scholarly journals Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP.

1994 ◽  
Vol 126 (1) ◽  
pp. 53-64 ◽  
Author(s):  
K Sandvig ◽  
M Ryd ◽  
O Garred ◽  
E Schweda ◽  
P K Holm ◽  
...  
Keyword(s):  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Golgi Apparatus ◽  
Butyric Acid ◽  
Golgi Complex ◽  
Shiga Toxin ◽  
Acid Treatment ◽  
Retrograde Transport ◽  
A431 Cells ◽  
Golgi Network

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid-treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.

scholarly journals Importance of glycolipid synthesis for butyric acid-induced sensitization to shiga toxin and intracellular sorting of toxin in A431 cells.

1996 ◽  
Vol 7 (9) ◽  
pp. 1391-1404 ◽  
Author(s):  
K Sandvig ◽  
O Garred ◽  
A van Helvoort ◽  
G van Meer ◽  
B van Deurs
Keyword(s):  
Golgi Apparatus ◽  
Butyric Acid ◽  
Shiga Toxin ◽  
Acyl Chain ◽  
Fumonisin B1 ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
A431 Cells ◽  
Glycolipid Synthesis ◽  
Intracellular Sorting

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (> 1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have not investigated the importance of this change by testing whether glycolipid synthesis is required for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inhibition of N-acyltransferase with fumonisin B1, by inhibition of glucosylceramide synthetase by PDMP or PPMP, or by inhibition of serine palmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid-induced change in sensitivity and the increase in the fraction of cell-associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoroalanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length of glycosphingolipids is important for intracellular sorting and translocation of Shiga toxin to the cytosol.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol

1998 ◽  
Vol 111 (7) ◽  
pp. 951-965 ◽  
Author(s):  
D. Drecktrah ◽  
P. de Figueiredo ◽  
R.M. Mason ◽  
W.J. Brown
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Nordihydroguaiaretic Acid ◽  
Retrograde Transport ◽  
Lipoxygenase Inhibitor ◽  
Golgi Stack ◽  
Golgi Network ◽  
In Vivo Effects

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

scholarly journals Syntaxin 5-Dependent Retrograde Transport to thetrans-Golgi Network Is Required for Adeno-Associated Virus Transduction

2014 ◽  
Vol 89 (3) ◽  
pp. 1673-1687 ◽  
Author(s):  
Mathieu E. Nonnenmacher ◽  
Jean-Christophe Cintrat ◽  
Daniel Gillet ◽  
Thomas Weber
Keyword(s):  
Gene Delivery ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Adeno Associated Virus ◽  
Viral Attachment ◽  
Golgi Network ◽  
Endosomal System

ABSTRACTIntracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward thetrans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.IMPORTANCEGene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to thetrans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

scholarly journals Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans ‐Golgi network but not the Golgi apparatus in Exo2‐treated cells

EMBO Reports ◽  
2004 ◽  
Vol 5 (6) ◽  
pp. 596-601 ◽  
Author(s):  
Yan Feng ◽  
Ashutosh P Jadhav ◽  
Chiara Rodighiero ◽  
Yukako Fujinaga ◽  
Tomas Kirchhausen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cholera Toxin ◽  
Retrograde Transport ◽  
Trans Golgi Network ◽  
Golgi Network

scholarly journals Retrograde Shiga Toxin Trafficking Is Regulated by ARHGAP21 and Cdc42

2009 ◽  
Vol 20 (20) ◽  
pp. 4303-4312 ◽  
Author(s):  
Heidi Hehnly ◽  
Katrina Marie Longhini ◽  
Ji-Long Chen ◽  
Mark Stamnes
Keyword(s):  
Golgi Apparatus ◽  
Shiga Toxin ◽  
Rho Gtpases ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Protein Translation ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Food Borne ◽  
Cytoskeletal Dynamics

Shiga-toxin–producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus.

scholarly journals IMPROVEMENT OF ORGANOLEPTIC INDICATORS OF CHEESE PRODUCTS BY CORRECTING FATTY ACID COMPOSITION OF FAT PHASE

Food systems ◽  
2020 ◽  
Vol 3 (2) ◽  
pp. 29-34
Author(s):  
О. V. Lepilkina ◽  
V. A. Mordvinova ◽  
E. V. Topnikova ◽  
I. L. Ostroukhova ◽  
E. S. Danilova
Keyword(s):  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Acid Composition ◽  
Butyric Acid ◽  
Milk Fat ◽  
Water Soluble ◽  
Organoleptic Characteristics ◽  
Fat Composition ◽  
Cheese Products ◽  
Vegetable Fat

