membrane receptors committed for internalization and trafficking pathways are sorted into coated vesicles. Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and elicit the generation of intracellular second messenger cyclic guanosine 3',5'-monophosphate (cGMP), which lowers blood pressure and incidence of heart failure. After ligand binding, the receptor is rapidly internalized, sequestrated, and redistributed into intracellular locations. Thus, NPRA is considered a dynamic cellular macromolecule that traverses different subcellular locations through its lifetime. The utilization of pharmacologic and molecular perturbants has helped in delineating the pathways of endocytosis, trafficking, down-regulation, and degradation of membrane receptors in intact cells. This review describes the investigation of the mechanisms of internalization, trafficking, and redistribution of NPRA compared with other cell surface receptors from the plasma membrane into the cell interior. The roles of different short-signal peptide sequence motifs in the internalization and trafficking of other membrane receptors have been briefly reviewed and their potential significance in the internalization and trafficking of NPRA is discussed. PMID:26151885

Physiological Roles of Plant Post-Golgi Transport Pathways in Membrane Trafficking.

PubMed

Uemura, Tomohiro

2016-10-01

Membrane trafficking is the fundamental system through which proteins are sorted to their correct destinations in eukaryotic cells. Key regulators of this system include RAB GTPases and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Interestingly, the numbers of RAB GTPases and SNAREs involved in post-Golgi transport pathways in plant cells are larger than those in animal and yeast cells, suggesting that plants have evolved unique and complex post-Golgi transport pathways. The trans-Golgi network (TGN) is an important organelle that acts as a sorting station in the post-Golgi transport pathways of plant cells. The TGN also functions as the early endosome, which is the first compartment to receive endocytosed proteins. Several endocytosed proteins on the plasma membrane (PM) are initially targeted to the TGN/EE, then recycled back to the PM or transported to the vacuole for degradation. The recycling and degradation of the PM localized proteins is essential for the development and environmental responses in plant. The present review describes the post-Golgi transport pathways that show unique physiological functions in plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Regulating the Efficacy of Inhibition Through Trafficking of γ-Aminobutyric Acid Type A Receptors.

PubMed

Vien, Thuy N; Moss, Stephen J; Davies, Paul A

2016-11-01

Trafficking of anesthetic-sensitive receptors within the plasma membrane, or from one cellular component to another, occurs continuously. Changes in receptor trafficking have implications in altering anesthetic sensitivity. γ-Aminobutyric acid type A receptors (GABAARs) are anion-permeable ion channels and are the major class of receptor in the adult mammalian central nervous system that mediates inhibition. GABAergic signaling allows for precise synchronized firing of action potentials within brain circuits that is critical for cognition, behavior, and consciousness. This precision depends upon tightly controlled trafficking of GABAARs into the membrane. General anesthetics bind to and allosterically enhance GABAARs by prolonging the open state of the receptor and thereby altering neuronal and brain circuit activity. Subunit composition and GABAAR localization strongly influence anesthetic end points; therefore, changes in GABAAR trafficking could have significant consequences to anesthetic sensitivity. GABAARs are not static membrane structures but are in a constant state of flux between extrasynaptic and synaptic locations and are continually endocytosed and recycled from and to the membrane. Neuronal activity, posttranslational modifications, and some naturally occurring and synthetic compounds can influence the expression and trafficking of GABAARs. In this article, we review GABAARs, their trafficking, and how phosphorylation of GABAAR subunits can influence the surface expression and function of the receptor. Ultimately, alterations of GABAAR trafficking could modify anesthetic end points, both unintentionally through pathologic processes but potentially as a therapeutic target to adjust anesthetic-sensitive GABAARs.

Lubiprostone targets prostanoid signaling and promotes ion transporter trafficking, mucus exocytosis, and contractility.

PubMed

Jakab, Robert L; Collaco, Anne M; Ameen, Nadia A

2012-11-01

Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery. Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca(2+) activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na-K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined. Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na-K-ATPase in enterocytes; and (4) increased mucus exocytosis in goblet cells. These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; and enhance mucus-mobilization and mucosal contractility.

Lubiprostone targets prostanoid signaling and promotes ion transporter trafficking, mucus exocytosis and contractility

PubMed Central

Jakab, Robert L.; Collaco, Anne M.; Ameen, Nadia A.

2012-01-01

Background and Aim Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery. Methods Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca2+ activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na-K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined. Results Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na-K-ATPase in enterocytes; (4) and increased mucus exocytosis in goblet cells. Conclusion These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; enhance mucus-mobilization and mucosal contractility. PMID:22923315

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein.

PubMed

Zhi, Pei; Chia, Pei Zhi Cheryl; Chia, Cheryl; Gleeson, Paul A

2011-09-01

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the

BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia

PubMed Central

2017-01-01

Primary cilia perceive the extracellular environment through receptors localized in the ciliary membrane, but mechanisms directing specific proteins to this domain are poorly understood. To address this question, we knocked down proteins potentially important for ciliary membrane targeting and determined how this affects the ciliary trafficking of fibrocystin, polycystin-2, and smoothened. Our analysis showed that fibrocystin and polycystin-2 are dependent on IFT20, GMAP210, and the exocyst complex, while smoothened delivery is largely independent of these components. In addition, we found that polycystin-2, but not smoothened or fibrocystin, requires the biogenesis of lysosome-related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with the role of BLOC-1 in sorting from the endosome, we find that disrupting the recycling endosome reduces ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary assembly and highlights the complexity of pathways taken to the cilium. PMID:28576874

Stability with large step sizes for multistep discretizations of stiff ordinary differential equations

NASA Technical Reports Server (NTRS)

Majda, George

1986-01-01

One-leg and multistep discretizations of variable-coefficient linear systems of ODEs having both slow and fast time scales are investigated analytically. The stability properties of these discretizations are obtained independent of ODE stiffness and compared. The results of numerical computations are presented in tables, and it is shown that for large step sizes the stability of one-leg methods is better than that of the corresponding linear multistep methods.

Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.

PubMed

Au, Catherine E; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Kearney, Robert E; Smith, Charles E; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Simon, Paul H G; Mandato, Craig; Nilsson, Tommy; Bergeron, John J M

2015-08-01

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body. © 2015 The Authors.

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

PubMed

Brast, Sabine; Grabner, Alexander; Sucic, Sonja; Sitte, Harald H; Hermann, Edwin; Pavenstädt, Hermann; Schlatter, Eberhard; Ciarimboli, Giuliano

2012-03-01

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

PubMed

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François

2012-08-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

PubMed

Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid Turnover of Stereocilia Membrane Proteins: Evidence from the Trafficking and Mobility of Plasma Membrane Ca2+-ATPase 2

PubMed Central

Grati, M'hamed; Schneider, Mark E.; Lipkow, Karen; Strehler, Emanuel E.; Wenthold, Robert J.; Kachar, Bechara

2007-01-01

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of ∼5–7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01– 0.005 μm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that ∼60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1– 0.2 μm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal. PMID:16763047

Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

PubMed Central

Bowen, Aaron B; Bourke, Ashley M; Hiester, Brian G; Hanus, Cyril

2017-01-01

Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network. PMID:28875935

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells.

PubMed

Maxson, Julia E; Enns, Caroline A; Zhang, An-Sheng

2009-02-19

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release.

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells

PubMed Central

Maxson, Julia E.; Enns, Caroline A.

2009-01-01

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release. PMID:19029439

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

PubMed

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture

PubMed Central

Rice, William L.; Li, Wei; Mamuya, Fahmy; McKee, Mary; Păunescu, Teodor G.; Lu, Hua A. Jenny

2015-01-01

In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro. PMID:26147297

Involvement of Dynein and Spectrin with Early Melanosome Transport and Melanosomal Protein Trafficking

PubMed Central

Watabe, Hidenori; Valencia, Julio C.; Le Pape, Elodie; Yamaguchi, Yuji; Nakamura, Masayuki; Rouzaud, François; Hoashi, Toshihiko; Kawa, Yoko; Mizoguchi, Masako; Hearing, Vincent J.

2007-01-01

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, TYR and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system which is involved with the sorting of cargo from the plasma membrane. These results provide important clues towards understanding the processes involved with early events in melanosome formation and transport. PMID:17687388

Protein kinesis: The dynamics of protein trafficking and stability

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

NONE

The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

PubMed Central

Gillespie, Eugene J.; Ho, Chi-Lee C.; Balaji, Kavitha; Clemens, Daniel L.; Deng, Gang; Wang, Yao E.; Elsaesser, Heidi J.; Tamilselvam, Batcha; Gargi, Amandeep; Dixon, Shandee D.; France, Bryan; Chamberlain, Brian T.; Blanke, Steven R.; Cheng, Genhong; de la Torre, Juan Carlos; Brooks, David G.; Jung, Michael E.; Colicelli, John; Damoiseaux, Robert; Bradley, Kenneth A.

2013-01-01

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease. PMID:24191014

Protein trafficking during plant innate immunity.

PubMed

Wang, Wen-Ming; Liu, Peng-Qiang; Xu, Yong-Ju; Xiao, Shunyuan

2016-04-01

Plants have evolved a sophisticated immune system to fight against pathogenic microbes. Upon detection of pathogen invasion by immune receptors, the immune system is turned on, resulting in production of antimicrobial molecules including pathogenesis-related (PR) proteins. Conceivably, an efficient immune response depends on the capacity of the plant cell's protein/membrane trafficking network to deploy the right defense-associated molecules in the right place at the right time. Recent research in this area shows that while the abundance of cell surface immune receptors is regulated by endocytosis, many intracellular immune receptors, when activated, are partitioned between the cytoplasm and the nucleus for induction of defense genes and activation of programmed cell death, respectively. Vesicle transport is an essential process for secretion of PR proteins to the apoplastic space and targeting of defense-related proteins to the plasma membrane or other endomembrane compartments. In this review, we discuss the various aspects of protein trafficking during plant immunity, with a focus on the immunity proteins on the move and the major components of the trafficking machineries engaged. © 2015 Institute of Botany, Chinese Academy of Sciences.

Morphological Study on Porous Silicon Carbide Membrane Fabricated by Double-Step Electrochemical Etching

NASA Astrophysics Data System (ADS)

Omiya, Takuma; Tanaka, Akira; Shimomura, Masaru

2012-07-01

The structure of porous silicon carbide membranes that peeled off spontaneously during electrochemical etching was studied. They were fabricated from n-type 6H SiC(0001) wafers by a double-step electrochemical etching process in a hydrofluoric electrolyte. Nanoporous membranes were obtained after double-step etching with current densities of 10-20 and 60-100 mA/cm2 in the first and second steps, respectively. Microporous membranes were also fabricated after double-step etching with current densities of 100 and 200 mA/cm2. It was found that the pore diameter is influenced by the etching current in step 1, and that a higher current is required in step 2 when the current in step 1 is increased. During the etching processes in steps 1 and 2, vertical nanopore and lateral crack formations proceed, respectively. The influx pathway of hydrofluoric solution, expansion of generated gases, and transfer limitation of positive holes to the pore surface are the key factors in the peeling-off mechanism of the membrane.

Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy.

PubMed

Zhou, Changqian; Ma, Kaili; Gao, Ruize; Mu, Chenglong; Chen, Linbo; Liu, Qiangqiang; Luo, Qian; Feng, Du; Zhu, Yushan; Chen, Quan

2017-02-01

Autophagy requires diverse membrane sources and involves membrane trafficking of mATG9, the only membrane protein in the ATG family. However, the molecular regulation of mATG9 trafficking for autophagy initiation remains unclear. Here we identified two conserved classic adaptor protein sorting signals within the cytosolic N-terminus of mATG9, which mediate trafficking of mATG9 from the plasma membrane and trans-Golgi network (TGN) via interaction with the AP1/2 complex. Src phosphorylates mATG9 at Tyr8 to maintain its endocytic and constitutive trafficking in unstressed conditions. In response to starvation, phosphorylation of mATG9 at Tyr8 by Src and at Ser14 by ULK1 functionally cooperate to promote interactions between mATG9 and the AP1/2 complex, leading to redistribution of mATG9 from the plasma membrane and juxta-nuclear region to the peripheral pool for autophagy initiation. Our findings uncover novel mechanisms of mATG9 trafficking and suggest a coordination of basal and stress-induced autophagy.

Discrete adjoint of fractional step Navier-Stokes solver in generalized coordinates

NASA Astrophysics Data System (ADS)

Wang, Mengze; Mons, Vincent; Zaki, Tamer

2017-11-01

Optimization and control in transitional and turbulent flows require evaluation of gradients of the flow state with respect to the problem parameters. Using adjoint approaches, these high-dimensional gradients can be evaluated with a similar computational cost as the forward Navier-Stokes simulations. The adjoint algorithm can be obtained by discretizing the continuous adjoint Navier-Stokes equations or by deriving the adjoint to the discretized Navier-Stokes equations directly. The latter algorithm is necessary when the forward-adjoint relations must be satisfied to machine precision. In this work, our forward model is the fractional step solution to the Navier-Stokes equations in generalized coordinates, proposed by Rosenfeld, Kwak & Vinokur. We derive the corresponding discrete adjoint equations. We also demonstrate the accuracy of the combined forward-adjoint model, and its application to unsteady wall-bounded flows. This work has been partially funded by the Office of Naval Research (Grant N00014-16-1-2542).

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

PubMed Central

Sanchez, Marco A.; Tran, Khoa D.; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M.

2016-01-01

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

PubMed Central

Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C.; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C.; Alonso, Miguel A.; Correas, Isabel; Cox, Susan; Ridley, Anne J.

2016-01-01

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

Analysis of SCAP N-glycosylation and Trafficking in Human Cells.

PubMed

Cheng, Chunming; Guo, Jeffrey Yunhua; Geng, Feng; Wu, Xiaoning; Cheng, Xiang; Li, Qiyue; Guo, Deliang

2016-11-08

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121[W

PubMed Central

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François

2012-01-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383

Wherever I may roam: protein and membrane trafficking in P. falciparum-infected red blood cells.

PubMed

Deponte, Marcel; Hoppe, Heinrich C; Lee, Marcus C S; Maier, Alexander G; Richard, Dave; Rug, Melanie; Spielmann, Tobias; Przyborski, Jude M

2012-12-01

Quite aside from its immense importance as a human pathogen, studies in recent years have brought to light the fact that the malaria parasite Plasmodium falciparum is an interesting eukaryotic model system to study protein trafficking. Studying parasite cell biology often reveals an overrepresentation of atypical cell biological features, possibly driven by the parasites' need to survive in an unusual biological niche. Malaria parasites possess uncommon cellular compartments to which protein traffic must be directed, including secretory organelles such as rhoptries and micronemes, a lysosome-like compartment referred to as the digestive vacuole and a complex (four membrane-bound) plastid, the apicoplast. In addition, the parasite must provide proteins to extracellular compartments and structures including the parasitophorous vacuole, the parasitophorous vacuolar membrane, the Maurer's clefts and both cytosol and plasma membrane of the host cell, the mature human red blood cell. Although some of these unusual destinations are possessed by other cell types, only Plasmodium parasites contain them all within one cell. Here we review what is known about protein and membrane transport in the P. falciparum-infected cell, highlighting novel features of these processes. A growing body of evidence suggests that this parasite is a real "box of tricks" with regards to protein traffic. Possibly, these tricks may be turned against the parasite by exploiting them as novel therapeutic targets. Copyright © 2012 Elsevier B.V. All rights reserved.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

NASA Astrophysics Data System (ADS)

Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R.; Oker-Blom, Christian; Vattulainen, Ilpo; Vuento, Matti

2009-12-01

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter SCD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane.

Hydrogen-rich saline controls remifentanil-induced hypernociception and NMDA receptor NR1 subunit membrane trafficking through GSK-3β in the DRG in rats.

PubMed

Zhang, Linlin; Shu, Ruichen; Wang, Chunyan; Wang, Haiyun; Li, Nan; Wang, Guolin

2014-07-01

Although NMDAR trafficking mediated by GSK-3β involvement in transmission of pronociceptive messages in the spinal cord has been confirmed by our previous studies, whether NMDAR trafficking is implicated in peripheral sensitization remains equivocal. It is demonstrated that inflammation is associated with spinal NMDAR-containing nociceptive neurons activation and the maintenance of opioid induced pain hypersensitivity. However, whether and how hydrogen-rich saline, as an effective anti-inflammatory drug, could prevent hyperalgesia through affecting peripheral sensitization caused by NMDAR activation remains to be explored. To test these effects, hydrogen-rich saline (2.5, 5 or 10 ml/kg) was administrated intraperitoneally after remifentanil infusion, NMDAR antagonist MK-801 or GSK-3β inhibitor TDZD-8 was administrated intravenously before remifentanil infusion in rats. We examined time course of hydrogen concentration in blood after hydrogen-rich saline administration. Mechanical and thermal hyperalgesia were evaluated by measuring PWT and PWL for 48 post-infusion hours, respectively. Western blotting and real-time qPCR assay were applied to analyze the NR1 membrane trafficking, GSK-3β expression and activity in DRG. Inflammatory mediators (TNF-α, IL-1β, and IL-6) expressions in DRG were also analyzed. We found that NR1 membrane trafficking in DRG increased, possibly due to GSK-3β activation after remifentanil infusion. We also discovered that hydrogen-rich saline not 2.5 ml/kg but 5 and 10 ml/kg could dose-dependently attenuate mechanical and thermal hyperalgesia without affecting baseline nociceptive threshold, reduce expressions of inflammatory mediators (TNF-α, IL-1β, and IL-6) and decrease NR1 trafficking mediated by GSK-3β, and minimal effective concentration was observed to be higher than 10 μmol/L, namely peak concentration in arterial blood after administration of HRS 2.5 ml/kg without any influence on hyperalgesia. Our results indicated that

Exo- and endocytotic trafficking of SCAMP2.

PubMed

Toyooka, Kiminori; Matsuoka, Ken

2009-12-01

Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane (PM) and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photoswitching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the PM and cell plate moved into the BY-2 cells within several minutes, but did not spread around PM. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, PM and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.

Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

PubMed

Inada, Noriko; Betsuyaku, Shigeyuki; Shimada, Takashi L; Ebine, Kazuo; Ito, Emi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Takano, Yoshitaka; Fukuda, Hiroo; Nakano, Akihiko; Ueda, Takashi

2016-09-01

RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

No Evidence for Spontaneous Lipid Transfer at ER-PM Membrane Contact Sites.

PubMed

Merklinger, Elisa; Schloetel, Jan-Gero; Spitta, Luis; Thiele, Christoph; Lang, Thorsten

2016-04-01

Non-vesicular lipid transport steps play a crucial role in lipid trafficking and potentially include spontaneous exchange. Since membrane contact facilitates this lipid transfer, it is most likely to occur at membrane contact sites (MCS). However, to date it is unknown whether closely attached biological membranes exchange lipids spontaneously. We have set up a system for studying the exchange of lipids at MCS formed between the endoplasmic reticulum (ER) and the plasma membrane. Contact sites were stably anchored and the lipids cholesterol and phosphatidylcholine (PC) were not capable of transferring spontaneously into the opposed bilayer. We conclude that physical contact between two associated biological membranes is not sufficient for transfer of the lipids PC and cholesterol.

A moving view: subcellular trafficking processes in pattern recognition receptor-triggered plant immunity.

PubMed

Ben Khaled, Sara; Postma, Jelle; Robatzek, Silke

2015-01-01

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

PubMed

Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

2011-02-17

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics. Copyright © 2011 Elsevier Inc. All rights reserved.

Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors

PubMed Central

Goo, Marisa S.; Scudder, Samantha L.; Patrick, Gentry N.

2015-01-01

Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses. PMID:26528125

The Regulation of Vesicle Trafficking by Small GTPases and Phospholipids during Pollen Tube Growth

USDA-ARS?s Scientific Manuscript database

Polarized and directional growth of pollen tubes is the only means by which immotile sperm of flowering plants reach the deeply embedded female gametes for fertilization. Vesicle trafficking is among the most critical cellular activities for pollen tube growth. Vesicle trafficking maintains membrane...

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

PubMed

Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

2018-03-01

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTPα/Fyn Signaling Pathway

PubMed Central

Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J.

2015-01-01

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTPα at Tyr789, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. PMID:25951993

Receptor Tyrosine Kinase Ubiquitination and De-Ubiquitination in Signal Transduction and Receptor Trafficking

PubMed Central

Critchley, William R.; Pellet-Many, Caroline; Ringham-Terry, Benjamin; Zachary, Ian C.; Ponnambalam, Sreenivasan

2018-01-01

Receptor tyrosine kinases (RTKs) are membrane-based sensors that enable rapid communication between cells and their environment. Evidence is now emerging that interdependent regulatory mechanisms, such as membrane trafficking, ubiquitination, proteolysis and gene expression, have substantial effects on RTK signal transduction and cellular responses. Different RTKs exhibit both basal and ligand-stimulated ubiquitination, linked to trafficking through different intracellular compartments including the secretory pathway, plasma membrane, endosomes and lysosomes. The ubiquitin ligase superfamily comprising the E1, E2 and E3 enzymes are increasingly implicated in this post-translational modification by adding mono- and polyubiquitin tags to RTKs. Conversely, removal of these ubiquitin tags by proteases called de-ubiquitinases (DUBs) enables RTK recycling for another round of ligand sensing and signal transduction. The endocytosis of basal and activated RTKs from the plasma membrane is closely linked to controlled proteolysis after trafficking and delivery to late endosomes and lysosomes. Proteolytic RTK fragments can also have the capacity to move to compartments such as the nucleus and regulate gene expression. Such mechanistic diversity now provides new opportunities for modulating RTK-regulated cellular responses in health and disease states. PMID:29543760

Performance and membrane fouling of a step-fed submerged membrane sequencing batch reactor treating swine biogas digestion slurry.

