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 Shiga Toxin Facilitates Its Retrograde Transport by Modifying Microtubule Dynamics

2006 ◽  
Vol 17 (10) ◽  
pp. 4379-4389 ◽  
Author(s):  
Heidi Hehnly ◽  
David Sheff ◽  
Mark Stamnes
Keyword(s):  
Shiga Toxin ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Host Cells ◽  
Mammalian Host ◽  
Golgi Stack ◽  
Toxin Binding ◽  
Cytoskeletal Dynamics ◽  
Induced Changes

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.

scholarly journals The Mitogen-activated Protein Kinase p38 Links Shiga Toxin-dependent Signaling and Trafficking

2008 ◽  
Vol 19 (1) ◽  
pp. 95-104 ◽  
Author(s):  
Sébastien Wälchli ◽  
Sigrid S. Skånland ◽  
Tone F. Gregers ◽  
Silje U. Lauvrak ◽  
Maria L. Torgersen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Golgi Apparatus ◽  
Shiga Toxin ◽  
Intracellular Transport ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Early Endosomes ◽  
Chemical Inhibitors ◽  
Mitogen Activated Protein ◽  
Interfering Rna

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca2+-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca2+levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca2+levels. Intracellular transport of Stx is Ca2+dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca2+and p38, to regulate its trafficking to the Golgi apparatus.

Recovery of protein secretion after brefeldin A treatment of rat lacrimal glands: effect of cAMP

1996 ◽  
Vol 271 (3) ◽  
pp. C783-C793 ◽  
Author(s):  
P. Robin ◽  
B. Rossignol ◽  
M. N. Raymond
Keyword(s):  
Golgi Apparatus ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Specific Protein ◽  
Dependent Manner ◽  
Lacrimal Glands ◽  
Extracellular Stimuli ◽  
Few Data

In exocrine cells, the discharge of secretory granule contents in response to extracellular stimuli has been widely documented. However, few data are available concerning the effect of these stimuli on the steps of the secretory pathway preceding protein exocytosis. To obtain more data on this subject, we used brefeldin A (BFA) to perturb intracellular protein transit. When, after exposure of the lacrimal gland lobules to 10 microM BFA, which led to a complete dismantling of the Golgi apparatus and fully inhibited the secretion of newly synthesized proteins, the drug concentration was lowered to 100 nM, a restoration of protein secretion was observed in a secretagogue-dependent manner. Secretagogues increasing the adenosine 3',5'-cyclic monophosphate (cAMP) level facilitated the recovery of protein secretion and Golgi apparatus restructuring, whereas other secretagogues, involving the calcium pathway, did not. Furthermore, the cAMP effect was prevented by H-89, a specific protein kinase A inhibitor. These effects of cAMP are due to neither BFA degradation nor BFA excretion from the cells. We conclude from these results that in rat lacrimal glands the recovery from the dramatic damage caused by BFA is promoted by a cAMP-dependent mechanism and further suggest a role of cAMP in the regulation of the Golgi structure and/or function.

scholarly journals The transmembrane protein p23 contributes to the organization of the Golgi apparatus

2000 ◽  
Vol 113 (6) ◽  
pp. 1043-1057 ◽  
Author(s):  
M. Rojo ◽  
G. Emery ◽  
V. Marjomaki ◽  
A.W. McDowall ◽  
R.G. Parton ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Ectopic Expression ◽  
Retrograde Transport ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
Constant Diameter ◽  
Golgi Network

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.

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 Rab6 Coordinates a Novel Golgi to ER Retrograde Transport Pathway in Live Cells

1999 ◽  
Vol 147 (4) ◽  
pp. 743-760 ◽  
Author(s):  
Jamie White ◽  
Ludger Johannes ◽  
Frédéric Mallard ◽  
Andreas Girod ◽  
Stephan Grill ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Live Cells ◽  
Cell Periphery ◽  
Wild Type ◽  
Transport Pathway ◽  
Toxin B ◽  
Kdel Receptor

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

Targeting of Shiga Toxin B-Subunit to Retrograde Transport Route in Association with Detergent-resistant Membranes

2001 ◽  
Vol 12 (8) ◽  
pp. 2453-2468 ◽  
Author(s):  
Thomas Falguières ◽  
Frédéric Mallard ◽  
Carole Baron ◽  
Daniel Hanau ◽  
Clifford Lingwood ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Shiga Toxin ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Toxin B ◽  
B Subunit ◽  
Early Endosomes ◽  
Golgi Network ◽  
Triton X

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to thetrans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

scholarly journals Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane.