The aim of the study was to establish the possibility of improving the organoleptic characteristics of cheese products by introducing butyric acid into the composition of the used vegetable fat composition. Cheese products made using two fat compositions consisting of refined, deodorized vegetable fats, including those modified by interesterification, were studied. The experimental fat composition contained in its composition additional butyric acid added to the composition of triglycerides. The study of cheese products was carried out in the process of ripening and storage for 120 days in comparison with cheese, the fat phase of which is represented by milk fat. It was found that the degree of proteolysis, estimated by the ratio of total water-soluble nitrogen to total nitrogen, does not depend on the nature of the fat in the protein matrix. Milk fat in cheeses is more subjected to lipolysis than vegetable fats in cheese products. The addition of butyric acid to the vegetable composition in an amount of 0.8% increased the acidity of the fat phase by 0.1 mmol/100 g. According to the assessment of organoleptic characteristics, cheese with milk fat had the most pronounced cheese flavor and aroma, cheese product without butyric acid in fat phase had the least pronounced ones. A cheese product with a fat composition containing added butyric acid was close to cheese with milk fat in terms of the severity of cheese taste, rheological characteristics and the spectrum of volatile flavoring substances. It is concluded that the taste and aroma of cheese products have a positive effect on adjusting the fatty acid composition of vegetable fat compositions by introducing butyric acid into their composition.

A hypothesis on the traffic of MG160, a medial Golgi sialoglycoprotein, from the trans-Golgi network to the Golgi cisternae

1994 ◽  
Vol 107 (3) ◽  
pp. 529-537 ◽  
Author(s):  
P.A. Johnston ◽  
A. Stieber ◽  
N.K. Gonatas
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Trans Golgi Network ◽  
Covalently Linked ◽  
Golgi Network

We have reported that MG160, an intrinsic membrane sialoglycoprotein of the Golgi apparatus (GA), resides in the medial cisternae of the organelle (Gonatas et al. (1989) J. Biol. Chem. 264, 646–653). In order to resolve the question whether MG160 acquires sialic acid residues in the trans cisternae or trans-Golgi network (TGN) prior to its retrograde transport, we have examined the effects of brefeldin A (BFA) on the post-translational processing of MG160, and the distribution of internalized wheat germ agglutinin covalently linked with HRP (WGA-HRP), which labels the TGN (Gonatas et al. (1977) J. Cell Biol. 73, 1–13). In BFA-treated PC12 cells, MG160 acquires resistance to endo H, but fails to be sialylated. This effect occurs in parallel with the redistribution of MG160 into an ER compartment dispersed throughout the cytoplasm including the nuclear envelope, and the collapse of the WGA-HRP-labelled TGN into vesicles and tubules surrounding the centriole. These results suggest that MG160 is not sialylated in BFA-treated cells because it is sequestered from the sialyltransferase enzyme(s), presumably located in the TGN, and provide evidence supporting the hypothesis for a retrograde transport pathway that recycles resident GA proteins, including MG160, between the Golgi cisternae and the TGN. To examine further the above hypothesis we studied cells treated with BFA and then allowed to recover from the effect of the drug for various lengths of time. After 15 minutes of recovery, cisternae of the Golgi apparatus, typically found in the pericentriolar region, are labeled by both MG160 and WGA-HRP.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A role for calmodulin in organelle membrane tubulation.

1995 ◽  
Vol 6 (7) ◽  
pp. 871-887 ◽  
Author(s):  
P de Figueiredo ◽  
W J Brown
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Coated Vesicle ◽  
Calmodulin Antagonists ◽  
Membrane Tubulation ◽  
Membrane Tubules ◽  
Potent Drug ◽  
Uniform Diameter ◽  
Golgi Network

Membrane tubules of uniform diameter (60-80 nm) and variable lengths have been seen to extend from the main bodies of the Golgi complex, trans Golgi network (TGN), and endosomes. In the case of endosomes, these tubules appear to mediate membrane and receptor recycling events. Brefeldin A (BFA) is a potent drug that completely blocks coated vesicle formation from the Golgi complex and TGN, but at the same time causes the enhanced formation of membrane tubules from these same organelles. Recently, experiments have shown that calmodulin antagonists inhibit the transport of receptors out of endosomes, perhaps by inhibiting the formation of recycling tubules. Using the potent calmodulin-specific antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (C-1), we found that the recycling of transferrin from endosomes to the cell surface was significantly inhibited, resulting in the formation of enlarged endosomal vacuoles. In addition, these same calmodulin antagonists also potently inhibited the formation of BFA-stimulated membrane tubules from the Golgi complex, TGN, and endosomes. In the case of the Golgi complex, failure to form tubules resulted in the inhibition of BFA-stimulated retrograde transport to the endoplasmic reticulum. These results suggest that calmodulin is a general regulator of membrane tubulation and is capable of influencing the morphology of several organelles.

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