PubMed

Han, Zhiying; Chen, Shixia; Lin, Xiaochang; Yu, Hongjun; Duan, Li'an; Ye, Zhangying; Jia, Yanbo; Zhu, Songming; Liu, Dezhao

2018-01-02

To identify the performance of step-fed submerged membrane sequencing batch reactor (SMSBR) treating swine biogas digestion slurry and to explore the correlation between microbial metabolites and membrane fouling within this novel reactor, a lab-scale step-fed SMSBR was operated under nitrogen loading rate of 0.026, 0.052 and 0.062 g NH 4 + -N (gVSS·d) -1 . Results show that the total removal efficiencies for NH 4 + -N, total nitrogen and chemical oxygen demand in the reactor (>94%, >89% and >97%, respectively) were high during the whole experiment. However, the cycle removal efficiency of NH 4 + -N decreased significantly when the nitrogen loading rate was increased to 0.062 g NH 4 + -N (gVSS·d) -1 . The total removal efficiency of total phosphorus in the step-fed SMSBR was generally higher than 75%, though large fluctuations were observed during the experiments. In addition, the concentrations of microbial metabolites, i.e., soluble microbial products (SMP) and extracellular polymeric substances (EPS) from activated sludge increased as nitrogen loading rate increased, both showing quadratic equation correlations with viscosity of the mixed liquid in the step-fed SMSBR (both R 2 > 0.90). EPS content was higher than SMP content, while protein (PN) was detected as the main component in both SMP and EPS. EPS PN was found to be well correlated with transmembrane pressure, membrane flux and the total membrane fouling resistance. Furthermore, the three-dimensional excitation-emission matrix fluorescence spectroscopy results suggested the tryptophan-like protein as one of the main contributors to the membrane fouling. Overall, this study showed that the step-fed SMSBR could be used to treat swine digestion slurry at nitrogen loading rate of 0.052 g NH 4 + -N (gVSS·d) -1 , and the control strategy of membrane fouling should be developed based on reducing the tryptophan-like PN in EPS.

Analysis of Actin-Based Intracellular Trafficking in Pollen Tubes.

PubMed

Jiang, Yuxiang; Zhang, Meng; Huang, Shanjin

2017-01-01

Underlying rapid and directional pollen tube growth is the active intracellular trafficking system that carries materials necessary for cell wall synthesis and membrane expansion to the expanding point of the pollen tube. The actin cytoskeleton has been shown to control various intracellular trafficking events in the pollen tube, but the underlying cellular and molecular mechanisms remain poorly understood. To better understand how the actin cytoskeleton is involved in the regulation of intracellular trafficking events, we need to establish assays to visualize and quantify the distribution and dynamics of organelles, vesicles, or secreted proteins. In this chapter, we introduce methods regarding the visualization and quantification of the distribution and dynamics of organelles or vesicles in pollen tubes.

Are Rab Proteins the Link Between Golgi Organization and Membrane Trafficking?

PubMed Central

Liu, Shijie; Storrie, Brian

2014-01-01

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Sacchromyces cerevisiae, the Golgi cisternae are physically separated from one another while, in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18 and Rab43. In yeast, these include Ypt1, Ypt32 and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport. PMID:22581368

GPCR Signaling and Trafficking: The Long and Short of It

PubMed Central

Pavlos, Nathan J.; Friedman, Peter A.

2016-01-01

Emerging findings disclose unexpected components of G protein-coupled receptor (GPCR) signaling and cell biology. Select GPCRs exhibit classical signaling that is restricted to cell membranes and newly described persistent signaling that depends on internalization of the GPCR bound to β-arrestins. Termination of non-canonical endosomal signaling requires intraluminal acidification and sophisticated protein trafficking machineries. Recent studies reveal the structural determinants of the trafficking chaperones. This review summarizes advances in GPCR signaling and trafficking with a focus on the parathyroid hormone receptor as prototype, and the actin-SNX27-retromer tubule complex, an endosomal sorting hub responsible for recycling and preservation of cell surface receptors. The findings are integrated into a model of PTHR trafficking with implications for signal transduction, bone growth, and mineral-ion metabolism. PMID:27889227

Brain Region-Specific Trafficking of the Dopamine Transporter

PubMed Central

Block, Ethan R.; Nuttle, Jacob; Balcita-Pedicino, Judith Joyce; Caltagarone, John; Watkins, Simon C.

2015-01-01

The dopamine (DA) transporter (DAT) controls dopaminergic neurotransmission by removing extracellular DA. Although DA reuptake is proposed to be regulated by DAT traffic to and from the cell surface, the membrane trafficking system involved in the endocytic cycling of DAT in the intact mammalian brain has not been characterized. Hence, we performed immunolabeling and quantitative analysis of the subcellular and regional distribution of DAT using the transgenic knock-in mouse expressing hemagglutinin (HA) epitope-tagged DAT (HA-DAT) and by using a combination of electron microscopy and a novel method for immunofluorescence labeling of HA-DAT in acute sagittal brain slices. Both approaches demonstrated that, in midbrain somatodendritic regions, HA-DAT was present in the plasma membrane, endoplasmic reticulum, and Golgi complex, with a small fraction in early and recycling endosomes and an even smaller fraction in late endosomes and lysosomes. In the striatum and in axonal tracts between the midbrain and striatum, HA-DAT was detected predominantly in the plasma membrane, and quantitative analysis revealed increased DAT density in striatal compared with midbrain plasma membranes. Endosomes were strikingly rare and lysosomes were absent in striatal axons, in which there was little intracellular HA-DAT. Acute administration of amphetamine in vivo (60 min) or to slices ex vivo (10–60 min) did not result in detectable changes in DAT distribution. Altogether, these data provide evidence for regional differences in DAT plasma membrane targeting and retention and suggest a surprisingly low level of endocytic trafficking of DAT in the striatum along with limited DAT endocytic activity in somatodendritic areas. SIGNIFICANCE STATEMENT The dopamine transporter (DAT) is the key regulator of the dopamine neurotransmission in the CNS. In the present study, we developed a new approach for studying DAT localization and dynamics in intact neurons in acute sagittal brain slices from

Erythropoietin Receptor Signaling Is Membrane Raft Dependent

PubMed Central

McGraw, Kathy L.; Fuhler, Gwenny M.; Johnson, Joseph O.; Clark, Justine A.; Caceres, Gisela C.; Sokol, Lubomir; List, Alan F.

2012-01-01

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. PMID:22509308

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

PubMed Central

Sciacca, Michele F.M.; Kotler, Samuel A.; Brender, Jeffrey R.; Chen, Jennifer; Lee, Dong-kuk; Ramamoorthy, Ayyalusamy

2012-01-01

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ1–40, defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ1–40 is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer’s disease. PMID:22947931

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue.

PubMed

Varkevisser, R; Houtman, M J C; Linder, T; de Git, K C G; Beekman, H D M; Tidwell, R R; Ijzerman, A P; Stary-Weinzinger, A; Vos, M A; van der Heyden, M A G

2013-07-01

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. hERG and K(IR)2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr . Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K(IR)2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K(IR)2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. Drug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS. © 2013 The British Pharmacological Society.

Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila.

PubMed

Morelli, Elena; Ginefra, Pierpaolo; Mastrodonato, Valeria; Beznoussenko, Galina V; Rusten, Tor Erik; Bilder, David; Stenmark, Harald; Mironov, Alexandre A; Vaccari, Thomas

2014-01-01

How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

PubMed

Chen, Yi-Yung; Rosario, Fredrick J; Shehab, Majida Abu; Powell, Theresa L; Gupta, Madhulika B; Jansson, Thomas

2015-12-01

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. © 2015 Authors; published by Portland Press Limited.

Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure.

PubMed

Szymański, Jędrzej; Janikiewicz, Justyna; Michalska, Bernadeta; Patalas-Krawczyk, Paulina; Perrone, Mariasole; Ziółkowski, Wiesław; Duszyński, Jerzy; Pinton, Paolo; Dobrzyń, Agnieszka; Więckowski, Mariusz R

2017-07-20

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca 2 + handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.

Superresolution imaging of viral protein trafficking

PubMed Central

Salka, Kyle; Bhuvanendran, Shivaprasad; Yang, David

2015-01-01

The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses. PMID:25724304

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue

PubMed Central

Varkevisser, R; Houtman, M J C; Linder, T; de Git, K C G; Beekman, H D M; Tidwell, R R; IJzerman, A P; Stary-Weinzinger, A; Vos, M A; van der Heyden, M A G

2013-01-01

Background and Purpose Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. Experimental Approach hERG and KIR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. Key Results Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and KIR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not KIR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. Conclusions and Implications Drug-induced trafficking defects can be minimized if certain chemical features are avoided or ‘synthesized out’; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS. PMID:23586323

Ras trafficking, localization and compartmentalized signalling

PubMed Central

Prior, Ian A.; Hancock, John F.

2012-01-01

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity.

PubMed

Gómez-Suaga, Patricia; Rivero-Ríos, Pilar; Fdez, Elena; Blanca Ramírez, Marian; Ferrer, Isidro; Aiastui, Ana; López De Munain, Adolfo; Hilfiker, Sabine

2014-12-20

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in an Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pulldown assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients when compared with healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition.

PubMed

Wibowo, Ardian S; Singh, Mirage; Reeder, Kristen M; Carter, Joshua J; Kovach, Alexander R; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E

2013-09-17

Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy.

PubMed

Nooh, Mohammed M; Bahouth, Suleiman W

2017-01-01

G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β 1 -adrenergic receptor (β 1 -AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β 1 -AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review. © 2017 Elsevier Inc. All rights reserved.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR

PubMed Central

Rosario, Fredrick J.; Shehab, Majida Abu; Powell, Theresa L.; Gupta, Madhulika B.; Jansson, Thomas

2015-01-01

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (–72%, P<0.0001) and SNAT-1 (–42%, P<0.05) and SNAT-2 (–31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. PMID:26374858

Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

PubMed

Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

2015-01-01

Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi.

SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation

PubMed Central

Saraceno, C; Marcello, E; Di Marino, D; Borroni, B; Claeysen, S; Perroy, J; Padovani, A; Tramontano, A; Gardoni, F; Di Luca, M

2014-01-01

A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP. PMID:25429624

Numerical calculation on a two-step subdiffusion behavior of lateral protein movement in plasma membranes

NASA Astrophysics Data System (ADS)

Sumi, Tomonari; Okumoto, Atsushi; Goto, Hitoshi; Sekino, Hideo

2017-10-01

A two-step subdiffusion behavior of lateral movement of transmembrane proteins in plasma membranes has been observed by using single-molecule experiments. A nested double-compartment model where large compartments are divided into several smaller ones has been proposed in order to explain this observation. These compartments are considered to be delimited by membrane-skeleton "fences" and membrane-protein "pickets" bound to the fences. We perform numerical simulations of a master equation using a simple two-dimensional lattice model to investigate the heterogeneous diffusion dynamics behavior of transmembrane proteins within plasma membranes. We show that the experimentally observed two-step subdiffusion process can be described using fence and picket models combined with decreased local diffusivity of transmembrane proteins in the vicinity of the pickets. This allows us to explain the two-step subdiffusion behavior without explicitly introducing nested double compartments.

A model for assessing the risk of human trafficking on a local level

NASA Astrophysics Data System (ADS)

Colegrove, Amanda

Human trafficking is a human rights violation that is difficult to quantify. Models for estimating the number of victims of trafficking presented by previous researchers depend on inconsistent, poor quality data. As an intermediate step to help current efforts by nonprofits to combat human trafficking, this project presents a model that is not dependent on quantitative data specific to human trafficking, but rather profiles the risk of human trafficking at the local level through causative factors. Businesses, indicated by the literature, were weighted based on the presence of characteristics that increase the likelihood of trafficking in persons. The mean risk was calculated by census tract to reveal the multiplicity of risk levels in both rural and urban settings. Results indicate that labor trafficking may be a more diffuse problem in Missouri than sex trafficking. Additionally, spatial patterns of risk remained largely the same regardless of adjustments made to the model.

Isoflurane unveils a critical role of glutamate transporter type 3 in regulating hippocampal GluR1 trafficking and context-related learning and memory in mice.

PubMed

Cao, J; Wang, Z; Mi, W; Zuo, Z

2014-07-11

Glutamate transporter type 3 (EAAT3) may play a role in cognition. Isoflurane enhances EAAT3 trafficking to the plasma membrane. Thus, we used isoflurane to determine how EAAT3 might regulate learning and memory and the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, such as GluR1, to the plasma membrane, a fundamental biochemical process for learning and memory. Here, isoflurane increased EAAT3 but did not change GluR1 levels in the plasma membrane of wild-type mouse hippocampus. Isoflurane increased protein phosphatase activity in the wild-type and EAAT3(-/-) mouse hippocampus. Also, isoflurane reduced GluR1 in the plasma membrane and decreased phospho-GluR1 in EAAT3(-/-) mice. The phosphatase inhibitor okadaic acid attenuated these effects. Finally, isoflurane inhibited context-related fear conditioning in EAAT3(-/-) mice but not in wild-type mice. Thus, isoflurane may increase GluR1 trafficking to the plasma membrane via EAAT3 and inhibit GluR1 trafficking via protein phosphatase. Lack of EAAT3 effects leads to decreased GluR1 trafficking and impaired cognition after isoflurane exposure in EAAT3(-/-) mice. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

PubMed

Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

1994-08-01

The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. These results illustrate the internalization and intracellular

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L.; Guttentag, Susan; Hubbard, Michael J.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o− WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells. PMID:21525008

ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L; Guttentag, Susan; Hubbard, Michael J; Rubenstein, Ronald C

2011-06-17

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Permeability and kinetic coefficients for mesoscale BCF surface step dynamics: Discrete two-dimensional deposition-diffusion equation analysis

DOE PAGES

Zhao, Renjie; Evans, James W.; Oliveira, Tiago J.

2016-04-08

Here, a discrete version of deposition-diffusion equations appropriate for description of step flow on a vicinal surface is analyzed for a two-dimensional grid of adsorption sites representing the stepped surface and explicitly incorporating kinks along the step edges. Model energetics and kinetics appropriately account for binding of adatoms at steps and kinks, distinct terrace and edge diffusion rates, and possible additional barriers for attachment to steps. Analysis of adatom attachment fluxes as well as limiting values of adatom densities at step edges for nonuniform deposition scenarios allows determination of both permeability and kinetic coefficients. Behavior of these quantities is assessedmore » as a function of key system parameters including kink density, step attachment barriers, and the step edge diffusion rate.« less

Permeability and kinetic coefficients for mesoscale BCF surface step dynamics: Discrete two-dimensional deposition-diffusion equation analysis

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

Zhao, Renjie; Evans, James W.; Oliveira, Tiago J.

Here, a discrete version of deposition-diffusion equations appropriate for description of step flow on a vicinal surface is analyzed for a two-dimensional grid of adsorption sites representing the stepped surface and explicitly incorporating kinks along the step edges. Model energetics and kinetics appropriately account for binding of adatoms at steps and kinks, distinct terrace and edge diffusion rates, and possible additional barriers for attachment to steps. Analysis of adatom attachment fluxes as well as limiting values of adatom densities at step edges for nonuniform deposition scenarios allows determination of both permeability and kinetic coefficients. Behavior of these quantities is assessedmore » as a function of key system parameters including kink density, step attachment barriers, and the step edge diffusion rate.« less

Devastating consequences of sex trafficking on women's health.

PubMed

McTavish, Fr James

2017-11-01

Sex trafficking has devastating consequences on the physical and mental well-being of millions of women around the world. These trafficking victims often come in contact with medical personnel, and these encounters with suitably prepared staff can be a step toward healing of the victims. The Catholic Church, especially through Pope Francis, is making strenuous efforts to curb the spread of sex trafficking. Same-sex feelings and behavior may arise post-trafficking in individuals, although this does not appear to be mentioned thus far in the literature. Here, we are most likely dealing with a type of "pseudo-lesbianism" post-trauma. The trafficking survivor can be helped to understand some of the likely roots of her feelings such as anti-male sentiments following abuse. She needs to be patiently, and expertly, accompanied to process the trauma she has experienced, and learn how to meet her genuine needs for female affection and affirmation in healthy, chaste, and non-erotic ways. Around the world, millions of female victims of human trafficking are forced into sex "work," often resulting in serious physical and mental-health problems. Healthcare staff should be alert to spot victims of sex trafficking and be ready to assist them. The Catholic Church, especially through Pope Francis, has been vocal in denouncing this form of modern slavery. Some female victims of sex trafficking may experience same-sex feelings afterward. Healing for such young women involves helping them to process their traumatic experiences, as well as patiently accompanying them as they seek to develop healthy, chaste friendships with other females and males.

Membrane traffic and muscle: lessons from human disease.

PubMed

Dowling, James J; Gibbs, Elizabeth M; Feldman, Eva L

2008-07-01

Like all mammalian tissues, skeletal muscle is dependent on membrane traffic for proper development and homeostasis. This fact is underscored by the observation that several human diseases of the skeletal muscle are caused by mutations in gene products of the membrane trafficking machinery. An examination of these diseases and the proteins that underlie them is instructive both in terms of determining disease pathogenesis and of understanding the normal aspects of muscle biology regulated by membrane traffic. This review highlights our current understanding of the trafficking genes responsible for human myopathies.

Human telomerase: biogenesis, trafficking, recruitment, and activation.

PubMed

Schmidt, Jens C; Cech, Thomas R

2015-06-01

Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric DNA in eukaryotes. Recent work has begun to reveal key aspects of the assembly of the human telomerase complex, its intracellular trafficking involving Cajal bodies, and its recruitment to telomeres. Once telomerase has been recruited to the telomere, it appears to undergo a separate activation step, which may include an increase in its repeat addition processivity. This review covers human telomerase biogenesis, trafficking, and activation, comparing key aspects with the analogous events in other species. © 2015 Schmidt and Cech Published by Cold Spring Harbor Laboratory Press.

Signal dependent transport of a membrane cargo from early endosomes to recycling endosomes.

PubMed

Mahmoud, Ismail S; Louber, Jade; Dower, Steve K; Verhagen, Anne M; Gleeson, Paul A

2017-08-01

Many membrane cargoes undergo endocytosis and intracellular recycling to the plasma membrane via the early endosomes and the recycling endosomes. However whether specific sorting signals are required for transport from early endosomes to recycling endosomes is not known and the current view is that transport to the recycling endosomes is by a passive default process. Here we show that the cytoplasmic tail of the neonatal Fc receptor (FcRn) contains discrete signals for endocytosis and for sorting to the recycling endosomes. The FcRn cytoplasmic tail has previously been shown to contain the unusual WISL motif for AP2/clathrin-mediated endocytosis. By analysing FcRn mutants and CD8/FcRn chimeric molecules, we have identified an extended WISL sequence (GLPAPWISL) which promotes sorting from the early endosomes to the recycling endosomes. The insertion of GLPAPWISL into the cytoplasmic tail of CD8 resulted in efficient endocytosis and trafficking to the recycling endosomes, with only low levels detected in the late endosomes. Replacement of the highly conserved GLAPAP sequence within the GLPAPWISL motif with alanine residues resulted in endocytosis of the CD8/FcRn chimera to the early endosomes which was then trafficked predominantly to the late endosomes rather than the recycling endosomes. These studies demonstrate that signals within the cytoplasmic domains of membrane cargo can mediate active transport from early to recycling endosomes. Copyright © 2017 Elsevier GmbH. All rights reserved.

Single-step electrochemical functionalization of double-walled carbon nanotube (DWCNT) membranes and the demonstration of ionic rectification

PubMed Central

2013-01-01

Carbon nanotube (CNT) membranes allow the mimicking of natural ion channels for applications in drug delivery and chemical separation. Double-walled carbon nanotube membranes were simply functionalized with dye in a single step instead of the previous two-step functionalization. Non-faradic electrochemical impedance spectra indicated that the functionalized gatekeeper by single-step modification can be actuated to mimic the protein channel under bias. This functional chemistry was proven by a highly efficient ion rectification, wherein the highest experimental rectification factor of ferricyanide was up to 14.4. One-step functionalization by electrooxidation of amine provides a simple and promising functionalization chemistry for the application of CNT membranes. PMID:23758999

Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6.

PubMed

Cayouette, Sylvie; Bousquet, Simon M; Francoeur, Nancy; Dupré, Emilie; Monet, Michaël; Gagnon, Hugo; Guedri, Youssef B; Lavoie, Christine; Boulay, Guylain

2010-07-01

TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface. 2010 Elsevier B.V. All rights reserved.

hERG trafficking inhibition in drug-induced lethal cardiac arrhythmia.

PubMed

Nogawa, Hisashi; Kawai, Tomoyuki

2014-10-15

Acquired long QT syndrome induced by non-cardiovascular drugs can cause lethal cardiac arrhythmia called torsades de points and is a significant problem in drug development. The prolongation of QT interval and cardiac action potential duration are mainly due to reduced physiological function of the rapidly activating voltage-dependent potassium channels encoded by human ether-a-go-go-related gene (hERG). Structurally diverse groups of drugs are known to directly inhibit hERG channel conductance. Therefore, the ability of acute hERG inhibition is routinely assessed at the preclinical stages in pharmaceutical testing. Recent findings indicated that chronic treatment with various drugs not only inhibits hERG channels but also decreases hERG channel expression in the plasma membrane of cardiomyocytes, which has become another concern in safety pharmacology. The mechanisms involve the disruption of hERG trafficking to the surface membrane or the acceleration of hERG protein degradation. From this perspective, we present a brief overview of mechanisms of drug-induced trafficking inhibition and pathological regulation. Understanding of drug-induced hERG trafficking inhibition may provide new strategies for predicting drug-induced QT prolongation and lethal cardiac arrhythmia in pharmaceutical drug development. Copyright © 2014 Elsevier B.V. All rights reserved.

Trafficking-deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction.

PubMed

Biliczki, Peter; Girmatsion, Zenawit; Brandes, Ralf P; Harenkamp, Sabine; Pitard, Bruno; Charpentier, Flavien; Hébert, Terence E; Hohnloser, Stefan H; Baró, Isabelle; Nattel, Stanley; Ehrlich, Joachim R

2009-12-01

KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function. We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers. Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed. KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes. The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially

Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants.

PubMed

Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

2015-09-01

Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg(2+) as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. © 2015 American Society of Plant Biologists. All Rights Reserved.

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons

PubMed Central

Soo Hoo, Linda; Banna, Chris D.; Radeke, Carolyn M.; Sharma, Nikunj; Albertolle, Mary E.; Low, Seng Hui; Weimbs, Thomas; Vandenberg, Carol A.

2016-01-01

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons. PMID:27662481

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

PubMed

Soo Hoo, Linda; Banna, Chris D; Radeke, Carolyn M; Sharma, Nikunj; Albertolle, Mary E; Low, Seng Hui; Weimbs, Thomas; Vandenberg, Carol A

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons.

Medical education and human trafficking: using simulation.

PubMed

Stoklosa, Hanni; Lyman, Michelle; Bohnert, Carrie; Mittel, Olivia

2017-01-01

Healthcare providers have the potential to play a crucial role in human trafficking prevention, identification, and intervention. However, trafficked patients are often unidentified due to lack of education and preparation available to healthcare professionals at all levels of training and practice. To increase victim identification in healthcare settings, providers need to be educated about the issue of trafficking and its clinical presentations in an interactive format that maximizes learning and ultimately patient-centered outcomes. In 2014, University of Louisville School of Medicine created a simulation-based medical education (SBME) curriculum to prepare students to recognize victims and intervene on their behalf. The authors share the factors that influenced the session's development and incorporation into an already full third year medical curriculum and outline the development process. The process included a needs assessment for the education intervention, development of objectives and corresponding assessment, implementation of the curriculum, and finally the next steps of the module as it develops further. Additional alternatives are provided for other medical educators seeking to implement similar modules at their home institution. It is our hope that the description of this process will help others to create similar interactive educational programs and ultimately help trafficking survivors receive the care they need. HCP: Healthcare professional; M-SIGHT: Medical student instruction in global human trafficking; SBME: Simulation-based medical education; SP: Standardized patient; TIC: Trauma-informed care.