1992 ◽  
Vol 119 (6) ◽  
pp. 1459-1468 ◽  
Author(s):  
A Cooper ◽  
H Bussey
Keyword(s):  
Golgi Apparatus ◽  
Membrane Protein ◽  
Secretory Pathway ◽  
Vacuolar Membrane ◽  
Unexpected Finding ◽  
Type I ◽  
Dependent Manner ◽  
Wild Type ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

We have investigated the localization of Kex1p, a type I transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. Indirect immunofluorescence demonstrated the presence of Kex1p in a punctate organelle resembling the yeast Golgi apparatus as identified by Kex2p and Sec7p (Franzusoff, A., K. Redding, J. Crosby, R. S. Fuller, and R. Schekman. 1991. J. Cell Biol. 112:27-37). Glycosylation studies of Kex1p were consistent with a Golgi location, as Kex1p was progressively N-glycosylated in an MNN1-dependent manner. To address the basis of Kex1p targeting to the Golgi apparatus, we examined the cellular location of a series of carboxy-terminal truncations of the protein. The results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the Golgi apparatus. Deletions of the retention region or overproduction of wild-type Kex1p led to mislocalization of Kex1p to the vacuolar membrane. This unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane.

Ochratoxin A challenges the intestinal epithelial cell integrity: results obtained in model experiments with Caco-2 cells

2019 ◽  
Vol 12 (4) ◽  
pp. 399-407 ◽  
Author(s):  
A. Alizadeh ◽  
P. Akbari ◽  
S. Varasteh ◽  
S. Braber ◽  
H. Malekinejad ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ochratoxin A ◽  
Lucifer Yellow ◽  
Intestinal Barrier ◽  
Clinical Importance ◽  
Hazardous Substances ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Cell Integrity ◽  
Barrier Integrity

Contamination of human and animal diets with different mycotoxins have gained significant attention over the past decade. The intestinal barrier is the first site of exposure and a primary target for nutritional contaminants and hazardous substances including mycotoxins. In this study, the potential impact of ochratoxin A (OTA) on intestinal barrier integrity was highlighted using a human intestinal Caco-2 cell line. Cell viability following OTA exposure was determined by lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, markers of barrier integrity, such as transepithelial electrical resistance (TEER) as well as the permeability of Lucifer Yellow (LY) and fluorescein isothiocyanate (FITC)-dextran, were assessed. Furthermore, the protein expression of different tight junction (TJ) proteins, as main constituents of barrier integrity, was evaluated by Western blot. Results show that OTA reduces TEER values in a concentration- and time-dependent manner and increase the permeability of LY through the intestinal epithelial layer, while the cell viability did not change significantly. However, the damage was not severe enough to change the permeability to larger molecules, such as FITC-dextran. OTA exposure down-regulated the expression of TJ proteins claudin-1, -3 and -4 and up-regulated the expression of zona occludens 1. The observation that OTA can disrupt the epithelial barrier is of clinical importance as it may lead to an increased passage of luminal antigens into the systemic circulation.

scholarly journals Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552

2016 ◽  
Vol 310 (7) ◽  
pp. C542-C557 ◽  
Author(s):  
Jia Wang ◽  
Liang Han ◽  
James Sinnett-Smith ◽  
Li-Li Han ◽  
Jan V. Stevens ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Nuclear Localization ◽  
Cross Talk ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Ang Ii ◽  
Potent Inducer

Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation ( P < 0.01), peaked at 4 h ( P < 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser552 in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser552 in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo.

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