Live Cell Imaging of the Endocytosis and the Intracellular Trafficking of Multifunctional Lipid Nanoparticles

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

Zhang, Tieqiao; Danthi, S. N.; Xie, Jianwu

Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug deliverymore » system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.« less

Live cell imaging of the endocytosis and the intracellular trafficking of multifunctional lipid nanoparticles

NASA Astrophysics Data System (ADS)

Zhang, Tieqiao; Danthi, S. Narasimhan; Xie, Jianwu; Hu, Dehong; Lu, Peter; Li, King

2006-02-01

Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug delivery system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.

Neutrophil Membrane Cholesterol Content is a Key Factor in Cystic Fibrosis Lung Disease.

PubMed

White, Michelle M; Geraghty, Patrick; Hayes, Elaine; Cox, Stephen; Leitch, William; Alfawaz, Bader; Lavelle, Gillian M; McElvaney, Oliver J; Flannery, Ryan; Keenan, Joanne; Meleady, Paula; Henry, Michael; Clynes, Martin; Gunaratnam, Cedric; McElvaney, Noel G; Reeves, Emer P

2017-09-01

Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n=48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays. Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Human trafficking: Role of oral health care providers.

PubMed

Nuzzolese, E

2014-11-30

Trafficking in human beings is a modern form of slavery and is a well-known phenomenon throughout the European Union and beyond. After drug dealing and the weapons industry, human trafficking is the second largest criminal activity in the world today and it is a growing crime. The aim of governmental and non-governmental agencies, which are either directly or indirectly involved in combating trafficking in human beings, is the identification and referral of victims of trafficking and also to encourage self-referrals. Identification is the most important step to provide protection and assistance to victims of trafficking. Victims often have a variety of physical and mental health needs, including psychological trauma, injuries from violence, head and neck trauma, sexually transmitted infections and other gynaecological problems, dental/oral problems and have poor nutrition. The author's experience in the field of community dentistry in presented within. Volunteer dental services are offered to non-European Union patients held in a centre for asylum seekers in Bari (Italy). Dental professionals can, in fact, contribute to the identification, assistance and protection of trafficked persons, as well as offering forensic services to assist the police investigation in order to identify crimes and find the criminal organizations behind them. As for domestic violence and child abuse cases, there are ethical concerns involved in the identification and protection of the trafficked persons, as well as the need for interdisciplinary work and awareness. Adequate training in behavioural science and intercultural learning is paramount in order to avoid misunderstandings and increase sensitivity.

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

PubMed

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

Economics of human trafficking.

PubMed

Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

2010-01-01

Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.

Identifying Human Trafficking Victims on a Psychiatry Inpatient Service: a Case Series.

PubMed

Nguyen, Phuong T; Lamkin, Joanna; Coverdale, John H; Scott, Samuel; Li, Karen; Gordon, Mollie R

2018-06-01

Human trafficking is a serious and prevalent human rights violation that closely intersects with mental health. Limited empirical attention has been paid to the presentations and identification of trafficking victims in psychiatric settings. The primary goal of this paper is to describe the varied presentations of trafficking victims on an urban inpatient psychiatric unit. A literature review was conducted to identify relevant empirical articles to inform our examination of cases. Adult inpatient cases meeting criteria for known or possible human trafficking were systematically identified and illustrative cases were described. Six cases were identified including one male and five females. Two had been labor trafficked and four were suspected or confirmed to have been sex trafficked. The cases demonstrated a tremendous diversity of demographic and psychiatric identifying factors. These cases indicate the importance of routinely screening for trafficking victims in inpatient psychiatry settings. Identification of cases is a requisite step in providing informed and evidence-based treatments and enabling the secondary prevention of re-exploitation. Additional research is warranted given the limited current empirical research on this topic area.

Global initiatives to tackle organ trafficking and transplant tourism.

PubMed

Bagheri, Alireza; Delmonico, Francis L

2013-11-01

The increasing gap between organ supply and demand has opened the door for illegal organ sale, trafficking of human organs, tissues and cells, as well as transplant tourism. Currently, underprivileged and vulnerable populations in resource-poor countries are a major source of organs for rich patient-tourists who can afford to purchase organs at home or abroad. This paper presents a summary of international initiatives, such as World Health Organization's Principle Guidelines, The Declaration of Istanbul, Asian Task Force Recommendations, as well as UNESCO's and the United Nation's initiatives against trafficking of human organs, tissues, cells, and transplant tourism. Beyond the summary, it calls for more practical measures to be taken to implement the existing guidelines and recommendations, in order to prevent exploitation of the poor as organ providers. The paper suggests that an international legally binding agreement in criminalizing organ trafficking would be a step forward to bring a change in the global picture of organ trafficking and transplant tourism.

Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

PubMed Central

Martin, Gregory M.; Chen, Pei-Chun; Devaraneni, Prasanna; Shyng, Show-Ling

2013-01-01

ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed. PMID:24399968

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model.

PubMed

Sasamoto, Kouhei; Marunaka, Rie; Niisato, Naomi; Sun, Hongxin; Taruno, Akiyuki; Pezzotti, Giuseppe; Yamamoto, Toshiro; Kanamura, Narisato; Zhu, Wenliang; Nishio, Kyosuke; Inui, Toshio; Eaton, Douglas C; Marunaka, Yoshinori

2017-01-01

Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active

Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing.

PubMed

Bartusch, Christina; Döring, Tatjana; Prange, Reinhild

2017-06-21

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

UNC-108/Rab2 Regulates Postendocytic Trafficking in Caenorhabditis elegans

PubMed Central

Chun, Denise K.; McEwen, Jason M.; Burbea, Michelle

2008-01-01

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking. PMID:18434599

Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

PubMed

Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

2017-07-28

The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery?

PubMed Central

Cox, Adrienne D.; Der, Channing J.; Philips, Mark R.

2015-01-01

RAS proteins require membrane association for their biological activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTIs) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anti-cancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated post-translational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. PMID:25878363

Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

PubMed

Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

2015-08-01

ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Medical education and human trafficking: using simulation

PubMed Central

Stoklosa, Hanni; Lyman, Michelle; Bohnert, Carrie; Mittel, Olivia

2017-01-01

ABSTRACT Healthcare providers have the potential to play a crucial role in human trafficking prevention, identification, and intervention. However, trafficked patients are often unidentified due to lack of education and preparation available to healthcare professionals at all levels of training and practice. To increase victim identification in healthcare settings, providers need to be educated about the issue of trafficking and its clinical presentations in an interactive format that maximizes learning and ultimately patient-centered outcomes. In 2014, University of Louisville School of Medicine created a simulation-based medical education (SBME) curriculum to prepare students to recognize victims and intervene on their behalf. The authors share the factors that influenced the session’s development and incorporation into an already full third year medical curriculum and outline the development process. The process included a needs assessment for the education intervention, development of objectives and corresponding assessment, implementation of the curriculum, and finally the next steps of the module as it develops further. Additional alternatives are provided for other medical educators seeking to implement similar modules at their home institution. It is our hope that the description of this process will help others to create similar interactive educational programs and ultimately help trafficking survivors receive the care they need. Abbreviations: HCP: Healthcare professional; M-SIGHT: Medical student instruction in global human trafficking; SBME: Simulation-based medical education; SP: Standardized patient; TIC: Trauma-informed care PMID:29228882

The “Tail” of Connexin43: An Unexpected Journey from Alternative Translation to Trafficking

PubMed Central

Basheer, Wassim; Shaw, Robin

2015-01-01

With each heartbeat, Connexin43 (Cx43) cell-cell communication gap junctions are needed to rapidly spread and coordinate excitation signals for an effective heart contraction. The correct formation and delivery of channels to their respective membrane subdomain is referred to as protein trafficking. Altered Cx43 trafficking is a dangerous complication of diseased myocardium which contributes to the arrhythmias of sudden cardiac death. Cx43 has also been found to regulate many other cellular processes that cannot be explained by cell-cell communication. We recently identified the existence of up to six endogenous internally translated Cx43 N-terminal truncated isoforms from the same full-length mRNA molecule. This is the first evidence that alternative translation is possible for human ion channels and in human heart. Interestingly, we found that these internally translated isoforms, more specifically the 20 kDa isoform (GJA1-20k), is important for delivery of Cx43 to its respective membrane subdomain. This review covers recent advances in Cx43 trafficking and potential importance of alternatively translated Cx43 truncated isoforms. PMID:26526689

Pathogen trafficking pathways and host phosphoinositide metabolism.

PubMed

Weber, Stefan S; Ragaz, Curdin; Hilbi, Hubert

2009-03-01

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.

Revisiting caveolin trafficking: the end of the caveosome

PubMed Central

Howes, Mark T.

2010-01-01

In this issue, a study by Hayer et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003086) provides insights into the trafficking of caveolins, the major membrane proteins of caveolae. As well as providing evidence for ubiquitin-mediated endosomal sorting and degradation of caveolin in multivesicular bodies (MVBs), the new findings question the existence of a unique organelle proposed nine years ago, the caveosome. PMID:21041440

Discrete maximal regularity of time-stepping schemes for fractional evolution equations.

PubMed

Jin, Bangti; Li, Buyang; Zhou, Zhi

2018-01-01

In this work, we establish the maximal [Formula: see text]-regularity for several time stepping schemes for a fractional evolution model, which involves a fractional derivative of order [Formula: see text], [Formula: see text], in time. These schemes include convolution quadratures generated by backward Euler method and second-order backward difference formula, the L1 scheme, explicit Euler method and a fractional variant of the Crank-Nicolson method. The main tools for the analysis include operator-valued Fourier multiplier theorem due to Weis (Math Ann 319:735-758, 2001. doi:10.1007/PL00004457) and its discrete analogue due to Blunck (Stud Math 146:157-176, 2001. doi:10.4064/sm146-2-3). These results generalize the corresponding results for parabolic problems.

Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

PubMed Central

Lee, Byung Ha; Gauna, Adrienne E.; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M.; Kawai, Toshihisa; Cha, Seunghee

2013-01-01

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS. PMID:23382834

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

PubMed Central

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

PubMed

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

PubMed Central

Greune, Lilo; Jarosch, Kevin-André; Steil, Daniel; Zhang, Wenlan; He, Xiaohua; Lloubes, Roland; Fruth, Angelika; Kim, Kwang Sik; Schmidt, M. Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge

2017-01-01

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors

VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration.

PubMed

Williams, B Blairanne; Cantrell, V Ashley; Mundell, Nathan A; Bennett, Andrea C; Quick, Rachel E; Jessen, Jason R

2012-05-01

Planar cell polarity (PCP) describes the polarized orientation of cells within the plane of a tissue. Unlike epithelial PCP, the mechanisms underlying PCP signaling in migrating cells remain undefined. Here, the establishment of PCP must be coordinated with dynamic changes in cell adhesion and extracellular matrix (ECM) organization. During gastrulation, the membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is required for PCP and convergence and extension cell movements. We report that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell-surface availability of MMP14 in manner that is dependent on focal adhesion kinase. We demonstrate that zebrafish trilobite/vangl2 mutant embryos exhibit increased Mmp14 activity and decreased ECM. Furthermore, in vivo knockdown of Mmp14 partially rescues the Vangl2 loss-of-function convergence and extension phenotype. This study identifies a mechanism linking VANGL2 with MMP14 trafficking and suggests that establishment of PCP in migrating gastrula cells requires regulated proteolytic degradation or remodeling of the ECM. Our findings implicate matrix metalloproteinases as downstream effectors of PCP and suggest a broadly applicable mechanism whereby VANGL2 affects diverse morphogenetic processes.

Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

PubMed Central

Macabuag, Natsuko

2015-01-01

N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants. PMID:26511252

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle

PubMed Central

Peterson, Soren J.; Krasnow, Mark A.

2015-01-01

SUMMARY To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains including inside the cell. PMID:25557078

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.

PubMed

Schmidlin, F; Dery, O; DeFea, K O; Slice, L; Patierno, S; Sternini, C; Grady, E F; Bunnett, N W

2001-07-06

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

Ultra-fast consensus of discrete-time multi-agent systems with multi-step predictive output feedback

NASA Astrophysics Data System (ADS)

Zhang, Wenle; Liu, Jianchang

2016-04-01

This article addresses the ultra-fast consensus problem of high-order discrete-time multi-agent systems based on a unified consensus framework. A novel multi-step predictive output mechanism is proposed under a directed communication topology containing a spanning tree. By predicting the outputs of a network several steps ahead and adding this information into the consensus protocol, it is shown that the asymptotic convergence factor is improved by a power of q + 1 compared to the routine consensus. The difficult problem of selecting the optimal control gain is solved well by introducing a variable called convergence step. In addition, the ultra-fast formation achievement is studied on the basis of this new consensus protocol. Finally, the ultra-fast consensus with respect to a reference model and robust consensus is discussed. Some simulations are performed to illustrate the effectiveness of the theoretical results.

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

PubMed

Tani, Motohiro; Kuge, Osamu

2012-12-01

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. © 2012 Blackwell Publishing Ltd.

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site

PubMed Central

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-01-01

ABSTRACT Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1–PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. PMID:26430213

Plasma membrane-associated platforms: dynamic scaffolds that organize membrane-associated events.

PubMed

Astro, Veronica; de Curtis, Ivan

2015-03-10

Specialized regions of the plasma membrane dedicated to diverse cellular processes, such as vesicle exocytosis, extracellular matrix remodeling, and cell migration, share a few cytosolic scaffold proteins that associate to form large plasma membrane-associated platforms (PMAPs). PMAPs organize signaling events and trafficking of membranes and molecules at specific membrane domains. On the basis of the intrinsic disorder of the proteins constituting the core of these PMAPs and of the dynamics of these structures at the periphery of motile cells, we propose a working model for the assembly and turnover of these platforms. Copyright © 2015, American Association for the Advancement of Science.

ARF1 and SAR1 GTPases in Endomembrane Trafficking in Plants

PubMed Central

Cevher-Keskin, Birsen

2013-01-01

Small GTPases largely control membrane traffic, which is essential for the survival of all eukaryotes. Among the small GTP-binding proteins, ARF1 (ADP-ribosylation factor 1) and SAR1 (Secretion-Associated RAS super family 1) are commonly conserved among all eukaryotes with respect to both their functional and sequential characteristics. The ARF1 and SAR1 GTP-binding proteins are involved in the formation and budding of vesicles throughout plant endomembrane systems. ARF1 has been shown to play a critical role in COPI (Coat Protein Complex I)-mediated retrograde trafficking in eukaryotic systems, whereas SAR1 GTPases are involved in intracellular COPII-mediated protein trafficking from the ER to the Golgi apparatus. This review offers a summary of vesicular trafficking with an emphasis on the ARF1 and SAR1 expression patterns at early growth stages and in the de-etiolation process. PMID:24013371

Force dependence of phagosome trafficking in retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Daniel, Rebekah; Koll, Andrew T.; Altman, David

2014-09-01

Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

Drive the Car(go)s-New Modalities to Control Cargo Trafficking in Live Cells.

PubMed

Mondal, Payel; Khamo, John S; Krishnamurthy, Vishnu V; Cai, Qi; Zhang, Kai

2017-01-01

Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.

Biological membranes

PubMed Central

Watson, Helen

2015-01-01

Biological membranes allow life as we know it to exist. They form cells and enable separation between the inside and outside of an organism, controlling by means of their selective permeability which substances enter and leave. By allowing gradients of ions to be created across them, membranes also enable living organisms to generate energy. In addition, they control the flow of messages between cells by sending, receiving and processing information in the form of chemical and electrical signals. This essay summarizes the structure and function of membranes and the proteins within them, and describes their role in trafficking and transport, and their involvement in health and disease. Techniques for studying membranes are also discussed. PMID:26504250

Regulation of intracellular trafficking and secretion of adiponectin by myosin II.

PubMed

Bedi, Deepa; Dennis, John C; Morrison, Edward E; Braden, Tim D; Judd, Robert L

2017-08-19

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanisms Underlying Activation of α₁-Adrenergic Receptor-Induced Trafficking of AQP5 in Rat Parotid Acinar Cells under Isotonic or Hypotonic Conditions.

PubMed

Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Shono, Masayuki; Ishikawa, Yasuko

2016-06-28

Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α₁-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La(3+), implying the participation of store-operated Ca(2+) channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La(3+), suggesting the involvement of extracellular Ca(2+) entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca(2+)/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca(2+) entry.

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

PubMed

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-11-15

Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. © 2015. Published by The Company of Biologists Ltd.

Resolving the homology—function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

PubMed Central

Klinger, Christen M.; Ramirez-Macias, Inmaculada; Herman, Emily K.; Turkewitz, Aaron P.; Field, Mark C.; Dacks, Joel B.

2016-01-01

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage. PMID:27444378

Continuous sweep versus discrete step protocols for studying effects of wearable robot assistance magnitude.

PubMed

Malcolm, Philippe; Rossi, Denise Martineli; Siviy, Christopher; Lee, Sangjun; Quinlivan, Brendan Thomas; Grimmer, Martin; Walsh, Conor J

2017-07-12

Different groups developed wearable robots for walking assistance, but there is still a need for methods to quickly tune actuation parameters for each robot and population or sometimes even for individual users. Protocols where parameters are held constant for multiple minutes have traditionally been used for evaluating responses to parameter changes such as metabolic rate or walking symmetry. However, these discrete protocols are time-consuming. Recently, protocols have been proposed where a parameter is changed in a continuous way. The aim of the present study was to compare effects of continuously varying assistance magnitude with a soft exosuit against discrete step conditions. Seven participants walked on a treadmill wearing a soft exosuit that assists plantarflexion and hip flexion. In Continuous-up, peak exosuit ankle moment linearly increased from approximately 0 to 38% of biological moment over 10 min. Continuous-down was the opposite. In Discrete, participants underwent five periods of 5 min with steady peak moment levels distributed over the same range as Continuous-up and Continuous-down. We calculated metabolic rate for the entire Continuous-up and Continuous-down conditions and the last 2 min of each Discrete force level. We compared kinematics, kinetics and metabolic rate between conditions by curve fitting versus peak moment. Reduction in metabolic rate compared to Powered-off was smaller in Continuous-up than in Continuous-down at most peak moment levels, due to physiological dynamics causing metabolic measurements in Continuous-up and Continuous-down to lag behind the values expected during steady-state testing. When evaluating the average slope of metabolic reduction over the entire peak moment range there was no significant difference between Continuous-down and Discrete. Attempting to correct the lag in metabolics by taking the average of Continuous-up and Continuous-down removed all significant differences versus Discrete. For kinematic and

P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery.

PubMed

Davis, Thomas P; Sanchez-Covarubias, Lucy; Tome, Margaret E

2014-01-01

The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription. © 2014 Elsevier Inc. All rights reserved.

Integration of Nine Steps into One Membrane Reactor To Produce Synthesis Gases for Ammonia and Liquid Fuel.

PubMed

Li, Wenping; Zhu, Xuefeng; Chen, Shuguang; Yang, Weishen

2016-07-18

The synthesis of ammonia and liquid fuel are two important chemical processes in which most of the energy is consumed in the production of H2 /N2 and H2 /CO synthesis gases from natural gas (methane). Here, we report a membrane reactor with a mixed ionic-electronic conducting membrane, in which the nine steps for the production of the two types of synthesis gases are shortened to one step by using water, air, and methane as feeds. In the membrane reactor, there is no direct CO2 emission and no CO or H2 S present in the ammonia synthesis gas. The energy consumption for the production of the two synthesis gases can be reduced by 63 % by using this membrane reactor. This promising membrane reactor process has been successfully demonstrated by experiment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

NASA In-step: Permeable Membrane Experiment

NASA Technical Reports Server (NTRS)

1992-01-01

Viewgraphs on the Permeable Membrane Experiment are presented. An experiment overview is given. The Membrane Phase Separation Experiment, Membrane Diffusion Interference Experiment, and Membrane Wetting Experiment are described. Finally, summary and conclusions are discussed.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

PubMed

Peterson, Soren J; Krasnow, Mark A

2015-01-15

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

Golgi- and Trans-Golgi Network-Mediated Vesicle Trafficking Is Required for Wax Secretion from Epidermal Cells1[W][OPEN

PubMed Central

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

2014-01-01

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

Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes

USDA-ARS?s Scientific Manuscript database

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better un...

Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

PubMed

Giudice, Jimena; Loehr, James A; Rodney, George G; Cooper, Thomas A

2016-11-15

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

PubMed Central

Farr, Glen A.; Hull, Michael; Stoops, Emily H.; Bateson, Rosalie; Caplan, Michael J.

2015-01-01

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking. PMID:26424804

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

PubMed Central

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Cruz-Moreno, Beatriz; Leppla, Stephen H.; Nizet, Victor

2017-01-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies. PMID:28945820

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

PubMed

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Schwartz, Ruth; Chin, Stephen; Zhu, Lin; Cruz-Moreno, Beatriz; Liu, Janet Z; Aguilar, Bernice; Hollands, Andrew; Leppla, Stephen H; Nizet, Victor; Bier, Ethan

2017-09-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Multiple Dileucine-like Motifs Direct VGLUT1 Trafficking

PubMed Central

Foss, Sarah M.; Li, Haiyan; Santos, Magda S.; Edwards, Robert H.

2013-01-01

The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation. PMID:23804088

Multiple dileucine-like motifs direct VGLUT1 trafficking.

PubMed

Foss, Sarah M; Li, Haiyan; Santos, Magda S; Edwards, Robert H; Voglmaier, Susan M

2013-06-26

The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation.

Prolonged morphine treatment alters δ opioid receptor post-internalization trafficking

PubMed Central

Ong, E W; Xue, L; Olmstead, M C; Cahill, C M

2015-01-01

BACKGROUND AND PURPOSE The δ opioid receptor (DOP receptor) undergoes internalization both constitutively and in response to agonists. Previous work has shown that DOP receptors traffic from intracellular compartments to neuronal cell membranes following prolonged morphine treatment. Here, we examined the effects of prolonged morphine treatment on the post-internalization trafficking of DOP receptors. EXPERIMENTAL APPROACH Using primary cultures of dorsal root ganglia neurons, we measured the co-localization of endogenous DOP receptors with post-endocytic compartments following both prolonged and acute agonist treatments. KEY RESULTS A departure from the constitutive trafficking pathway was observed following acute DOP receptor agonist-induced internalization by deltorphin II. That is, the DOP receptor underwent distinct agonist-induced post-endocytic sorting. Following prolonged morphine treatment, constitutive DOP receptor trafficking was augmented. SNC80 following prolonged morphine treatment also caused non-constitutive DOP receptor agonist-induced post-endocytic sorting. The μ opioid receptor (MOP receptor) agonist DAMGO induced DOP receptor internalization and trafficking following prolonged morphine treatment. Finally, all of the alterations to DOP receptor trafficking induced by both DOP and MOP receptor agonists were inhibited or absent when those agonists were co-administered with a DOP receptor antagonist, SDM-25N. CONCLUSIONS AND IMPLICATIONS The results support the hypothesis that prolonged morphine treatment induces the formation of MOP–DOP receptor interactions and subsequent augmentation of the available cell surface DOP receptors, at least some of which are in the form of a MOP/DOP receptor species. The pharmacology and trafficking of this species appear to be unique compared to those of its individual constituents. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other

Prolonged morphine treatment alters δ opioid receptor post-internalization trafficking.

PubMed

Ong, E W; Xue, L; Olmstead, M C; Cahill, C M

2015-01-01

The δ opioid receptor (DOP receptor) undergoes internalization both constitutively and in response to agonists. Previous work has shown that DOP receptors traffic from intracellular compartments to neuronal cell membranes following prolonged morphine treatment. Here, we examined the effects of prolonged morphine treatment on the post-internalization trafficking of DOP receptors. Using primary cultures of dorsal root ganglia neurons, we measured the co-localization of endogenous DOP receptors with post-endocytic compartments following both prolonged and acute agonist treatments. A departure from the constitutive trafficking pathway was observed following acute DOP receptor agonist-induced internalization by deltorphin II. That is, the DOP receptor underwent distinct agonist-induced post-endocytic sorting. Following prolonged morphine treatment, constitutive DOP receptor trafficking was augmented. SNC80 following prolonged morphine treatment also caused non-constitutive DOP receptor agonist-induced post-endocytic sorting. The μ opioid receptor (MOP receptor) agonist DAMGO induced DOP receptor internalization and trafficking following prolonged morphine treatment. Finally, all of the alterations to DOP receptor trafficking induced by both DOP and MOP receptor agonists were inhibited or absent when those agonists were co-administered with a DOP receptor antagonist, SDM-25N. The results support the hypothesis that prolonged morphine treatment induces the formation of MOP-DOP receptor interactions and subsequent augmentation of the available cell surface DOP receptors, at least some of which are in the form of a MOP/DOP receptor species. The pharmacology and trafficking of this species appear to be unique compared to those of its individual constituents. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The Authors. British

SNARE-mediated trafficking of {alpha}{sub 5}{beta}{sub 1} integrin is required for spreading in CHO cells

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

Skalski, Michael; Coppolino, Marc G.

2005-10-07

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cellmore » spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of {alpha}{sub 5}{beta}{sub 1} integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.« less

Membrane order in the plasma membrane and endocytic recycling compartment.

PubMed

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Role of LRRK2 in the regulation of dopamine receptor trafficking

PubMed Central

Sanna, Simona; Taymans, Jean Marc; Morari, Michele; Brugnoli, Alberto; Frassineti, Martina; Masala, Alessandra; Esposito, Sonia; Galioto, Manuela; Valle, Cristiana; Carri, Maria Teresa; Biosa, Alice; Greggio, Elisa; Crosio, Claudia

2017-01-01

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson’s disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD. PMID:28582422

Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

PubMed

Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2014-07-01

Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking

PubMed Central

Iles, Lakesla R.; Bartholomeusz, Geoffrey

2017-01-01

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor–guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors. PMID:28883040

Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking.

PubMed

Selyunin, Andrey S; Iles, Lakesla R; Bartholomeusz, Geoffrey; Mukhopadhyay, Somshuvra

2017-10-02

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors. © 2017 Selyunin et al.

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.

Integration of cellular ubiquitin and membrane traffic systems: focus on deubiquitylases.

PubMed

Clague, Michael J; Urbé, Sylvie

2017-06-01

The cell is comprised of integrated multilevel protein networks or systems. The ubiquitin, protein homeostasis and membrane trafficking systems are highly integrated. Here, we look at the influence of reversible ubiquitylation on membrane trafficking and organelle dynamics. We review the regulation of endocytic sorting, selective autophagy and the secretory pathway by ubiquitin signals, with a particular focus on detailing the contribution of deubiquitylating enzymes. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

A novel approach for blood purification: mixed-matrix membranes combining diffusion and adsorption in one step.

PubMed

Tijink, Marlon S L; Wester, Maarten; Sun, Junfen; Saris, Anno; Bolhuis-Versteeg, Lydia A M; Saiful, Saiful; Joles, Jaap A; Borneman, Zandrie; Wessling, Matthias; Stamatialis, Dimitris F

2012-07-01

Hemodialysis is a commonly used blood purification technique in patients requiring kidney replacement therapy. Sorbents could increase uremic retention solute removal efficiency but, because of poor biocompatibility, their use is often limited to the treatment of patients with acute poisoning. This paper proposes a novel membrane concept for combining diffusion and adsorption of uremic retention solutes in one step: the so-called mixed-matrix membrane (MMM). In this concept, adsorptive particles are incorporated in a macro-porous membrane layer whereas an extra particle-free membrane layer is introduced on the blood-contacting side of the membrane to improve hemocompatibility and prevent particle release. These dual-layer mixed-matrix membranes have high clean-water permeance and high creatinine adsorption from creatinine model solutions. In human plasma, the removal of creatinine and of the protein-bound solute para-aminohippuric acid (PAH) by single and dual-layer membranes is in agreement with the removal achieved by the activated carbon particles alone, showing that under these experimental conditions the accessibility of the particles in the MMM is excellent. This study proves that the combination of diffusion and adsorption in a single step is possible and paves the way for the development of more efficient blood purification devices, excellently combining the advantages of both techniques. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Legislating thresholds for drug trafficking: a policy development case study from New South Wales, Australia.

PubMed

Hughes, Caitlin Elizabeth; Ritter, Alison; Cowdery, Nicholas

2014-09-01

Legal thresholds are used in many parts of the world to define the quantity of illicit drugs over which possession is deemed "trafficking" as opposed to "possession for personal use". There is limited knowledge about why or how such laws were developed. In this study we analyse the policy processes underpinning the introduction and expansion of the drug trafficking legal threshold system in New South Wales (NSW), Australia. A critical legal and historical analysis was undertaken sourcing data from legislation, Parliamentary Hansard debates, government inquiries, police reports and research. A timeline of policy developments was constructed from 1970 until 2013 outlining key steps including threshold introduction (1970), expansion (1985), and wholesale revision (1988). We then critically analysed the drivers of each step and the roles played by formal policy actors, public opinion, research/data and the drug trafficking problem. We find evidence that while justified as a necessary tool for effective law enforcement of drug trafficking, their introduction largely preceded overt police calls for reform or actual increases in drug trafficking. Moreover, while the expansion from one to four thresholds had the intent of differentiating small from large scale traffickers, the quantities employed were based on government assumptions which led to "manifest problems" and the revision in 1988 of over 100 different quantities. Despite the revisions, there has remained no further formal review and new quantities for "legal highs" continue to be added based on assumption and an uncertain evidence-base. The development of legal thresholds for drug trafficking in NSW has been arbitrary and messy. That the arbitrariness persists from 1970 until the present day makes it hard to conclude the thresholds have been well designed. Our narrative provides a platform for future policy reform. Copyright © 2014 Elsevier B.V. All rights reserved.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

PubMed

Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

The State, the family and language of 'social evils': re-stigmatising victims of trafficking in Vietnam.

PubMed

Vijeyarasa, Ramona

2010-08-01

The Vietnamese Government continues to take steps to address trafficking in women and girls. However, rather than perceiving trafficking as a violation of human rights, greater attention is given by the government to its effects on society and social morals, particularly where victims have engaged in sex work in destination countries. Trafficked returnees are directly implicated in the State's approach to defining sex work as a 'social evil'. This approach reproduces the socio-economic inequality involved in trafficking and further marginalises trafficked women. Simultaneously, although Vietnamese women are often drawn into trafficking due to family obligations, they frequently face dishonour upon their return or are forced to hide the truth of their experience of being trafficked. This paper argues that the language of 'social evils' and the responses of the State and family undermine the ability of trafficked returnees to reintegrate. This is heightened where returnees are deemed to be transmitters of HIV infection, hence suffering human trafficking, sex-work and HIV/AIDS-related stigma. I also reflect upon whether the approach of service providers exacerbates stigma, particularly in the context of shelter rehabilitation and present several recommendations for reform, the most pressing being the need to eliminate the language of 'social evils'.

Trafficking in persons: a health concern?

PubMed

Zimmerman, Cathy; Kiss, Ligia; Houssain, Mazeda; Watts, Charlotte

2009-01-01

Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care.

A Scale-Invariant ``Discrete-Time'' Balitsky--Kovchegov Equation

NASA Astrophysics Data System (ADS)

Bialas, A.; Peschanski, R.

2005-06-01

We consider a version of QCD dipole cascading corresponding to a finite number n of discrete Δ Y steps of branching in rapidity. Using the discretization scheme preserving the holomorphic factorizability and scale-invariance in position space of the dipole splitting function, we derive an exact recurrence formula from step to step which plays the rôle of a ``discrete-time'' Balitsky--Kovchegov equation. The BK solutions are recovered in the limit n=∞ and Δ Y=0.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

PubMed

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

PubMed Central

Hoogenraad, Casper C.; Popa, Ioana; Futai, Kensuke; Sanchez-Martinez, Emma; Wulf, Phebe S.; van Vlijmen, Thijs; Dortland, Bjorn R.; Oorschot, Viola; Govers, Roland; Monti, Maria; Heck, Albert J. R.; Sheng, Morgan; Klumperman, Judith; Rehmann, Holger; Jaarsma, Dick; Kapitein, Lukas C.; van der Sluijs, Peter

2010-01-01

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking. PMID:20098723

Human Trafficking

ERIC Educational Resources Information Center

Wilson, David McKay

2011-01-01

The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

Manipulation of host membranes by bacterial effectors.

PubMed

Ham, Hyeilin; Sreelatha, Anju; Orth, Kim

2011-07-18

Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

PubMed

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-05-18

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Tomlinson, Darren C.; Harrison, Michael A.; Ponnambalam, Sreenivasan

2015-01-01

VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR–VEGF complexes with membrane trafficking along the endosome–lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR–VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. PMID:26285805

One-step fabrication of porous GaN crystal membrane and its application in energy storage

NASA Astrophysics Data System (ADS)

Zhang, Lei; Wang, Shouzhi; Shao, Yongliang; Wu, Yongzhong; Sun, Changlong; Huo, Qin; Zhang, Baoguo; Hu, Haixiao; Hao, Xiaopeng

2017-03-01

Single-crystal gallium nitride (GaN) membranes have great potential for a variety of applications. However, fabrication of single-crystalline GaN membranes remains a challenge owing to its chemical inertness and mechanical hardness. This study prepares large-area, free-standing, and single-crystalline porous GaN membranes using a one-step high-temperature annealing technique for the first time. A promising separation model is proposed through a comprehensive study that combines thermodynamic theories analysis and experiments. Porous GaN crystal membrane is processed into supercapacitors, which exhibit stable cycling life, high-rate capability, and ultrahigh power density, to complete proof-of-concept demonstration of new energy storage application. Our results contribute to the study of GaN crystal membranes into a new stage related to the elelctrochemical energy storage application.

Eeyarestatin 1 Interferes with Both Retrograde and Anterograde Intracellular Trafficking Pathways

PubMed Central

Aletrari, Mina-Olga; McKibbin, Craig; Williams, Helen; Pawar, Vidya; Pietroni, Paola; Lord, J. Michael; Flitsch, Sabine L.; Whitehead, Roger; Swanton, Eileithyia; High, Stephen; Spooner, Robert A.

2011-01-01

Background The small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation. Methodology and Principal Findings Using cytotoxicity measurements, we found no role for ESI as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking. Conclusions and Significance ESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed. PMID:21799938

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

PubMed

Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

2006-01-24

Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

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.

Metabotropic Glutamate Receptors in the Trafficking of Ionotropic Glutamate and GABAA Receptors at Central Synapses

PubMed Central

Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

2006-01-01

The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABAA receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABAA receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca2+ concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABAA receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABAA receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves. PMID:18615134

Human Trafficking in Ethiopia: A Scoping Review to Identify Gaps in Service Delivery, Research, and Policy.

PubMed

Beck, Dana C; Choi, Kristen R; Munro-Kramer, Michelle L; Lori, Jody R

2017-12-01

The purpose of this review is to integrate evidence on human trafficking in Ethiopia and identify gaps and recommendations for service delivery, research and training, and policy. A scoping literature review approach was used to systematically search nursing, medical, psychological, law, and international databases and synthesize information on a complex, understudied topic. The search yielded 826 articles, and 39 met the predetermined criteria for inclusion in the review. Trafficking in Ethiopia has occurred internally and externally in the form of adult and child labor and sex trafficking. There were also some reports of organ trafficking and other closely related human rights violations, such as child marriage, child soldiering, and exploitative intercountry adoption. Risk factors for trafficking included push factors (poverty, political instability, economic problems, and gender discrimination) and pull factors (demand for cheap labor). Trafficking was associated with poor health and economic outcomes for victims. Key recommendations for service delivery, research and training, and policy are identified, including establishing comprehensive services for survivor rehabilitation and reintegration, conducting quantitative health outcomes research, and reforming policy around migration and trafficking. Implementing the recommendations identified by this review will allow policy makers, researchers, and practitioners to take meaningful steps toward confronting human trafficking in Ethiopia.

Membrane order in the plasma membrane and endocytic recycling compartment

PubMed Central

Iaea, David B.; Maxfield, Frederick R.

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles. PMID:29125865

A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

PubMed

Gottschalk, Elinor Y; Meneses, Patricio I

2015-09-01

The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of

Disruption of endolysosomal trafficking pathways in glioma cells by methuosis-inducing indole-based chalcones.

PubMed

Mbah, Nneka E; Overmeyer, Jean H; Maltese, William A

2017-06-01

Methuosis is a form of non-apoptotic cell death involving massive vacuolization of macropinosome-derived endocytic compartments, followed by a decline in metabolic activity and loss of membrane integrity. To explore the induction of methuosis as a potential therapeutic strategy for killing cancer cells, we have developed small molecules (indole-based chalcones) that trigger this form of cell death in glioblastoma and other cancer cell lines. Here, we report that in addition to causing fusion and expansion of macropinosome compartments, the lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), disrupts vesicular trafficking at the lysosomal nexus, manifested by impaired degradation of EGF and LDL receptors, defective processing of procathepsins, and accumulation of autophagosomes. In contrast, secretion of the ectodomain derived from a prototypical type-I membrane glycoprotein, β-amyloid precursor protein, is increased rather than diminished. A closely related MOMIPP analog, which causes substantial vacuolization without reducing cell viability, also impedes cathepsin processing and autophagic flux, but has more modest effects on receptor degradation. A third analog, which causes neither vacuolization nor loss of viability, has no effect on endolysosomal trafficking. The results suggest that differential cytotoxicity of structurally similar indole-based chalcones is related, at least in part, to the severity of their effects on endolysosomal trafficking pathways.

PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.

PubMed

Lu, Wei; Ziff, Edward B

2005-08-04

PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

PubMed Central

Pelayo, Juan-Carlos; Poole, Daniel P; Steinhoff, Martin; Cottrell, Graeme S; Bunnett, Nigel W

2011-01-01

Abstract Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK1R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nm, 10 min) induced interaction of NK1R and β-arrestin at the plasma membrane, and the SP–NK1R–β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK1R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H+ATPase inhibitor bafilomycin A1, which prevent endosomal SP degradation, suppressed NK1R recycling by >50%. Preincubation of neurones with SP (10 nm, 5 min) desensitized Ca2+ transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK1R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP–NK1R–β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK1R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling. PMID:21878523

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion

PubMed Central

Hui, Enfu; Johnson, Colin P.; Yao, Jun; Dunning, F. Mark; Chapman, Edwin R.

2009-01-01

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined; previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes, but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion. PMID:19703397

31 CFR 536.311 - Narcotics trafficking.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 536.311... OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

PubMed Central

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

2016-01-01

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

PubMed

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

2016-06-09

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

PubMed

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

IKKβ and NFκB transcription govern lymphoma cell survival through AKT-induced plasma membrane trafficking of GLUT1

PubMed Central

Sommermann, Thomas; O’Neill, Kathleen; Plas, David R.; Cahir-McFarland, Ellen

2011-01-01

All cancer cells require increased nutrient uptake to support proliferation. Here we investigated the signals that govern glucose uptake in B-cell lymphomas and determined that the protein kinase IKKβ induced GLUT1 membrane trafficking in both viral and spontaneous B-cell lymphomas. IKKβ induced AKT activity, while IKKβ-driven NFκB transcription was required for GLUT1 surface localization downstream of AKT. Activated NFκB promoted AKT-mediated phosphorylation of the GLUT1 regulator, AKT Substrate 160kD (AS160), but was not required for AKT phosphorylation of the mammalian target of rapamycin (mTOR) regulator Tuberous Sclerosis 2 (TSC2). In Epstein Barr virus (EBV) transformed B-cells, NFκB inhibition repressed glucose uptake and induced caspase-independent cell death associated with autophagy. After NFκB inhibition, an alternate carbon source ameliorated both autophagy and cell death, whereas autophagy inhibitors specifically accelerated cell death. Taken together, the results suggest that NFκB signaling establishes a metabolic program supporting proliferation and apoptosis resistance by driving glucose import. PMID:21987722

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity*

PubMed Central

Xu, Jian; Kurup, Pradeep; Bartos, Jason A.; Patriarchi, Tommaso; Hell, Johannes W.; Lombroso, Paul J.

2012-01-01

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr1472. Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr402. In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr402. STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr402 and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP. PMID:22544749

Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

PubMed

Bao, Lan

2015-09-30

Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

PubMed

Jia, Hao-Ran; Wang, Hong-Yin; Yu, Zhi-Wu; Chen, Zhan; Wu, Fu-Gen

2016-03-16

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

31 CFR 536.311 - Narcotics trafficking.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity undertaken...

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

PubMed Central

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

2015-01-01

Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

Ascorbic acid increases SVCT2 localization at the plasma membrane by accelerating its trafficking from early secretory compartments and through the endocytic-recycling pathway.

PubMed

Covarrubias-Pinto, A; Acuña, A I; Boncompain, G; Papic, E; Burgos, P V; Perez, F; Castro, M A

2018-05-20

Ascorbic acid (Asc) is an antioxidant molecule essential for physiological functions. The concentration of extracellular Asc increases during synaptic transmission and renal reabsorption. These phenomena induce an increase of the Sodium-dependent-Vitamin-C-transporter 2 (SVCT2) at plasma membrane (PM) localization, as we previously demonstrated in neuronal and non-neuronal cells. Hence, the aim of this study was to evaluate intracellular SVCT2 trafficking kinetics in response to Asc. We observed two peaks of SVCT2 localization and function at the PM (at 5-10 min, "acute response", and 30-60 min, "post-acute response") when cells were incubated with Asc. We defined that the post-acute response was dependent on SVCT2 located in early secretory compartments, and its trafficking was abolished with Tunicamycin and Brefeldin A treatment. Moreover, using the RUSH system to retain and synchronize cargo secretion through the secretory pathway we demonstrated that the post-acute response increases SVCT2 trafficking kinetics from the ER to the PM suggesting the retention of SVCT2 at the early secretory pathway when Asc is absent. However, these observations do not explain the increased SVCT2 levels at the PM during the "acute" response, suggesting the involvement of a faster mechanism in close proximity with the PM. To investigate the possible role of endosomal compartments, we tested the effect of endocytosis inhibition. Expression of dominant-negative (DN) versions of the GTPase-dynamin II and clathrin-accessory protein AP180 showed a significant increase in SVCT2 levels at the PM. Moreover, expression of Rab11-DN, a GTPase implicated in cargo protein recycling from endosomes to the PM showed a similar outcome, strongly indicating that Asc impacts SVCT2 trafficking during the acute response. Therefore, our results revealed two mechanisms by which Asc modulates SVCT2 levels at the PM, one at the early secretory pathway and another at the endocytic compartments. We

Intracellular trafficking of silicon particles and logic-embedded vectors

NASA Astrophysics Data System (ADS)

Ferrati, Silvia; Mack, Aaron; Chiappini, Ciro; Liu, Xuewu; Bean, Andrew J.; Ferrari, Mauro; Serda, Rita E.

2010-08-01

Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 μm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments.Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon

Metabotropic glutamate receptors in the trafficking of ionotropic glutamate and GABA(A) receptors at central synapses.

PubMed

Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

2006-01-01

The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABA(A) receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABA(A) receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca(2+) concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABA(A) receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABA(A) receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves.

UNC93B1 mediates differential trafficking of endosomal TLRs

PubMed Central

Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

2013-01-01

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999

A one-step strategy for ultra-fast and low-cost mass production of plastic membrane microfluidic chips.

PubMed

Hu, Chong; Lin, Sheng; Li, Wanbo; Sun, Han; Chen, Yangfan; Chan, Chiu-Wing; Leung, Chung-Hang; Ma, Dik-Lung; Wu, Hongkai; Ren, Kangning

2016-10-05

An ultra-fast, extremely cost-effective, and environmentally friendly method was developed for fabricating flexible microfluidic chips with plastic membranes. With this method, we could fabricate plastic microfluidic chips rapidly (within 12 seconds per piece) at an extremely low cost (less than $0.02 per piece). We used a heated perfluoropolymer perfluoroalkoxy (often called Teflon PFA) solid stamp to press a pile of two pieces of plastic membranes, low density polyethylene (LDPE) and polyethylene terephthalate (PET) coated with an ethylene-vinyl acetate copolymer (EVA). During the short period of contact with the heated PFA stamp, the pressed area of the membranes permanently bonded, while the LDPE membrane spontaneously rose up at the area not pressed, forming microchannels automatically. These two regions were clearly distinguishable even at the micrometer scale so we were able to fabricate microchannels with widths down to 50 microns. This method combines the two steps in the conventional strategy for microchannel fabrication, generating microchannels and sealing channels, into a single step. The production is a green process without using any solvent or generating any waste. Also, the chips showed good resistance against the absorption of Rhodamine 6G, oligonucleotides, and green fluorescent protein (GFP). We demonstrated some typical microfluidic manipulations with the flexible plastic membrane chips, including droplet formation, on-chip capillary electrophoresis, and peristaltic pumping for quantitative injection of samples and reagents. In addition, we demonstrated convenient on-chip detection of lead ions in water samples by a peristaltic-pumping design, as an example of the application of the plastic membrane chips in a resource-limited environment. Due to the high speed and low cost of the fabrication process, this single-step method will facilitate the mass production of microfluidic chips and commercialization of microfluidic technologies.

Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins

PubMed Central

Kozyulina, Polina Y.; Loskutov, Yuriy V.; Kozyreva, Varvara K.; Rajulapati, Anuradha; Ice, Ryan J.; Jones, Brandon. C.; Pugacheva, Elena N.

2014-01-01

The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of pro-metastatic protein, NEDD9, in breast cancer (BC) cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and co-localizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand/integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Re-expression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9 depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. PMID:25319010

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

PubMed

Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

2014-04-01

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Classifying the molecular functions of Rab GTPases in membrane trafficking using deep convolutional neural networks.

PubMed

Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

2018-06-13

Deep learning has been increasingly used to solve a number of problems with state-of-the-art performance in a wide variety of fields. In biology, deep learning can be applied to reduce feature extraction time and achieve high levels of performance. In our present work, we apply deep learning via two-dimensional convolutional neural networks and position-specific scoring matrices to classify Rab protein molecules, which are main regulators in membrane trafficking for transferring proteins and other macromolecules throughout the cell. The functional loss of specific Rab molecular functions has been implicated in a variety of human diseases, e.g., choroideremia, intellectual disabilities, cancer. Therefore, creating a precise model for classifying Rabs is crucial in helping biologists understand the molecular functions of Rabs and design drug targets according to such specific human disease information. We constructed a robust deep neural network for classifying Rabs that achieved an accuracy of 99%, 99.5%, 96.3%, and 97.6% for each of four specific molecular functions. Our approach demonstrates superior performance to traditional artificial neural networks. Therefore, from our proposed study, we provide both an effective tool for classifying Rab proteins and a basis for further research that can improve the performance of biological modeling using deep neural networks. Copyright © 2018 Elsevier Inc. All rights reserved.

Human trafficking and the healthcare professional.

PubMed

Barrows, Jeffrey; Finger, Reginald

2008-05-01

Despite the legislation passed in the 19th century outlawing human slavery, it is more widespread today than at the conclusion of the civil war. Modern human slavery, termed human trafficking, comes in several forms. The most common type of human trafficking is sex trafficking, the sale of women and children into prostitution. Labor trafficking is the sale of men, women, and children into hard labor for which they receive little or no compensation. Other forms of trafficking include child soldiering, war brides, and organ removal. Healthcare professionals play a critical role in both finding victims of human trafficking while they are still in captivity, as well as caring for their mental and physical needs upon release. Those working in the healthcare profession need to be educated regarding how a trafficking victim may present, as well as their unique healthcare needs.

Blue-light-activated phototropin2 trafficking from the cytoplasm to Golgi/post-Golgi vesicles.

PubMed

Aggarwal, Chhavi; Banaś, Agnieszka Katarzyna; Kasprowicz-Maluśki, Anna; Borghetti, Carolina; Labuz, Justyna; Dobrucki, Jerzy; Gabryś, Halina

2014-07-01

Phototropins are plasma membrane-localized UVA/blue light photoreceptors which mediate phototropism, inhibition of primary hypocotyl elongation, leaf positioning, chloroplast movements, and stomatal opening. Blue light irradiation activates the C-terminal serine/threonine kinase domain of phototropin which autophosphorylates the receptor. Arabidopsis thaliana encodes two phototropins, phot1 and phot2. In response to blue light, phot1 moves from the plasma membrane into the cytosol and phot2 translocates to the Golgi complex. In this study the molecular mechanism and route of blue-light-induced phot2 trafficking are demonstrated. It is shown that Atphot2 behaves in a similar manner when expressed transiently under 35S or its native promoter. The phot2 kinase domain but not blue-light-mediated autophosphorylation is required for the receptor translocation. Using co-localization and western blotting, the receptor was shown to move from the cytoplasm to the Golgi complex, and then to the post-Golgi structures. The results were confirmed by brefeldin A (an inhibitor of the secretory pathway) which disrupted phot2 trafficking. An association was observed between phot2 and the light chain2 of clathrin via bimolecular fluorescence complementation. The fluorescence was observed at the plasma membrane. The results were confirmed using co-immunoprecipitation. However, tyrphostin23 (an inhibitor of clathrin-mediated endocytosis) and wortmannin (a suppressor of receptor endocytosis) were not able to block phot2 trafficking, indicating no involvement of receptor endocytosis in the formation of phot2 punctuate structures. Protein turnover studies indicated that the receptor was continuously degraded in both darkness and blue light. The degradation of phot2 proceeded via a transport route different from translocation to the Golgi complex. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

PubMed Central

VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

2011-01-01

Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

To discuss illicit nuclear trafficking

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

Balatsky, Galya I; Severe, William R; Wallace, Richard K

2010-01-01

The Illicit nuclear trafficking panel was conducted at the 4th Annual INMM workshop on Reducing the Risk from Radioactive and Nuclear Materials on February 2-3, 2010 in Washington DC. While the workshop occurred prior to the Nuclear Security Summit, April 12-13 2010 in Washington DC, some of the summit issues were raised during the workshop. The Communique of the Washington Nuclear Security Summit stated that 'Nuclear terrorism is one of the most challenging threats to international security, and strong nuclear security measures are the most effective means to prevent terrorists, criminals, or other unauthorized actors from acquiring nuclear materials.' Themore » Illicit Trafficking panel is one means to strengthen nuclear security and cooperation at bilateral, regional and multilateral levels. Such a panel promotes nuclear security culture through technology development, human resources development, education and training. It is a tool which stresses the importance of international cooperation and coordination of assistance to improve efforts to prevent and respond to incidents of illicit nuclear trafficking. Illicit trafficking panel included representatives from US government, an international organization (IAEA), private industry and a non-governmental organization to discuss illicit nuclear trafficking issues. The focus of discussions was on best practices and challenges for addressing illicit nuclear trafficking. Terrorism connection. Workshop discussions pointed out the identification of terrorist connections with several trafficking incidents. Several trafficking cases involved real buyers (as opposed to undercover law enforcement agents) and there have been reports identifying individuals associated with terrorist organizations as prospective plutonium buyers. Some specific groups have been identified that consistently search for materials to buy on the black market, but no criminal groups were identified that specialize in nuclear materials or isotope

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

PubMed Central

Fischer, Martina; Jehmlich, Nico; Rose, Laura; Koch, Sophia; Laue, Michael; Renard, Bernhard Y.; Schmidt, Frank; Heuer, Dagmar

2015-01-01

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection. PMID:26042774

Multiscale computational models in physical systems biology of intracellular trafficking.

PubMed

Tourdot, Richard W; Bradley, Ryan P; Ramakrishnan, Natesan; Radhakrishnan, Ravi

2014-10-01

In intracellular trafficking, a definitive understanding of the interplay between protein binding and membrane morphology remains incomplete. The authors describe a computational approach by integrating coarse-grained molecular dynamics (CGMD) simulations with continuum Monte Carlo (CM) simulations of the membrane to study protein-membrane interactions and the ensuing membrane curvature. They relate the curvature field strength discerned from the molecular level to its effect at the cellular length-scale. They perform thermodynamic integration on the CM model to describe the free energy landscape of vesiculation in clathrin-mediated endocytosis. The method presented here delineates membrane morphologies and maps out the free energy changes associated with membrane remodeling due to varying coat sizes, coat curvature strengths, membrane bending rigidities, and tensions; furthermore several constraints on mechanisms underlying clathrin-mediated endocytosis have also been identified, Their CGMD simulations have revealed the importance of PIP2 for stable binding of proteins essential for curvature induction in the bilayer and have provided a molecular basis for the positive curvature induction by the epsin N-terminal homology (EIMTH) domain. Calculation of the free energy landscape for vesicle budding has identified the critical size and curvature strength of a clathrin coat required for nucleation and stabilisation of a mature vesicle.

A new theory for multistep discretizations of stiff ordinary differential equations: Stability with large step sizes

NASA Technical Reports Server (NTRS)

Majda, G.

1985-01-01

A large set of variable coefficient linear systems of ordinary differential equations which possess two different time scales, a slow one and a fast one is considered. A small parameter epsilon characterizes the stiffness of these systems. A system of o.d.e.s. in this set is approximated by a general class of multistep discretizations which includes both one-leg and linear multistep methods. Sufficient conditions are determined under which each solution of a multistep method is uniformly bounded, with a bound which is independent of the stiffness of the system of o.d.e.s., when the step size resolves the slow time scale, but not the fast one. This property is called stability with large step sizes. The theory presented lets one compare properties of one-leg methods and linear multistep methods when they approximate variable coefficient systems of stiff o.d.e.s. In particular, it is shown that one-leg methods have better stability properties with large step sizes than their linear multistep counter parts. The theory also allows one to relate the concept of D-stability to the usual notions of stability and stability domains and to the propagation of errors for multistep methods which use large step sizes.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

PubMed Central

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-01-01

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

PubMed

Tortosa, Elena; Hoogenraad, Casper C

2018-02-01

In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations*

PubMed Central

Martin, Gregory M.; Rex, Emily A.; Devaraneni, Prasanna; Denton, Jerod S.; Boodhansingh, Kara E.; DeLeon, Diva D.; Stanley, Charles A.; Shyng, Show-Ling

2016-01-01

ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the KATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. PMID:27573238

Psychological consequences of child trafficking: An historical cohort study of trafficked children in contact with secondary mental health services.

PubMed

Ottisova, Livia; Smith, Patrick; Shetty, Hitesh; Stahl, Daniel; Downs, Johnny; Oram, Sian

2018-01-01

Child trafficking is the recruitment and movement of people aged younger than 18 for the purposes of exploitation. Research on the mental health of trafficked children is limited, and little is known about the use of mental health services by this group. This study aimed to investigate the mental health and service use characteristics of trafficked children in contact with mental health services in England. The study employed an historical cohort design. Electronic health records of over 250,000 patients were searched to identify trafficked children, and a matched cohort of non-trafficked children was randomly selected. Data were extracted on the socio-demographic and clinical characteristics, abuse history, and trafficking experiences of the trafficked children. Logistic and linear random effects regression models were fitted to compare trafficked and non-trafficked children on their clinical profiles and service use characteristics. Fifty-one trafficked children were identified, 78% were female. The most commonly recorded diagnoses for trafficked children were post-traumatic stress disorder (PTSD) (22%) and affective disorders (22%). Records documented a high prevalence of physical violence (53%) and sexual violence (49%) among trafficked children. Trafficked children had significantly longer duration of contact with mental health services compared to non-trafficked controls (b = 1.66, 95% CI 1.09-2.55, p<0.02). No significant differences were found, however, with regards to pathways into care, prevalence of compulsory psychiatric admission, length of inpatient stays, or changes in global functioning. Child trafficking is associated with high levels of physical and sexual abuse and longer duration of contact with mental health services. Research is needed on most effective interventions to promote recovery for this vulnerable group.

Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1-Dependent Trafficking of Integrins.

PubMed

Kozyulina, Polina Y; Loskutov, Yuriy V; Kozyreva, Varvara K; Rajulapati, Anuradha; Ice, Ryan J; Jones, Brandon C; Pugacheva, Elena N

2015-03-01

The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand-integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. This study provides valuable new insight into the potential therapeutic benefit of NEDD9 depletion to reduce dissemination of tumor cells and discovers a new regulatory role of NEDD9 in promoting migration through modulation of CAV1-dependent trafficking of integrins. ©2014 American Association for Cancer Research.

Sex Trafficking Related Knowledge, Awareness, and Attitudes among Adolescent Female Students in Nepal: A Cross-Sectional Study.

PubMed

Shrestha, Roman; Karki, Pramila; Suwal, Asha; Copenhaver, Michael

2015-01-01

Sex trafficking has been a long-standing concern in Nepal. Very little has been achieved, however, in terms of actual reduction in the number of victims despite numerous anti-sex trafficking programs. This situation may be attributable to a lack of empirical evidence upon which to formulate anti-sexual trafficking interventions. This study aimed to assess sex trafficking-related knowledge, awareness and attitudes, and factors associated with sex trafficking awareness and attitudes towards the victims of sex trafficking and/or anti-sex trafficking campaigns among adolescent female students in Nepal. A cross-sectional study was conducted between August-September 2013 among 292 adolescent female students (>10 years old) using systematic random sampling from three high schools in Sindhupalchowk district, Nepal. As an initial step, descriptive analyses were employed to characterize the data and multivariate logistic regression analyses were used to explore factors associated with sex trafficking awareness and related attitudes. Seventy-six percent of sampled students reported that they were aware of sex trafficking and 94.6% indicated media (i.e., radio or television) as the primary sources of their knowledge. Fifty-one percent mentioned relatives/friends as mediators of sex trafficking, 60.4% reported promise for better jobs as the primary attraction behind sex trafficking, and 48.6% mentioned adolescent females as the most vulnerable group for sex trafficking. Over half (56.8%) of the respondents had positive attitudes towards the victims of sex trafficking and/or anti-sex trafficking campaigns. Age (OR = 3.38, 95% CI:2.51-4.55), parents' occupation (OR = 3.89, 95% CI:1.58-9.58), and having a radio/TV at home (OR = 6.67, 95% CI:3.99-9.54) were significantly associated with awareness, whereas being younger (OR = 0.67, 95% CI:0.55-0.79) and having joint-family (OR = 2.67, 95% CI:1.49-4.80) were significantly associated with having a positive attitudes towards the

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.

PubMed

Rao, Mangala; Peachman, Kristina K; Alving, Carl R; Rothwell, Stephen W

2003-12-01

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.

Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II.

PubMed

Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F; Bahnson, Brian J

2015-05-01

The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of discrete sites in Yip1A necessary for regulation of endoplasmic reticulum structure.

PubMed

Dykstra, Kaitlyn M; Ulengin, Idil; Delrose, Nicholas; Lee, Tina H

2013-01-01

The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.

Psychological consequences of child trafficking: An historical cohort study of trafficked children in contact with secondary mental health services

PubMed Central

Smith, Patrick; Shetty, Hitesh; Stahl, Daniel; Oram, Sian

2018-01-01

Background Child trafficking is the recruitment and movement of people aged younger than 18 for the purposes of exploitation. Research on the mental health of trafficked children is limited, and little is known about the use of mental health services by this group. This study aimed to investigate the mental health and service use characteristics of trafficked children in contact with mental health services in England. Methods & findings The study employed an historical cohort design. Electronic health records of over 250,000 patients were searched to identify trafficked children, and a matched cohort of non-trafficked children was randomly selected. Data were extracted on the socio-demographic and clinical characteristics, abuse history, and trafficking experiences of the trafficked children. Logistic and linear random effects regression models were fitted to compare trafficked and non-trafficked children on their clinical profiles and service use characteristics. Fifty-one trafficked children were identified, 78% were female. The most commonly recorded diagnoses for trafficked children were post-traumatic stress disorder (PTSD) (22%) and affective disorders (22%). Records documented a high prevalence of physical violence (53%) and sexual violence (49%) among trafficked children. Trafficked children had significantly longer duration of contact with mental health services compared to non-trafficked controls (b = 1.66, 95% CI 1.09–2.55, p<0.02). No significant differences were found, however, with regards to pathways into care, prevalence of compulsory psychiatric admission, length of inpatient stays, or changes in global functioning. Conclusions Child trafficking is associated with high levels of physical and sexual abuse and longer duration of contact with mental health services. Research is needed on most effective interventions to promote recovery for this vulnerable group. PMID:29518168

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth.

PubMed

Lyu, Junfang; Yang, Eun Ju; Head, Sarah A; Ai, Nana; Zhang, Baoyuan; Wu, Changjie; Li, Ruo-Jing; Liu, Yifan; Yang, Chen; Dang, Yongjun; Kwon, Ho Jeong; Ge, Wei; Liu, Jun O; Shim, Joong Sup

2017-11-28

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Physical health symptoms reported by trafficked women receiving post-trafficking support in Moldova: prevalence, severity and associated factors

PubMed Central

2012-01-01

Background Many trafficked people suffer high levels of physical, sexual and psychological abuse. Yet, there has been limited research on the physical health problems associated with human trafficking or how the health needs of women in post-trafficking support settings vary according to socio-demographic or trafficking characteristics. Methods We analysed the prevalence and severity of 15 health symptoms reported by 120 trafficked women who had returned to Moldova between December 2007 and December 2008 and were registered with the International Organisation for Migration Assistance and Protection Programme. Women had returned to Moldova an average of 5.9 months prior to interview (range 2-12 months). Results Headaches (61.7%), stomach pain (60.9%), memory problems (44.2%), back pain (42.5%), loss of appetite (35%), and tooth pain (35%) were amongst the most commonly reported symptoms amongst both women trafficked for sexual exploitation and women trafficked for labour exploitation. The prevalence of headache and memory problems was strongly associated with duration of exploitation. Conclusions Trafficked women who register for post-trafficking support services after returning to their country of origin are likely to have long-term physical and dental health needs and should be provided with access to comprehensive medical services. Health problems among women who register for post-trafficking support services after returning to their country of origin are not limited to women trafficked for sexual exploitation but are also experienced by victims of labour exploitation. PMID:22834807

Physical health symptoms reported by trafficked women receiving post-trafficking support in Moldova: prevalence, severity and associated factors.

PubMed

Oram, Siân; Ostrovschi, Nicolae V; Gorceag, Viorel I; Hotineanu, Mihai A; Gorceag, Lilia; Trigub, Carolina; Abas, Melanie

2012-07-26

Many trafficked people suffer high levels of physical, sexual and psychological abuse. Yet, there has been limited research on the physical health problems associated with human trafficking or how the health needs of women in post-trafficking support settings vary according to socio-demographic or trafficking characteristics. We analysed the prevalence and severity of 15 health symptoms reported by 120 trafficked women who had returned to Moldova between December 2007 and December 2008 and were registered with the International Organisation for Migration Assistance and Protection Programme. Women had returned to Moldova an average of 5.9 months prior to interview (range 2-12 months). Headaches (61.7%), stomach pain (60.9%), memory problems (44.2%), back pain (42.5%), loss of appetite (35%), and tooth pain (35%) were amongst the most commonly reported symptoms amongst both women trafficked for sexual exploitation and women trafficked for labour exploitation. The prevalence of headache and memory problems was strongly associated with duration of exploitation. Trafficked women who register for post-trafficking support services after returning to their country of origin are likely to have long-term physical and dental health needs and should be provided with access to comprehensive medical services. Health problems among women who register for post-trafficking support services after returning to their country of origin are not limited to women trafficked for sexual exploitation but are also experienced by victims of labour exploitation.

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

PubMed Central

Estin, Miriam L.; Thompson, Scott B.; Traxinger, Brianna; Fisher, Marlie H.; Friedman, Rachel S.; Jacobelli, Jordan

2017-01-01

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner. PMID:28320969

Efficient Preparation of Super Antifouling PVDF Ultrafiltration Membrane with One Step Fabricated Zwitterionic Surface.

PubMed

Zhao, Xinzhen; He, Chunju

2015-08-19

On the basis of the excellent fouling resistance of zwitterionic materials, the super antifouling polyvinylidene fluoride (PVDF) membrane was efficiently prepared though one-step sulfonation of PVDF and polyaniline blend membrane in situ. The self-doped sulfonated polyaniline (SPANI) was generated as a novel zwitterionic polymer to improve the antifouling property of PVDF ultrafiltration membrane used in sewage treatment. Surface attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, surface zeta potential, and water contact angle demonstrated the successful fabrication of zwitterionic interface by convenient sulfonation modification. The static adsorption fouling test showed the quantified adsorption mass of bovine serum albumin (BSA) pollutant on the PVDF/SPANI membrane surface decreases to 3(±2) μg/cm(2), and the water flux recovery ratio (FRR) values were no less than 95% for the three model pollutants of BSA, sodium alginate (SA), and humic acid (HA), which were corresponding hydrophobic, hydrophilic, and natural pollutants in sewage, respectively. This Research Article demonstrated the antifouling advantages of zwitterionic SPANI and aimed to provide a simple method for the large scale preparation of zwitterionic antifouling ultrafiltration membranes.

Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

PubMed Central

Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

2012-01-01

Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

Human trafficking and health: a cross-sectional survey of NHS professionals’ contact with victims of human trafficking

PubMed Central

Ross, Claire; Dimitrova, Stoyanka; Howard, Louise M; Dewey, Michael; Zimmerman, Cathy; Oram, Siân

2015-01-01

Objectives (1) To estimate the proportion of National Health Service (NHS) professionals who have come into contact with trafficked people and (2) to measure NHS professionals’ knowledge and confidence to respond to human trafficking. Design A cross-sectional survey. Setting Face-to-face mandatory child protection and/or vulnerable adults training sessions at 10 secondary healthcare provider organisations in England, and meetings of the UK College of Emergency Medicine. Participants 782/892 (84.4%) NHS professionals participated, including from emergency medicine, maternity, mental health, paediatrics and other clinical disciplines. Measures Self-completed questionnaire developed by an expert panel. Questionnaire asks about prior training and contact with potential victims of trafficking, perceived and actual human trafficking knowledge, confidence in responding to human trafficking, and interest in future human trafficking training. Results 13% participants reported previous contact with a patient they knew or suspected of having been trafficked; among maternity services professionals this was 20.4%. However, 86.8% (n=679) reported lacking knowledge of what questions to ask to identify potential victims and 78.3% (n=613) reported that they had insufficient training to assist trafficked people. 71% (n=556), 67.5% (n=528) and 53.4% (n=418) lacked confidence in making appropriate referrals for men, women and children, respectively, who had been trafficked. 95.3% (n=746) of respondents were unaware of the scale of human trafficking in the UK, and 76.5% (n=598) were unaware that calling the police could put patients in more danger. Psychometric analysis showed that subscales measuring perceived knowledge, actual knowledge and confidence to respond to human trafficking demonstrated good internal consistency (Cronbach's αs 0.93, 0.63 and 0.64, respectively) and internal correlations. Conclusions NHS professionals working in secondary care are in contact with potential

Understanding human trafficking in the United States.

PubMed

Logan, T K; Walker, Robert; Hunt, Gretchen

2009-01-01

The topic of modern-day slavery or human trafficking has received increased media and national attention. However, to date there has been limited research on the nature and scope of human trafficking in the United States. This article describes and synthesizes nine reports that assess the U.S. service organizations' legal representative knowledge of, and experience with, human trafficking cases, as well as information from actual cases and media reports. This article has five main goals: (a) to define what human trafficking is, and is not; (b) to describe factors identified as contributing to vulnerability to being trafficked and keeping a person entrapped in the situation; (c) to examine how the crime of human trafficking differs from other kinds of crimes in the United States; (d) to explore how human trafficking victims are identified; and, (e) to provide recommendations to better address human trafficking in the United States.

Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator and Drugs: Insights from Cellular Trafficking.

PubMed

Bridges, Robert J; Bradbury, Neil A

2018-01-01

The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein's absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.

Targeting GLP-1 receptor trafficking to improve agonist efficacy.

PubMed

Jones, Ben; Buenaventura, Teresa; Kanda, Nisha; Chabosseau, Pauline; Owen, Bryn M; Scott, Rebecca; Goldin, Robert; Angkathunyakul, Napat; Corrêa, Ivan R; Bosco, Domenico; Johnson, Paul R; Piemonti, Lorenzo; Marchetti, Piero; Shapiro, A M James; Cochran, Blake J; Hanyaloglu, Aylin C; Inoue, Asuka; Tan, Tricia; Rutter, Guy A; Tomas, Alejandra; Bloom, Stephen R

2018-04-23

Glucagon-like peptide-1 receptor (GLP-1R) activation promotes insulin secretion from pancreatic beta cells, causes weight loss, and is an important pharmacological target in type 2 diabetes (T2D). Like other G protein-coupled receptors, the GLP-1R undergoes agonist-mediated endocytosis, but the functional and therapeutic consequences of modulating GLP-1R endocytic trafficking have not been clearly defined. Here, we investigate a series of biased GLP-1R agonists with variable propensities for GLP-1R internalization and recycling. Compared to a panel of FDA-approved GLP-1 mimetics, compounds that retain GLP-1R at the plasma membrane produce greater long-term insulin release, which is dependent on a reduction in β-arrestin recruitment and faster agonist dissociation rates. Such molecules elicit glycemic benefits in mice without concomitant increases in signs of nausea, a common side effect of GLP-1 therapies. Our study identifies a set of agents with specific GLP-1R trafficking profiles and the potential for greater efficacy and tolerability as T2D treatments.

Illicit Trafficking of Natural Radionuclides

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

Friedrich, Steinhaeusler; Lyudmila, Zaitseva

2008-08-07

Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (<20% U 235) or highly enriched uranium (>20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from anmore » operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.« less

Illicit Trafficking of Natural Radionuclides

NASA Astrophysics Data System (ADS)

Friedrich, Steinhäusler; Lyudmila, Zaitseva

2008-08-01

Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (<20% U 235) or highly enriched uranium (>20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

PubMed

Martin, Gregory M; Rex, Emily A; Devaraneni, Prasanna; Denton, Jerod S; Boodhansingh, Kara E; DeLeon, Diva D; Stanley, Charles A; Shyng, Show-Ling

2016-10-14

ATP-sensitive potassium (K ATP ) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of K ATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by K ATP channel openers. Cross-linking experiments showed that K ATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the K ATP channel opener diazoxide. Our study expands the list of K ATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Preliminary Validation of the Sex Trafficking Attitudes Scale.

PubMed

Houston-Kolnik, Jaclyn D; Todd, Nathan R; Wilson, Midge

2016-09-01

This study presents the Sex Trafficking Attitudes Scale (STAS), assessing cognitive, behavioral, and affective attitudes toward the sex trafficking of women and girls. Across two studies, exploratory and confirmatory factor analyses revealed and confirmed six subscales: (a) Knowledge About Sex Trafficking, (b) Awareness of Sex Trafficking, (c) Attitudes Toward Ability to Leave Sex Trafficking, (d) Attitudes Toward Helping Survivors, (e) Empathic Reactions Toward Sex Trafficking, and (f) Efficacy to Reduce Sex Trafficking. Results showed support for convergent validity as the subscales were associated with related measures. The STAS holds promise to expand research and inform efforts to support trafficking survivors. © The Author(s) 2016.

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

PubMed Central

Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

2016-01-01

Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

Sex Trafficking Related Knowledge, Awareness, and Attitudes among Adolescent Female Students in Nepal: A Cross-Sectional Study

PubMed Central

2015-01-01

Background Sex trafficking has been a long-standing concern in Nepal. Very little has been achieved, however, in terms of actual reduction in the number of victims despite numerous anti-sex trafficking programs. This situation may be attributable to a lack of empirical evidence upon which to formulate anti-sexual trafficking interventions. This study aimed to assess sex trafficking-related knowledge, awareness and attitudes, and factors associated with sex trafficking awareness and attitudes towards the victims of sex trafficking and/or anti-sex trafficking campaigns among adolescent female students in Nepal. Methods A cross-sectional study was conducted between August–September 2013 among 292 adolescent female students (>10 years old) using systematic random sampling from three high schools in Sindhupalchowk district, Nepal. As an initial step, descriptive analyses were employed to characterize the data and multivariate logistic regression analyses were used to explore factors associated with sex trafficking awareness and related attitudes. Results Seventy-six percent of sampled students reported that they were aware of sex trafficking and 94.6% indicated media (i.e., radio or television) as the primary sources of their knowledge. Fifty-one percent mentioned relatives/friends as mediators of sex trafficking, 60.4% reported promise for better jobs as the primary attraction behind sex trafficking, and 48.6% mentioned adolescent females as the most vulnerable group for sex trafficking. Over half (56.8%) of the respondents had positive attitudes towards the victims of sex trafficking and/or anti-sex trafficking campaigns. Age (OR = 3.38, 95% CI:2.51–4.55), parents’ occupation (OR = 3.89, 95% CI:1.58–9.58), and having a radio/TV at home (OR = 6.67, 95% CI:3.99–9.54) were significantly associated with awareness, whereas being younger (OR = 0.67, 95% CI:0.55–0.79) and having joint-family (OR = 2.67, 95% CI:1.49–4.80) were significantly associated with

Disruption of clathrin-mediated trafficking causes centrosome overduplication and senescence.

PubMed

Olszewski, Maciej B; Chandris, Panagiotis; Park, Bum-Chan; Eisenberg, Evan; Greene, Lois E

2014-01-01

The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila.

PubMed

Deshpande, Mugdha; Rodal, Avital A

2016-02-01

Neurons require target-derived autocrine and paracrine growth factors to maintain proper identity, innervation, homeostasis and survival. Neuronal growth factor signaling is highly dependent on membrane traffic, both for the packaging and release of the growth factors themselves, and for regulation of intracellular signaling by their transmembrane receptors. Here, we review recent findings from the Drosophila larval neuromuscular junction (NMJ) that illustrate how specific steps of intracellular traffic and inter-organelle interactions impinge on signaling, particularly in the bone morphogenic protein, Wingless and c-Jun-activated kinase pathways, regulating elaboration and stability of NMJ arbors, construction of synapses and synaptic transmission and homeostasis. These membrane trafficking and signaling pathways have been implicated in human motor neuron diseases including amyotrophic lateral sclerosis and hereditary spastic paraplegia, highlighting their importance for neuronal health and survival. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

31 CFR 598.310 - Narcotics trafficking.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 598.310... General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity to cultivate, produce, manufacture, distribute, sell, finance, or transport narcotic drugs...

Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

PubMed Central

Whitman, Shannon D.; Dutch, Rebecca Ellis

2007-01-01

Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F. PMID:17328935

Identification of a novel trafficking pathway exporting a replication protein, Orc2 to nucleus via classical secretory pathway in Plasmodium falciparum.

PubMed

Sharma, Rahul; Sharma, Bhumika; Gupta, Ashish; Dhar, Suman Kumar

2018-05-01

Malaria parasites use an extensive secretory pathway to traffic a number of proteins within itself and beyond. In higher eukaryotes, Endoplasmic Reticulum (ER) membrane bound transcription factors such as SREBP are reported to get processed en route and migrate to nucleus under the influence of specific cues. However, a protein constitutively trafficked to the nucleus via classical secretory pathway has not been reported. Herein, we report the presence of a novel trafficking pathway in an apicomplexan, Plasmodium falciparum where a homologue of an Origin Recognition Complex 2 (Orc2) goes to the nucleus following its association with the ER. Our work highlights the unconventional role of ER in protein trafficking and reports for the first time an ORC homologue getting trafficked through such a pathway to the nucleus where it may be involved in DNA replication and other ancillary functions. Such trafficking pathways may have a profound impact on the cell biology of a malaria parasite and have significant implications in strategizing new antimalarials. Copyright © 2018 Elsevier B.V. All rights reserved.

Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2.

PubMed

Yang, Zhe; Follett, Jordan; Kerr, Markus C; Clairfeuille, Thomas; Chandra, Mintu; Collins, Brett M; Teasdale, Rohan D

2018-05-04

Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of sorting nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid-stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

31 CFR 598.310 - Narcotics trafficking.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 598.310 Section... Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity to cultivate, produce, manufacture, distribute, sell, finance, or transport narcotic drugs, controlled...

Human trafficking and health: a cross-sectional survey of NHS professionals' contact with victims of human trafficking.

PubMed

Ross, Claire; Dimitrova, Stoyanka; Howard, Louise M; Dewey, Michael; Zimmerman, Cathy; Oram, Siân

2015-08-20

(1) To estimate the proportion of National Health Service (NHS) professionals who have come into contact with trafficked people and (2) to measure NHS professionals' knowledge and confidence to respond to human trafficking. A cross-sectional survey. Face-to-face mandatory child protection and/or vulnerable adults training sessions at 10 secondary healthcare provider organisations in England, and meetings of the UK College of Emergency Medicine. 782/892 (84.4%) NHS professionals participated, including from emergency medicine, maternity, mental health, paediatrics and other clinical disciplines. Self-completed questionnaire developed by an expert panel. Questionnaire asks about prior training and contact with potential victims of trafficking, perceived and actual human trafficking knowledge, confidence in responding to human trafficking, and interest in future human trafficking training. 13% participants reported previous contact with a patient they knew or suspected of having been trafficked; among maternity services professionals this was 20.4%. However, 86.8% (n=679) reported lacking knowledge of what questions to ask to identify potential victims and 78.3% (n=613) reported that they had insufficient training to assist trafficked people. 71% (n=556), 67.5% (n=528) and 53.4% (n=418) lacked confidence in making appropriate referrals for men, women and children, respectively, who had been trafficked. 95.3% (n=746) of respondents were unaware of the scale of human trafficking in the UK, and 76.5% (n=598) were unaware that calling the police could put patients in more danger. Psychometric analysis showed that subscales measuring perceived knowledge, actual knowledge and confidence to respond to human trafficking demonstrated good internal consistency (Cronbach's αs 0.93, 0.63 and 0.64, respectively) and internal correlations. NHS professionals working in secondary care are in contact with potential victims of human trafficking, but lack knowledge and confidence in

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

PubMed

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Deregulation and Station Trafficking.

ERIC Educational Resources Information Center

Bates, Benjamin J.

To test whether the revocation of the Federal Communications Commission's "Anti-Trafficking" rule (requiring television station owners to keep a station for three years before transferring its license to another party) impacted station owner behavior, a study compared the behavior of television station "traffickers" (owners…

Human trafficking and the dental professional.

PubMed

O'Callaghan, Michael G

2012-05-01

"Human trafficking" is a term for a modern form of slavery. It is a criminal human rights violation and a significant health issue. Dental professionals can assist in recognizing victims of trafficking. The author conducted a PubMed search of the English-language literature through May 2011, which yielded no articles meeting the search criteria "dentistry" and "human trafficking prostitution." Given these results, the author reviewed articles published in medical journals, reports from both governmental and nongovernmental agencies and lay literature. The author examines the present state of human trafficking and provides information--including specific questions to ask--to help dentists identify victims. In addition, the author suggests means of notifying authorities and assisting trafficking victims. He also examines the health care needs of these patients. Human trafficking is a global problem, with thousands of victims in the United States, including many women and children. Dentists have a responsibility to act for the benefit of others, which includes detecting signs of abuse and neglect. Dental professionals are on the front lines with respect to encountering and identifying potential victims who seek dental treatment. Dentists can combat human trafficking by becoming informed and by maintaining vigilance in their practices.

Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

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

Kaellquist, Linda; Rosen, Hanna; Nordenfelt, Pontus

2010-11-15

Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 withmore » clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.« less

Intracellular Trafficking of Silicon Particles and Logic-Embedded Vectors

PubMed Central

Ferrati, Silvia; Mack, Aaron; Chiappini, Ciro; Liu, Xuewu; Bean, Andrew J.; Ferrari, Mauro; Serda, Rita E.

2010-01-01

Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 μm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments. PMID:20820744

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

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

Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito

2010-09-03

Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored.more » Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH{sub 2}-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with {gamma}-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.« less

31 CFR 598.310 - Narcotics trafficking.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Narcotics trafficking. 598.310 Section 598.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF... Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity to...

31 CFR 598.310 - Narcotics trafficking.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Narcotics trafficking. 598.310 Section 598.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF... Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity to...

31 CFR 598.310 - Narcotics trafficking.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Narcotics trafficking. 598.310 Section 598.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF... Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity to...

Discrete exterior calculus discretization of incompressible Navier-Stokes equations over surface simplicial meshes

NASA Astrophysics Data System (ADS)

Mohamed, Mamdouh S.; Hirani, Anil N.; Samtaney, Ravi

2016-05-01

A conservative discretization of incompressible Navier-Stokes equations is developed based on discrete exterior calculus (DEC). A distinguishing feature of our method is the use of an algebraic discretization of the interior product operator and a combinatorial discretization of the wedge product. The governing equations are first rewritten using the exterior calculus notation, replacing vector calculus differential operators by the exterior derivative, Hodge star and wedge product operators. The discretization is then carried out by substituting with the corresponding discrete operators based on the DEC framework. Numerical experiments for flows over surfaces reveal a second order accuracy for the developed scheme when using structured-triangular meshes, and first order accuracy for otherwise unstructured meshes. By construction, the method is conservative in that both mass and vorticity are conserved up to machine precision. The relative error in kinetic energy for inviscid flow test cases converges in a second order fashion with both the mesh size and the time step.

Gating characteristics control glutamate receptor distribution and trafficking in vivo.

PubMed

Petzoldt, Astrid G; Lee, Yü-Hien; Khorramshahi, Omid; Reynolds, Eric; Plested, Andrew J R; Herzel, Hanspeter; Sigrist, Stephan J

2014-09-08

Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Step Closer to Membrane Protein Multiplexed Nanoarrays Using Biotin-Doped Polypyrrole

PubMed Central

2015-01-01

Whether for fundamental biological research or for diagnostic and drug discovery applications, protein micro- and nanoarrays are attractive technologies because of their low sample consumption, high-throughput, and multiplexing capabilities. However, the arraying platforms developed so far are still not able to handle membrane proteins, and specific methods to selectively immobilize these hydrophobic and fragile molecules are needed to understand their function and structural complexity. Here we integrate two technologies, electropolymerization and amphipols, to demonstrate the electrically addressable functionalization of micro- and nanosurfaces with membrane proteins. Gold surfaces are selectively modified by electrogeneration of a polymeric film in the presence of biotin, where avidin conjugates can then be selectively immobilized. The method is successfully applied to the preparation of protein-multiplexed arrays by sequential electropolymerization and biomolecular functionalization steps. The surface density of the proteins bound to the electrodes can be easily tuned by adjusting the amount of biotin deposited during electropolymerization. Amphipols are specially designed amphipathic polymers that provide a straightforward method to stabilize and add functionalities to membrane proteins. Exploiting the strong affinity of biotin for streptavidin, we anchor distinct membrane proteins onto different electrodes via a biotin-tagged amphipol. Antibody-recognition events demonstrate that the proteins are stably immobilized and that the electrodeposition of polypyrrole films bearing biotin units is compatible with the protein-binding activity. Since polypyrrole films show good conductivity properties, the platform described here is particularly well suited to prepare electronically transduced bionanosensors. PMID:24476392

Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide

NASA Astrophysics Data System (ADS)

Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A.; Kopelman, Raoul

2013-10-01

Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers. Electronic supplementary information (ESI) available: Effect of Potassium depletion on F3 peptide subcellular localization, MTT

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

PubMed Central

Ho, Vincent K.; Angelotti, Timothy

2013-01-01

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins

Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

PubMed Central

Fox, Philip D.; Haberkorn, Christopher J.; Akin, Elizabeth J.; Seel, Peter J.; Krapf, Diego; Tamkun, Michael M.

2015-01-01

ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. PMID:25908859

Human Trafficking: How Nurses Can Make a Difference.

PubMed

Scannell, Meredith; MacDonald, Andrea E; Berger, Amanda; Boyer, Nichole

Human trafficking is a human rights violation and a global health problem. Victims of human trafficking have medical and mental health sequelae requiring specific healthcare interventions. Healthcare professionals may be the initial contact that these victims make outside the world of trafficking. Healthcare professionals are key agents in the identification of human trafficking, which is essential in eliminating this public health problem. Unfortunately, healthcare professionals are not always able to detect signs of human trafficking. Failure to detect results in missed opportunities to assist victims. This is a case report of a victim of human trafficking who presented to an emergency department with medical and mental health issues. Despite numerous encounters with different healthcare professionals, signs and symptoms of human trafficking were not identified. Skilled assessment made by a forensic nurse alerted the healthcare team to clear features of human trafficking associated with this person. Through this case report we illustrate the key role the nurse played in identifying signs of human trafficking. Improvement of human trafficking educational programs is highlighted as a key adjunct to improving detection and facilitating the proper treatment of victims.

Role of Occupational Therapy in Combating Human Trafficking.

PubMed

Gorman, Kathleen W; Hatkevich, Beth Ann

Human trafficking is a modern-day form of slavery that includes sex trafficking, labor trafficking, and trafficking of children. It is estimated that 35.8 million people are enslaved around the world. Because of the traumatic experiences that victims of human trafficking encounter, the needs of victims are extensive and require the services of several providers, including health care providers, for victims to transform into survivors and thrivers. Currently, the role of occupational therapy is minimal and unexplored. The profession of occupational therapy has the capacity of having a profound role in both providing client-centered care services to victims and survivors of human trafficking and partaking in preventive advocacy efforts to combat human trafficking. Further advocacy efforts are required to promote the profession of occupational therapy in combating human trafficking. Copyright © 2016 by the American Occupational Therapy Association, Inc.

Biopolitical management, economic calculation and "trafficked women".

PubMed

Berman, Jacqueline

2010-01-01

Narratives surrounding human trafficking, especially trafficking in women for sex work, employ gendered and racialized tropes that have among their effects, a shrouding of women's economic decision-making and state collusion in benefiting from their labour. This paper explores the operation of these narratives in order to understand the ways in which they mask the economics of trafficking by sensationalizing the sexual and criminal aspects of it, which in turn allows the state to pursue political projects under the guise of a benevolent concern for trafficked women and/or protection of its own citizens. This paper will explore one national example: Article 18 of Italian Law 40 (1998). I argue that its passage has led to an increase in cooperation with criminal prosecution of traffickers largely because it approaches trafficked women as capable of making decisions about how and what they themselves want to do. This paper will also consider a more global approach to trafficking embedded in the concept of "migration management", an International Organization for Migration (IOM) framework that is now shaping EU, US and other national immigration laws and policies that impact trafficking. It will also examine the inherent limitations of both the national and global approach as an occasion to unpack how Article 18 and Migration Management function as forms of biopolitical management that participate in the production of "trafficking victims" into a massified population to be managed, rather than engender a more engaged discussion of what constitutes trafficking and how to redress it.

Insulin-regulated Aminopeptidase Is a Key Regulator of GLUT4 Trafficking by Controlling the Sorting of GLUT4 from Endosomes to Specialized Insulin-regulated Vesicles

PubMed Central

Jordens, Ingrid; Molle, Dorothee; Xiong, Wenyong; Keller, Susanna R.

2010-01-01

Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway. PMID:20410133

Trafficking as a Human Rights Violation: Is South Africa's Curriculum Stuck in a Traffick Jam?

ERIC Educational Resources Information Center

du Preez, Petro; Simmonds, Shan

2013-01-01

Human trafficking is a form of modern day slavery and is often collectively referred to as a human rights violation. However, human trafficking is more complex than this suggests as this article attempts to demonstrate. It begins by describing the landscape of international trends in human trafficking, with particular attention to child…

HIV Trafficking Between Blood and Semen During Early Untreated HIV Infection.

PubMed

Chaillon, Antoine; Smith, Davey M; Vanpouille, Christophe; Lisco, Andrea; Jordan, Parris; Caballero, Gemma; Vargas, Milenka; Gianella, Sara; Mehta, Sanjay R

2017-01-01

Understanding the dynamics of HIV across anatomic compartments is important to design effective eradication strategies. In this study, we evaluated viral trafficking between blood and semen during primary HIV infection in 6 antiretroviral-naive men who have sex with men. Deep sequencing data of HIV env were generated from longitudinal blood plasma, peripheral blood mononuclear cells, and seminal plasma samples. The presence or absence of viral compartmentalization was assessed using tree-based Slatkin-Maddison and distance-based Fst methods. Phylogeographic analyses were performed using a discrete Bayesian asymmetric approach of diffusion with Markov jump count estimation to evaluate the gene flow between blood and semen during primary HIV infection. Levels of DNA from human herpesviruses and selected inflammatory cytokines were also measured on genital secretions collected at baseline to evaluate potential correlates of increased viral migration between anatomic compartments. We detected varying degrees of compartmentalization in all 6 individuals evaluated. None of them maintained viral compartmentalization between blood and seminal plasma throughout the analyzed time points. Phylogeographic analyses revealed that the HIV population circulating in blood plasma populated the seminal compartment during the earliest stages of infection. In our limited data set, we found no association between local inflammation or herpesvirus shedding at baseline and viral trafficking between semen and blood. The early spread of virus from blood plasma to genital tract and the complex viral interplay between these compartments suggest that viral eradication efforts will require monitoring viral subpopulations in anatomic sites and viral trafficking during the course of infection.

Health implications of human trafficking.

PubMed

Richards, Tiffany A

2014-01-01

Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population. © 2014 AWHONN.

Women trafficking: causes, concerns, care!

PubMed

Khowaja, Shaneela Sadaruddin; Tharani, Ambreen Jawed; Agha, Ajmal; Karamaliani, Rozina Sherali

2012-08-01

Pakistan is both a country of origin and destination as far as women trafficking is concerned. Poverty, gender discrimination, lack of education, and ignorance about legal rights are some of the underlying causes. Available data suggest several areas of concern, like, for instance: direct health effects, maladaptive coping leading to the use of illicit drugs, and inaccessibility to healthcare facilities. Therefore, numerous interventions would be required at three levels: the prevention of trafficking, the protection of victims and the prosecution of the traffickers.

CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane

PubMed Central

Li, Jingjing; Miao, Lianjie; Zhao, Chen; Shaikh Qureshi, Wasay Mohiuddin; Shieh, David; Guo, Hua; Lu, Yangyang; Hu, Saiyang; Huang, Alice; Zhang, Lu; Cai, Chen-leng; Wan, Leo Q.; Xin, Hongbo; Vincent, Peter; Singer, Harold A.; Zheng, Yi; Cleaver, Ondine; Fan, Zhen-Chuan

2017-01-01

The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo, and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation. PMID:28465335

Partition-based discrete-time quantum walks

NASA Astrophysics Data System (ADS)

Konno, Norio; Portugal, Renato; Sato, Iwao; Segawa, Etsuo

2018-04-01

We introduce a family of discrete-time quantum walks, called two-partition model, based on two equivalence-class partitions of the computational basis, which establish the notion of local dynamics. This family encompasses most versions of unitary discrete-time quantum walks driven by two local operators studied in literature, such as the coined model, Szegedy's model, and the 2-tessellable staggered model. We also analyze the connection of those models with the two-step coined model, which is driven by the square of the evolution operator of the standard discrete-time coined walk. We prove formally that the two-step coined model, an extension of Szegedy model for multigraphs, and the two-tessellable staggered model are unitarily equivalent. Then, selecting one specific model among those families is a matter of taste not generality.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Development of a membrane adsorber based capture step for the purification of yellow fever virus.

PubMed

Pato, Tânia P; Souza, Marta Cristina O; Silva, Andréa N M R; Pereira, Renata C; Silva, Marlon V; Caride, Elena; Gaspar, Luciane P; Freire, Marcos S; Castilho, Leda R

2014-05-19

Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose). Copyright © 2014 Elsevier Ltd. All rights reserved.

Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

PubMed Central

Wunderle, Lina; Knopf, Julia D.; Kühnle, Nathalie; Morlé, Aymeric; Hehn, Beate; Adrain, Colin; Strisovsky, Kvido; Freeman, Matthew; Lemberg, Marius K.

2016-01-01

Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes. PMID:27264103

The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb.

PubMed

Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye

2016-09-01

The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking. Copyright © 2016 Elsevier Inc. All rights reserved.

Time step rescaling recovers continuous-time dynamical properties for discrete-time Langevin integration of nonequilibrium systems.

PubMed

Sivak, David A; Chodera, John D; Crooks, Gavin E

2014-06-19

When simulating molecular systems using deterministic equations of motion (e.g., Newtonian dynamics), such equations are generally numerically integrated according to a well-developed set of algorithms that share commonly agreed-upon desirable properties. However, for stochastic equations of motion (e.g., Langevin dynamics), there is still broad disagreement over which integration algorithms are most appropriate. While multiple desiderata have been proposed throughout the literature, consensus on which criteria are important is absent, and no published integration scheme satisfies all desiderata simultaneously. Additional nontrivial complications stem from simulating systems driven out of equilibrium using existing stochastic integration schemes in conjunction with recently developed nonequilibrium fluctuation theorems. Here, we examine a family of discrete time integration schemes for Langevin dynamics, assessing how each member satisfies a variety of desiderata that have been enumerated in prior efforts to construct suitable Langevin integrators. We show that the incorporation of a novel time step rescaling in the deterministic updates of position and velocity can correct a number of dynamical defects in these integrators. Finally, we identify a particular splitting (related to the velocity Verlet discretization) that has essentially universally appropriate properties for the simulation of Langevin dynamics for molecular systems in equilibrium, nonequilibrium, and path sampling contexts.

One Step Biomass Gas Reforming-Shift Separation Membrane Reactor

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

Roberts, Michael J.; Souleimanova, Razima

2012-12-28

GTI developed a plan where efforts were concentrated in 4 major areas: membrane material development, membrane module development, membrane process development, and membrane gasifier scale-up. GTI assembled a team of researchers to work in each area. Task 1.1 Ceramic Membrane Synthesis and Testing was conducted by Arizona State University (ASU), Task 1.2 Metallic Membrane Synthesis and Testing was conducted by the U.S. National Energy Technology Laboratory (NETL), Task 1.3 was conducted by SCHOTT, and GTI was to test all membranes that showed potential. The initial focus of the project was concentrated on membrane material development. Metallic and glass-based membranes weremore » identified as hydrogen selective membranes under the conditions of the biomass gasification, temperatures above 700C and pressures up to 30 atmospheres. Membranes were synthesized by arc-rolling for metallic type membranes and incorporating Pd into a glass matrix for glass membranes. Testing for hydrogen permeability properties were completed and the effects of hydrogen sulfide and carbon monoxide were investigated for perspective membranes. The initial candidate membrane of Pd80Cu20 chosen in 2008 was selected for preliminary reactor design and cost estimates. Although the H2A analysis results indicated a $1.96 cost per gge H2 based on a 5A (micron) thick PdCu membrane, there was not long-term operation at the required flux to satisfy the go/no go decision. Since the future PSA case yielded a $2.00/gge H2, DOE decided that there was insufficient savings compared with the already proven PSA technology to further pursue the membrane reactor design. All ceramic membranes synthesized by ASU during the project showed low hydrogen flux as compared with metallic membranes. The best ceramic membrane showed hydrogen permeation flux of 0.03 SCFH/ft2 at the required process conditions while the metallic membrane, Pd80Cu20 showed a flux of 47.2 SCFH/ft2 (3 orders of magnitude difference). Results

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.

Child organ trafficking: global reality and inadequate international response.

PubMed

Bagheri, Alireza

2016-06-01

In organ transplantation, the demand for human organs has grown far faster than the supply of organs. This has opened the door for illegal organ trade and trafficking including from children. Organized crime groups and individual organ brokers exploit the situation and, as a result, black markets are becoming more numerous and organized organ trafficking is expanding worldwide. While underprivileged and vulnerable men and women in developing countries are a major source of trafficked organs, and may themselves be trafficked for the purpose of illegal organ removal and trade, children are at especial risk of exploitation. With the confirmed cases of children being trafficked for their organs, child organ trafficking, which once called a "modern urban legend", is a sad reality in today's world. By presenting a global picture of child organ trafficking, this paper emphasizes that child organ trafficking is no longer a myth but a reality which has to be addressed. It argues that the international efforts against organ trafficking and trafficking in human beings for organ removal have failed to address child organ trafficking adequately. This chapter suggests that more orchestrated international collaboration as well as development of preventive measure and legally binding documents are needed to fight child organ trafficking and to support its victims.

Human Trafficking in the Emergency Department

PubMed Central

Patel, Ronak B.; Ahn, Roy; Burke, Thomas F.

2010-01-01

Human trafficking continues to persist, affecting up to 200 million people worldwide. As clinicians in emergency departments commonly encounter victims of intimate partner violence, some of these encounters will be with trafficking victims. These encounters provide a rare opportunity for healthcare providers to intervene and help. This case report of a human trafficking patient from a teaching hospital illustrates the complexity in identifying these victims. Clinicians can better identify potential trafficking cases by increasing their awareness of this phenomenon, using qualified interpreters, isolating potential victims by providing privacy and using simple clear reassuring statements ensuring security. A multidisciplinary approach can then be mobilized to help these patients. PMID:21293753

Cigarette trafficking in five northeastern US cities.

PubMed

Davis, Kevin C; Grimshaw, Victoria; Merriman, David; Farrelly, Matthew C; Chernick, Howard; Coady, Micaela H; Campbell, Kelsey; Kansagra, Susan M

2014-05-01

Cigarette taxation is effective in reducing tobacco use in the USA. However, these benefits are reduced when taxes are unpaid. Cigarette trafficking (ie, the illegal importation of cigarettes into a high-tax jurisdiction from a lower-tax jurisdiction) is well documented in high-tax places like New York City (NYC), but the extent of trafficking in other northeastern cities is relatively unknown. To estimate the extent of cigarette trafficking in Boston, NYC, Philadelphia, Providence and Washington, DC, and project the benefits of reducing cigarette trafficking for recouping lost taxes and reducing smoking in these cities. Littered cigarette packs were collected from a random sample of Census tracts in five US cities. Data collection yielded 1439 total littered packs. The share of cigarette packs bearing proper local, known non-local, foreign or unknown, or no tax stamp was calculated for each city. These data were used to estimate tax revenue recovery if cigarette trafficking could be eliminated. We also estimated the extent to which eliminating cigarette trafficking would reduce cigarette consumption. Overall, 58.7% of packs did not have a proper local tax stamp, and 30.5-42.1% were attributed to trafficking. We estimate that eliminating cigarette trafficking would result in declines in youth smoking prevalence ranging from negligible in low-tax cities like Philadelphia to up to 9.3% in higher-tax NYC. We estimate that these five cities could recoup $680-729 million annually in cigarette tax revenue if cigarette trafficking was eliminated. Reducing cigarette trafficking would increase the effectiveness of tobacco taxes in reducing smoking and generate additional tax revenue, particularly in higher-taxed cities. Federal action to reduce cigarette trafficking, such as a track-and-trace system, is needed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

PubMed

Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

2017-01-01

In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

Trafficking and Trauma: Insight and Advice for the Healthcare System From Sex-trafficked Women Incarcerated on Rikers Island.

PubMed

Ravi, Anita; Pfeiffer, Megan R; Rosner, Zachary; Shea, Judy A

2017-12-01

Sex-trafficked persons experience significant trauma while exploited, resulting in complex health issues and barriers to health care. Incorporating survivor perspectives is critical in optimizing health care delivery for this population. We interviewed sex-trafficking survivors regarding their experiences with trauma while being trafficked and elicited advice about health care delivery. Qualitative interviews were conducted in New York City's Rikers Island jail from July to September 2015. In total, 21 English-speaking women who had experienced sex trafficking were the subjects of the study. Interview domains included: interpersonal violence, behavioral health, and health care delivery advice. Interviewees described experiencing severe and chronic trauma perpetrated by traffickers and sex buyers. Substance use was the primary method of coping with trauma. With regard to mental health, interviewees noted diagnoses of depression, anxiety and posttraumatic stress disorder, low self-esteem, and challenges in intimate relationships. Health care delivery themes included approaches to discussing trafficking in health care settings, concerns regarding sexual assault examinations, and suggestions for improving direct-services and prevention programming. With this perspective into the complex intersection of trauma and behavioral health that sex-trafficked women can experience, health care providers can better understand the context and recommendations regarding trauma-informed care practices for this population. Our results also offer several avenues for future studies with regard to discussing trafficking in clinical settings and an opportunity for stakeholders to incorporate survivor-based input to improve health care for this population.

The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

PubMed Central

Bucci, Cecilia; Alifano, Pietro; Cogli, Laura

2014-01-01

Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways. PMID:25295627

Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.

PubMed

Yudowski, Guillermo A; Puthenveedu, Manojkumar A; Henry, Anastasia G; von Zastrow, Mark

2009-06-01

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (approximately twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.

Assisting victims of human trafficking: strategies to facilitate identification, exit from trafficking, and the restoration of wellness.

PubMed

Hodge, David R

2014-04-01

Human trafficking is a pressing social justice concern. Social work is uniquely situated to address this problem. However, despite the profession's commitment to social justice, the scholarship to equip social workers to address this issue has been largely absent from professional discourse. To address this gap, this article helps social work practitioners to assist victims of human trafficking. After orienting readers to the scope and process of human trafficking, the topics of victim identification, exit from trafficking, and the restoration of psychological wellness are discussed. By equipping themselves in these three areas, practitioners can advance social justice on behalf of some of the most exploited people in the world.

Adaptive Discrete Hypergraph Matching.

PubMed

Yan, Junchi; Li, Changsheng; Li, Yin; Cao, Guitao

2018-02-01

This paper addresses the problem of hypergraph matching using higher-order affinity information. We propose a solver that iteratively updates the solution in the discrete domain by linear assignment approximation. The proposed method is guaranteed to converge to a stationary discrete solution and avoids the annealing procedure and ad-hoc post binarization step that are required in several previous methods. Specifically, we start with a simple iterative discrete gradient assignment solver. This solver can be trapped in an -circle sequence under moderate conditions, where is the order of the graph matching problem. We then devise an adaptive relaxation mechanism to jump out this degenerating case and show that the resulting new path will converge to a fixed solution in the discrete domain. The proposed method is tested on both synthetic and real-world benchmarks. The experimental results corroborate the efficacy of our method.

An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.

PubMed

Pan, Yuan; Laird, Joseph G; Yamaguchi, David M; Baker, Sheila A

2015-06-01

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane.

An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors

PubMed Central

Pan, Yuan; Laird, Joseph G.; Yamaguchi, David M.; Baker, Sheila A.

2015-01-01

Purpose. Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. Methods. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. Results. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. Conclusions. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane. PMID:26030105

An Arabidopsis Prenylated Rab Acceptor 1 Isoform, AtPRA1.B6, Displays Differential Inhibitory Effects on Anterograde Trafficking of Proteins at the Endoplasmic Reticulum1[W][OA

PubMed Central

Lee, Myoung Hui; Jung, Chanjin; Lee, Junho; Kim, Soo Youn; Lee, Yongjik; Hwang, Inhwan

2011-01-01

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aid the targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana) PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to the endoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In this study, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to various endomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat protein complex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescent protein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFP was inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(ΔC):mRFP was not affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency of sporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteins revealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels were controlled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER. PMID:21828250

Box C/D small nucleolar RNA (snoRNA) U60 regulates intracellular cholesterol trafficking.

PubMed

Brandis, Katrina A; Gale, Sarah; Jinn, Sarah; Langmade, Stephen J; Dudley-Rucker, Nicole; Jiang, Hui; Sidhu, Rohini; Ren, Aileen; Goldberg, Anna; Schaffer, Jean E; Ory, Daniel S

2013-12-13

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.

Investigating catalyst coated membrane equilibration time for polymer electrolyte membrane fuel cell manufacturing

NASA Astrophysics Data System (ADS)

Cote, Philippe

Mercedes-Benz Canada Inc., Fuel Cell Division, manufactures polymer electrolyte membrane fuel cell stacks for use in vehicles. The manufacturing line is being optimized for efficiency and quality control, in order to uphold the high standards of Mercedes-Benz Inc. vehicles. In an operating polymer electrolyte membrane fuel cell, the catalyst coated membrane facilitates the electrochemical reaction that generates electricity. This research examines the equilibration of catalyst coated membrane rolls to controlled temperature and humidity conditions, before they are used in the manufacturing of polymer electrolyte membrane fuel cells. Equilibration involves allowing the water content in the catalyst coated membrane to stabilize at the controlled conditions, in order to reduce mechanical stress in the material for better manufacturability. Initial equilibration measurements were conducted on discrete catalyst coated membrane samples using novel electronic conductivity measurements of the catalyst layer, and compared to ionic conductivity measurements of the membrane. Electronic conductivity measurements are easier to implement in the manufacturing environment than the more complex ionic conductivity measurements. When testing discrete catalyst coated membrane samples in an environmental chamber, the equilibration trends for the measured ionic and electronic conductivity signals were similar enough to permit us to adapt the electronic conductivity measurements for catalyst coated membrane in roll form. Equilibration measurements of catalyst coated membrane rolls were optimized to achieve a robust and repeatable procedure which could be used in the manufacturing environment at Mercedes-Benz Canada Inc., Fuel Cell Division.

Membrane trafficking: decoding vesicle identity with contrasting chemistries.

PubMed

Frost, Adam

2011-10-11

Proteins involved in membrane traffic must distinguish between different classes of vesicles. New work now shows that α-synuclein and ALPS motifs represent two extreme types of amphipathic helix that are tuned to detect both the curvature of transport vesicles as well as their bulk lipid content. Copyright © 2011 Elsevier Ltd. All rights reserved.

Trafficking in persons and development: towards greater policy coherence.

PubMed

Danailova-Trainor, Gergana; Laczko, Frank

2010-01-01

Poverty is often regarded as the "root cause" of trafficking, but the linkages between poverty, a lack of development and trafficking are complex. For example, there is some evidence to suggest that victims of cross-border trafficking are more likely to originate from middle-income rather than lower-income countries. Trafficking and development have tended to be treated as very separate policy areas and the assessment of the development impact of counter-trafficking programmes is still at an early stage. This paper outlines a possible framework for a more evidence-based approach to understanding the linkages between trafficking, trafficking policy and human development. The paper argues that the human development gains from greater mobility could be significantly enhanced if there was greater coherence between policies to combat trafficking and policies to promote development.

Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

NASA Technical Reports Server (NTRS)

Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

2002-01-01

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis.

PubMed

Song, Kai; Xue, Yiqun; Wang, Xiaohua; Wan, Yinglang; Deng, Xin; Lin, Jinxing

2017-06-01

Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFP A206K driven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFP A206K line was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFP A206K can be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana. Copyright © 2017 Elsevier GmbH. All rights reserved.

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

PubMed Central

Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

2014-01-01

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

Galerkin v. discrete-optimal projection in nonlinear model reduction

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

Carlberg, Kevin Thomas; Barone, Matthew Franklin; Antil, Harbir

Discrete-optimal model-reduction techniques such as the Gauss{Newton with Approximated Tensors (GNAT) method have shown promise, as they have generated stable, accurate solutions for large-scale turbulent, compressible ow problems where standard Galerkin techniques have failed. However, there has been limited comparative analysis of the two approaches. This is due in part to difficulties arising from the fact that Galerkin techniques perform projection at the time-continuous level, while discrete-optimal techniques do so at the time-discrete level. This work provides a detailed theoretical and experimental comparison of the two techniques for two common classes of time integrators: linear multistep schemes and Runge{Kutta schemes.more » We present a number of new ndings, including conditions under which the discrete-optimal ROM has a time-continuous representation, conditions under which the two techniques are equivalent, and time-discrete error bounds for the two approaches. Perhaps most surprisingly, we demonstrate both theoretically and experimentally that decreasing the time step does not necessarily decrease the error for the discrete-optimal ROM; instead, the time step should be `matched' to the spectral content of the reduced basis. In numerical experiments carried out on a turbulent compressible- ow problem with over one million unknowns, we show that increasing the time step to an intermediate value decreases both the error and the simulation time of the discrete-optimal reduced-order model by an order of magnitude.« less

Discretization vs. Rounding Error in Euler's Method

ERIC Educational Resources Information Center

Borges, Carlos F.

2011-01-01

Euler's method for solving initial value problems is an excellent vehicle for observing the relationship between discretization error and rounding error in numerical computation. Reductions in stepsize, in order to decrease discretization error, necessarily increase the number of steps and so introduce additional rounding error. The problem is…

Traveling waves in discretized Balitsky Kovchegov evolution

NASA Astrophysics Data System (ADS)

Marquet, C.; Peschanski, R.; Soyez, G.; Bialas, A.

2006-02-01

We study the asymptotic solutions of a version of the Balitsky-Kovchegov evolution with discrete steps in rapidity. We derive a closed iterative equation in momentum space. We show that it possesses traveling-wave solutions and extract their properties. We find no evidence for chaotic behaviour due to discretization.

Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry.

PubMed

DiGiuseppe, Stephen; Keiffer, Timothy R; Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Guion, Lucile G M; Müller, Martin; Sapp, Martin

2015-10-01

The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. In order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Blocking ESCRT-Mediated Envelopment Inhibits Microtubule-Dependent Trafficking of Alphaherpesviruses In Vitro

PubMed Central

Kharkwal, Himanshu; Smith, Caitlin G.

2014-01-01

ABSTRACT Herpes simplex virus (HSV) and, as reported here, pseudorabies virus (PRV) utilize the ESCRT apparatus to drive cytoplasmic envelopment of their capsids. Here, we demonstrate that blocking ESCRT-mediated envelopment using the dominant-negative inhibitor Vps4A-EQ (Vps4A in which glutamate [E] at position 228 in the ATPase active site is replaced by a glutamine [Q]) reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound green fluorescent protein (GFP)-labeled Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro; however, motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids. IMPORTANCE The alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm; however, the molecular mechanisms of trafficking are not well understood. In this study, we have used a cell-free system to examine the requirements for microtubule trafficking and have attempted to distinguish between the movement of so-called “naked” and

SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.

PubMed

Bowton, E; Saunders, C; Reddy, I A; Campbell, N G; Hamilton, P J; Henry, L K; Coon, H; Sakrikar, D; Veenstra-VanderWeele, J M; Blakely, R D; Sutcliffe, J; Matthies, H J G; Erreger, K; Galli, A

2014-10-14

Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission

Experiences of sex trafficking victims in Mumbai, India.

PubMed

Silverman, J G; Decker, M R; Gupta, J; Maheshwari, A; Patel, V; Willis, B M; Raj, A

2007-06-01

To explore mechanisms and contexts related to sex trafficking victimization among South Asian women and girls rescued from brothels in Mumbai, India. Records of residents at a major non-governmental organization providing rescue, shelter and care of minor girls and of women held against their will in brothels in Mumbai were systematically reviewed (n=160). Descriptive statistics were calculated, and demographic differences in trafficking mechanisms and pre-disposing contexts were explored. The majority of victims (51.9%) were trafficked as minors and by individuals previously known to them (59.7%). Traffickers most commonly lured victims via promises of economic opportunity (55.0%) or kidnapped individuals via use of drugs or force (26.3%). Victims were most often trafficked from public settings (e.g., markets, train stations; 50.9%) and via public transportation (94.9%). Almost half (49.4%) reported some type of family disruption as directly leading to their being trafficked; violence involving husbands or other family members (38.0%) and marital separation or abandonment (32.9%) were the most common forms of disruption reported. Differences in experiences of trafficking were identified based on age, nationality, education, and marital status; no differences were found based on religion. The interaction of poverty and gender-based mistreatment of women and girls in families heightens the risk of sex trafficking; further empirical research is needed on this critically understudied issue. Prevention efforts should work to improve economic opportunities and security for impoverished women and girls, educate communities regarding the tactics and identities of traffickers, as well as promote structural interventions to reduce trafficking.

Reduced discretization error in HZETRN

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

Slaba, Tony C., E-mail: Tony.C.Slaba@nasa.gov; Blattnig, Steve R., E-mail: Steve.R.Blattnig@nasa.gov; Tweed, John, E-mail: jtweed@odu.edu

2013-02-01

The deterministic particle transport code HZETRN is an efficient analysis tool for studying the effects of space radiation on humans, electronics, and shielding materials. In a previous work, numerical methods in the code were reviewed, and new methods were developed that further improved efficiency and reduced overall discretization error. It was also shown that the remaining discretization error could be attributed to low energy light ions (A < 4) with residual ranges smaller than the physical step-size taken by the code. Accurately resolving the spectrum of low energy light particles is important in assessing risk associated with astronaut radiation exposure.more » In this work, modifications to the light particle transport formalism are presented that accurately resolve the spectrum of low energy light ion target fragments. The modified formalism is shown to significantly reduce overall discretization error and allows a physical approximation to be removed. For typical step-sizes and energy grids used in HZETRN, discretization errors for the revised light particle transport algorithms are shown to be less than 4% for aluminum and water shielding thicknesses as large as 100 g/cm{sup 2} exposed to both solar particle event and galactic cosmic ray environments.« less

Human trafficking law and social structures.

PubMed

Wooditch, Alese

2012-08-01

Human trafficking has only recently emerged at the forefront of policy reform, even in developed nations. Yet, heightened awareness of the issue has not translated into effective policy as the majority of nations have ineffective antitrafficking practices; many countries have failed to criminalize human trafficking, whereas others do not actively enforce statutes in place. By applying Black's theory of law, this study offers a preliminary understanding into the variation of global prosecutorial efforts in human trafficking and adequacy of antitrafficking law. To isolate this relationship, the effects of trafficking markets are controlled. As with prior research, the study finds limited support for the theory. The article concludes with a discussion on the implications of the quantity of antitrafficking law and morphology association for policy development.

Committee opinion no. 507: human trafficking.

PubMed

2011-09-01

Human trafficking is a widespread problem with estimates ranging from 14,000 to 50,000 individuals trafficked into the United States annually. This hidden population involves the commercial sex industry, agriculture, factories, hotel and restaurant businesses, domestic workers, marriage brokers, and some adoption firms. Because 80% of trafficked individuals are women and girls, women’s health care providers may better serve their diverse patient population by increasing their awareness of this problem. The exploitation of people of any race, gender, sexual orientation, or ethnicity is unacceptable at any time, in any place. The members of the American College of Obstetricians and Gynecologists should be aware of this problem and strive to recognize and assist their patients who are victims or who have been victims of human trafficking.

Sex Trafficking of Women and Girls

PubMed Central

Deshpande, Neha A; Nour, Nawal M

2013-01-01

Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community. The costs to society include the degradation of human and women’s rights, poor public health, disrupted communities, and diminished social development. Victims of sex trafficking acquire adverse physical and psychological health conditions and social disadvantages. Thus, sex trafficking is a critical health issue with broader social implications that requires both medical and legal attention. Healthcare professionals can work to improve the screening, identification, and assistance of victims of sex trafficking in a clinical setting and help these women and girls access legal and social services. PMID:23687554

REGULATED VESICULAR TRAFFICKING OF SPECIFIC PCDH15 AND VLGR1 VARIANTS IN AUDITORY HAIR CELLS

PubMed Central

Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T.; Cosgrove, Dominic

2012-01-01

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal co-localization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular sub-pools. The apically trafficked pool co-localized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associates with membrane microdomains and SNAP25. Moreover, co-immunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions. PMID:23035094

MAS1 Receptor Trafficking Involves ERK1/2 Activation Through a β-Arrestin2-Dependent Pathway.

PubMed

Cerniello, Flavia M; Carretero, Oscar A; Longo Carbajosa, Nadia A; Cerrato, Bruno D; Santos, Robson A; Grecco, Hernán E; Gironacci, Mariela M

2017-11-01

The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R trafficking plays a critical function in signal termination and propagation and in R resensitization. We examined MAS1R internalization and trafficking on agonist stimulation and the role of β-arrestin2 in the activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and Akt after MAS1R stimulation. Human embryonic kidney 293T cells were transfected with the coding sequence for MAS1R-YFP (MAS1R fused to yellow fluorescent protein). MAS1R internalization was evaluated by measuring the MAS1R present in the plasma membrane after agonist stimulation using a ligand-binding assay. MAS1R trafficking was evaluated by its colocalization with trafficking markers. MAS1R internalization was blocked in the presence of shRNAcaveolin-1 and with dominant negatives for Eps15 (a protein involved in endocytosed Rs by clathrin-coated pits) and for dynamin. After stimulation, MAS1R colocalized with Rab11-a slow recycling vesicle marker-and not with Rab4-a fast recycling vesicle marker-or LysoTracker-a lysosome marker. Cells transfected with MAS1R showed an increase in Akt and ERK1/2 activation on angiotensin-(1-7) stimulation, which was blocked when the clathrin-coated pits pathway was blocked. Suppression of β-arrestin2 by shRNA reduced the angiotensin-(1-7)-induced ERK1/2 activation, whereas Akt activation was not modified. We conclude that on agonist stimulation, MAS1R is internalized through clathrin-coated pits and caveolae in a dynamin-dependent manner and is then slowly recycled back to the plasma membrane. MAS1R induced Akt and ERK1/2 activation from early endosomes, and the activation of ERK1/2 was mediated by β-arrestin2. Thus, MAS1R activity and density may be tightly controlled by the cell. © 2017 American Heart Association, Inc.

Female sex trafficking: conceptual issues, current debates, and future directions.

PubMed

Meshkovska, Biljana; Siegel, Melissa; Stutterheim, Sarah E; Bos, Arjan E R

2015-01-01

Female sex trafficking is a pressing concern. In this article, we provide a comprehensive overview of relevant issues regarding the concept of female sex trafficking and research in the field of human trafficking, drawing on a variety of disciplines, including economics, gender and sexuality studies, psychology, sociology, law, and social work. We discuss the debates surrounding the definition of human trafficking, compare and contrast it with human smuggling, and outline connections between female sex trafficking and the issue of sex work and prostitution. We further discuss the history and current estimations of female sex trafficking. We then outline the main actors in female sex trafficking, including trafficked persons, traffickers, clients, and service providers, and we overview the trafficking process from recruitment to identification, recovery, and (re)integration. Finally, we conclude with recommendations for future research that tie together the concepts of vulnerability, exploitation, and long-term recovery and (re)integration.

Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly

PubMed Central

Counihan, Natalie A.; Rawlinson, Stephen M.; Lindenbach, Brett D.

2011-01-01

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly. PMID:22028650

CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane.

PubMed

Li, Jingjing; Miao, Lianjie; Zhao, Chen; Shaikh Qureshi, Wasay Mohiuddin; Shieh, David; Guo, Hua; Lu, Yangyang; Hu, Saiyang; Huang, Alice; Zhang, Lu; Cai, Chen-Leng; Wan, Leo Q; Xin, Hongbo; Vincent, Peter; Singer, Harold A; Zheng, Yi; Cleaver, Ondine; Fan, Zhen-Chuan; Wu, Mingfu

2017-05-01

The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo , and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation. © 2017. Published by The Company of Biologists Ltd.

Rapid constitutive and ligand-activated endocytic trafficking of P2X receptor.

PubMed

Vacca, Fabrizio; Giustizieri, Michela; Ciotti, Maria Teresa; Mercuri, Nicola Biagio; Volonté, Cinzia

2009-05-01

P2X receptors mediate a variety of physiological actions, including smooth muscle contraction, neuro-endocrine secretion and synaptic transmission. Among P2X receptors, the P2X(3) subtype is expressed in sensory neurons of dorsal root- and trigeminal-ganglia, where it performs a well-recognized role in sensory and pain transmission. Recent evidence indicates that the strength of P2X(3)-mediated responses is modulated in vivo by altering the number of receptors at the plasma membrane. In the present study, we investigate the trafficking properties of P2X(3) receptor in transfected HEK293 cells and in primary cultures of dorsal root ganglion neurons, finding that P2X(3) receptor undergoes rapid constitutive and cholesterol-dependent endocytosis. We also show that endocytosis is accompanied by preferential targeting of the receptor to late endosomes/lysosomes, with subsequent degradation. Furthermore, we observe that at steady state the receptor localizes predominantly in lamp1-positive intracellular structures, with a minor fraction present at the plasma membrane. Finally, the level of functional receptor expressed on the cell surface is rapidly up-regulated in response to agonist stimulation, which also augments receptor endocytosis. The findings presented in this work underscore a very dynamic trafficking behavior of P2X(3) receptor and disclose a possible mechanism for the rapid modulation of ATP-mediated responses potentially relevant during physiological and pathological conditions.

Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

PubMed Central

Mrozowska, Paulina S.

2016-01-01

MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures. PMID:27138252

LRP1 influences trafficking of N-type calcium channels via interaction with the auxiliary α2δ-1 subunit

PubMed Central

Kadurin, Ivan; Rothwell, Simon W.; Lana, Beatrice; Nieto-Rostro, Manuela; Dolphin, Annette C.

2017-01-01

Voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit, which determines the main functional and pharmacological attributes of the channel. The CaV1 and CaV2 channels are associated with auxiliary β- and α2δ-subunits. The molecular mechanisms involved in α2δ subunit trafficking, and the effect of α2δ subunits on trafficking calcium channel complexes remain poorly understood. Here we show that α2δ-1 is a ligand for the Low Density Lipoprotein (LDL) Receptor-related Protein-1 (LRP1), a multifunctional receptor which mediates trafficking of cargoes. This interaction with LRP1 is direct, and is modulated by the LRP chaperone, Receptor-Associated Protein (RAP). LRP1 regulates α2δ binding to gabapentin, and influences calcium channel trafficking and function. Whereas LRP1 alone reduces α2δ-1 trafficking to the cell-surface, the LRP1/RAP combination enhances mature glycosylation, proteolytic processing and cell-surface expression of α2δ-1, and also increase plasma-membrane expression and function of CaV2.2 when co-expressed with α2δ-1. Furthermore RAP alone produced a small increase in cell-surface expression of CaV2.2, α2δ-1 and the associated calcium currents. It is likely to be interacting with an endogenous member of the LDL receptor family to have these effects. Our findings now provide a key insight and new tools to investigate the trafficking of calcium channel α2δ subunits. PMID:28256585

AKAP79/150 impacts intrinsic excitability of hippocampal neurons through phospho-regulation of A-type K+ channel trafficking.

PubMed

Lin, Lin; Sun, Wei; Kung, Faith; Dell'Acqua, Mark L; Hoffman, Dax A

2011-01-26

Kv4.2, as the primary α-subunit of rapidly inactivating, A-type voltage-gated K(+) (Kv) channels expressed in hippocampal CA1 pyramidal dendrites, plays a critical role in regulating their excitability. Activity-dependent trafficking of Kv4.2 relies on C-terminal protein kinase A (PKA) phosphorylation. A-kinase-anchoring proteins (AKAPs) target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. As part of a previous study, we showed that AKAP79/150 interacts with Kv4.2 complexes and that the two proteins colocalize in hippocampal neurons. However, the nature and functional consequence of their interaction has not been previously explored. Here, we report that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK (membrane-associated guanylate kinase)-binding domain. We show that AKAP79/150-anchored PKA activity controls Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring led to a decrease in neuronal excitability, while preventing dephosphorylation by the phosphatase calcineurin resulted in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting CA1 excitability.

Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling.

PubMed

Henmi, Yuji; Oe, Natsuko; Kono, Nozomu; Taguchi, Tomohiko; Takei, Kohji; Tanabe, Kenji

2016-03-01

EHD3 is localized on the tubular structures of early endosomes, and it regulates their trafficking pathway. However, the regulatory mechanism of EHD3-containing tubular structures remains poorly understood. An in vitro liposome co-sedimentation assay revealed that EHD3 interacted with phosphatidic acid through its helical domain and this interaction induced liposomal tubulations. Additionally, inhibiting phosphatidic acid synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor significantly reduced the number of EHD3-containing tubules and impaired their trafficking from early endosomes. These results suggest that EHD3 and phosphatidic acid cooperatively regulate membrane deformation and trafficking from early endosomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Membrane contact sites, ancient and central hubs of cellular lipid logistics.

PubMed

Jain, Amrita; Holthuis, Joost C M

2017-09-01

Membrane contact sites (MCSs) are regions where two organelles are closely apposed to facilitate molecular communication and promote a functional integration of compartmentalized cellular processes. There is growing evidence that MCSs play key roles in controlling intracellular lipid flows and distributions. Strikingly, even organelles connected by vesicular trafficking exchange lipids en bulk via lipid transfer proteins that operate at MCSs. Herein, we describe how MCSs developed into central hubs of lipid logistics during the evolution of eukaryotic cells. We then focus on how modern eukaryotes exploit MCSs to help solve a major logistical problem, namely to preserve the unique lipid mixtures of their early and late secretory organelles in the face of extensive vesicular trafficking. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann. Copyright © 2017. Published by Elsevier B.V.

Human trafficking and exploitation: A global health concern.

PubMed

Zimmerman, Cathy; Kiss, Ligia

2017-11-01

In this collection review, Cathy Zimmerman and colleague introduce the PLOS Medicine Collection on Human Trafficking, Exploitation and Health, laying out the magnitude of the global trafficking problem and offering a public health policy framework to guide responses to trafficking.

Connecting numbers to discrete quantification: a step in the child's construction of integer concepts.

PubMed

Slusser, Emily; Ditta, Annie; Sarnecka, Barbara

2013-10-01

The present study asks when young children understand that number words quantify over sets of discrete individuals. For this study, 2- to 4-year-old children were asked to extend the number word five or six either to a cup containing discrete objects (e.g., blocks) or to a cup containing a continuous substance (e.g., water). In Experiment 1, only children who knew the exact meanings of the words one, two and three extended higher number words (five or six) to sets of discrete objects. In Experiment 2, children who only knew the exact meaning of one extended higher number words to discrete objects under the right conditions (i.e., when the problem was first presented with the number words one and two). These results show that children have some understanding that number words pertain to discrete quantification from very early on, but that this knowledge becomes more robust as children learn the exact, cardinal meanings of individual number words. Copyright © 2013 Elsevier B.V. All rights reserved.

Activity-dependent trafficking of lysosomes in dendrites and dendritic spines.

PubMed

Goo, Marisa S; Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J; Ellisman, Mark H; Bloodgood, Brenda L; Patrick, Gentry N

2017-08-07

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. © 2017 Goo et al.

Activity-dependent trafficking of lysosomes in dendrites and dendritic spines

PubMed Central

Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J.; Ellisman, Mark H.

2017-01-01

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. PMID:28630145

Stability analysis of implicit time discretizations for the Compton-scattering Fokker-Planck equation

NASA Astrophysics Data System (ADS)

Densmore, Jeffery D.; Warsa, James S.; Lowrie, Robert B.; Morel, Jim E.

2009-09-01

The Fokker-Planck equation is a widely used approximation for modeling the Compton scattering of photons in high energy density applications. In this paper, we perform a stability analysis of three implicit time discretizations for the Compton-Scattering Fokker-Planck equation. Specifically, we examine (i) a Semi-Implicit (SI) scheme that employs backward-Euler differencing but evaluates temperature-dependent coefficients at their beginning-of-time-step values, (ii) a Fully Implicit (FI) discretization that instead evaluates temperature-dependent coefficients at their end-of-time-step values, and (iii) a Linearized Implicit (LI) scheme, which is developed by linearizing the temperature dependence of the FI discretization within each time step. Our stability analysis shows that the FI and LI schemes are unconditionally stable and cannot generate oscillatory solutions regardless of time-step size, whereas the SI discretization can suffer from instabilities and nonphysical oscillations for sufficiently large time steps. With the results of this analysis, we present time-step limits for the SI scheme that prevent undesirable behavior. We test the validity of our stability analysis and time-step limits with a set of numerical examples.

Step length after discrete perturbation predicts accidental falls and fall-related injury in elderly people with a range of peripheral neuropathy

PubMed Central

Allet, L; Kim, H; Ashton-Miller, JA; De Mott, T; Richardson, JK

2013-01-01

Aims Distal symmetric polyneuropathy increases fall risk due to inability to cope with perturbations. We aimed to 1) identify the frontal plane lower limb sensorimotor functions which are necessary for robustness to a discrete, underfoot perturbation during gait; and 2) determine whether changes in the post-perturbed step parameters could distinguish between fallers and non fallers. Methods Forty-two subjects (16 healthy old and 26 with diabetic PN) participated. Frontal plane lower limb sensorimotor functions were determined using established laboratory-based techniques. The subjects' most extreme alterations in step width or step length in response to a perturbation were measured. In addition, falls and fall-related injuries were prospectively recorded. Results Ankle proprioceptive threshold (APrT; p=.025) and hip abduction rate of torque generation (RTG; p=.041) independently predicted extreme step length after medial perturbation, with precise APrT and greater hip RTG allowing maintenance of step length. Fallers demonstrated greater extreme step length changes after medial perturbation than non fallers (percent change = 16.41±8.42 vs 11.0±4.95; p=.06) Conclusions The ability to rapidly generate frontal plane hip strength and/or precisely perceive motion at the ankle is needed to maintain a normal step length after perturbation, a parameter, which distinguishes between fallers and non fallers. PMID:24183899

Step length after discrete perturbation predicts accidental falls and fall-related injury in elderly people with a range of peripheral neuropathy.

PubMed

Allet, Lara; Kim, Hogene; Ashton-Miller, James; De Mott, Trina; Richardson, James K

2014-01-01

Distal symmetric polyneuropathy increases fall risk due to inability to cope with perturbations. We aimed to 1) identify the frontal plane lower limb sensorimotor functions which are necessary for robustness to a discrete, underfoot perturbation during gait; and 2) determine whether changes in the post-perturbed step parameters could distinguish between fallers and non fallers. Forty-two subjects (16 healthy old and 26 with diabetic PN) participated. Frontal plane lower limb sensorimotor functions were determined using established laboratory-based techniques. The subjects' most extreme alterations in step width or step length in response to a perturbation were measured. In addition, falls and fall-related injuries were prospectively recorded. Ankle proprioceptive threshold (APrT; p=.025) and hip abduction rate of torque generation (RTG; p=.041) independently predicted extreme step length after medial perturbation, with precise APrT and greater hip RTG allowing maintenance of step length. Injured subjects demonstrated greater extreme step length changes after medial perturbation than non-injured subjects (percent change = 18.5 ± 9.2 vs. 11.3 ± 4.57; p = .01). The ability to rapidly generate frontal plane hip strength and/or precisely perceive motion at the ankle is needed to maintain a normal step length after perturbation, a parameter which distinguishes between subjects sustaining a fall-related injury and those who did not. © 2014.

BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus*

PubMed Central

Kang, Eugene L.; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

2012-01-01

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (495DDISLL500) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules. PMID:23109336

Uncoordinated (UNC)119: coordinating the trafficking of myristoylated proteins.

PubMed

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in Caenorhabditis elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking ofmyristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transport myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

Uncoordinated (UNC)119: Coordinating the Trafficking of Myristoylated Proteins

PubMed Central

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D.; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in C. elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking of myristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transpot myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. PMID:23000199

RNA trafficking in parasitic plant systems

PubMed Central

LeBlanc, Megan; Kim, Gunjune; Westwood, James H.

2012-01-01

RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host–parasite connections and the potential significance of host RNAs for the parasite. Additional research on host–parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking. PMID:22936942

The Intracellular Trafficking Pathway of Transferrin

PubMed Central

Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

2011-01-01

Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

Membranes, mechanics, and intracellular transport

NASA Astrophysics Data System (ADS)

Parthasarathy, Raghuveer

2012-10-01

Cellular membranes are remarkable materials -- self-assembled, flexible, two-dimensional fluids. Understanding how proteins manipulate membrane curvature is crucial to understanding the transport of cargo in cells, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical-trap based assay involving dynamic deformation of biomimetic membranes, we have examined the behavior of Sar1, a key component of the COPII family of transport proteins. We find that Sar1 from yeast (S. cerevisiae) lowers membrane rigidity by up to 100% as a function of its concentration, thereby lowering the energetic cost of membrane deformation. Human Sar1 proteins can also lower the mechanical rigidity of the membranes to which they bind. However, unlike the yeast proteins, the rigidity is not a monotonically decreasing function of concentration but rather shows increased rigidity and decreased mobility at high concentrations that implies interactions between proteins. In addition to describing this study of membrane mechanics, I'll also discuss some topics relevant to a range of biophysical investigations, such as the insights provided by imaging methods and open questions in the dynamics of multicellular systems.

Human Trafficking, Mental Illness, and Addiction: Avoiding Diagnostic Overshadowing.

PubMed

Stoklosa, Hanni; MacGibbon, Marti; Stoklosa, Joseph

2017-01-01

This article reviews an emergency department-based clinical vignette of a trafficked patient with co-occurring pregnancy-related, mental health, and substance use disorder issues. The authors, including a survivor of human trafficking, draw on their backgrounds in addiction care, human trafficking, emergency medicine, and psychiatry to review the literature on relevant general health and mental health consequences of trafficking and propose an approach to the clinical complexities this case presents. In their discussion, the authors explicate the deleterious role of implicit bias and diagnostic overshadowing in trafficked patients with co-occurring addiction and mental illness. Finally, the authors propose a trauma-informed, multidisciplinary response to potentially trafficked patients. © 2017 American Medical Association. All Rights Reserved.

FM Dye Photo-Oxidation as a Tool for Monitoring Membrane Recycling in Inner Hair Cells

PubMed Central

Rizzoli, Silvio O.

2014-01-01

Styryl (FM) dyes have been used for more than two decades to investigate exo- and endocytosis in conventional synapses. However, they are difficult to use in the inner hair cells of the auditory pathway (IHCs), as FM dyes appear to penetrate through mechanotransducer channels into the cytosol of IHCs, masking endocytotic uptake. To solve this problem we applied to IHCs the FM dye photo-oxidation technique, which renders the dyes into electron microscopy markers. Photo-oxidation allowed the unambiguous identification of labeled organelles, despite the presence of FM dye in the cytosol. This enabled us to describe the morphologies of several organelles that take up membrane in IHCs, both at rest and during stimulation. At rest, endosome-like organelles were detected in the region of the cuticular plate. Larger tubulo-cisternal organelles dominated the top and nuclear regions. Finally, the basal region, where the IHC active zones are located, contained few labeled organelles. Stimulation increased significantly membrane trafficking in the basal region, inducing the appearance of labeled vesicles and cistern-like organelles. The latter were replaced by small, synaptic-like vesicles during recovery after stimulation. In contrast, no changes in membrane trafficking were induced by stimulation in the cuticular plate region or in the top and nuclear regions. We conclude that synaptic vesicle recycling takes place mostly in the basal region of the IHCs. Other organelles participate in abundant constitutive membrane trafficking throughout the rest of the IHC volume. PMID:24505482

Update: What Nurses Need to Know about Human Trafficking.

PubMed

Washburn, Joy

Nurses are key people who interact with victims of human trafficking in healthcare and other settings. This article provides a current overview of human trafficking, explains legal definitions, elements for protocols in healthcare settings when trafficking is suspected, nursing roles and responses, interview tools, resources, public health recommendations, and nursing education approaches to address human trafficking.