scholarly journals Transport of Bacterial Lipopolysaccharide to the Golgi Apparatus

1999 ◽  
Vol 190 (4) ◽  
pp. 523-534 ◽  
Author(s):  
Nathalie Thieblemont ◽  
Samuel D. Wright
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Hela Cells ◽  
Brefeldin A ◽  
Cellular Responses ◽  
Epithelial Cell Line ◽  
Cholera Toxin B Subunit ◽  
B Subunit ◽  
Cell Line Hela ◽  
Tetramethylrhodamine Isothiocyanate

Addition of lipopolysaccharide (LPS) to cells in the form of LPS–soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)–dextran, LysoTracker™ Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)–ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY–ceramide and TRITC (tetramethylrhodamine isothiocyanate)–labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS–sCD14 complexes in a CD14-dependent fashion: BODIPY–LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A–treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.

scholarly journals Capacity of the Golgi Apparatus for Cargo Transport Prior to Complete Assembly

2006 ◽  
Vol 17 (9) ◽  
pp. 4105-4117 ◽  
Author(s):  
Shu Jiang ◽  
Sung W. Rhee ◽  
Paul A. Gleeson ◽  
Brian Storrie
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Hela Cells ◽  
Mammalian Cells ◽  
De Novo ◽  
Brefeldin A ◽  
Cargo Transport ◽  
Kinetics Of

In yeast, particular emphasis has been given to endoplasmic reticulum (ER)-derived, cisternal maturation models of Golgi assembly while in mammalian cells more emphasis has been given to golgins as a potentially stable assembly framework. In the case of de novo Golgi formation from the ER after brefeldin A/H89 washout in HeLa cells, we found that scattered, golgin-enriched, structures formed early and contained golgins including giantin, ranging across the entire cis to trans spectrum of the Golgi apparatus. These structures were incompetent in VSV-G cargo transport. Second, we compared Golgi competence in cargo transport to the kinetics of addition of various glycosyltransferases and glycosidases into nascent, golgin-enriched structures after drug washout. Enzyme accumulation was sequential with trans and then medial glycosyltransferases/glycosidases found in the scattered, nascent Golgi. Involvement in cargo transport preceded full accumulation of enzymes or GPP130 into nascent Golgi. Third, during mitosis, we found that the formation of a golgin-positive acceptor compartment in early telophase preceded the accumulation of a Golgi glycosyltransferase in nascent Golgi structures. We conclude that during mammalian Golgi assembly components fit into a dynamic, first-formed, multigolgin-enriched framework that is initially cargo transport incompetent. Resumption of cargo transport precedes full Golgi assembly.

scholarly journals Capacity of the Golgi Apparatus for Biogenesis from the Endoplasmic Reticulum

2003 ◽  
Vol 14 (12) ◽  
pp. 5011-5018 ◽  
Author(s):  
Sapna Puri ◽  
Adam D. Linstedt
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Self Assembly ◽  
De Novo ◽  
Brefeldin A ◽  
The Other ◽  
Matrix Proteins ◽  
Er Export ◽  
Golgi Matrix

It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.

scholarly journals cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

2012 ◽  
Vol 23 (16) ◽  
pp. 3203-3214 ◽  
Author(s):  
Yoko Ito ◽  
Tomohiro Uemura ◽  
Keiko Shoda ◽  
Masaru Fujimoto ◽  
Takashi Ueda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Fluorescent Proteins ◽  
Plant Cells ◽  
Brefeldin A ◽  
Eukaryotic Cells ◽  
Er Exit Sites ◽  
Intermediate Compartment ◽  
Golgi Protein ◽  
And Function

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

Brefeldin A-induced disassembly of the Golgi apparatus is followed by disruption of the endoplasmic reticulum in plant cells

1994 ◽  
Vol 45 (10) ◽  
pp. 1347-1351 ◽  
Author(s):  
Janey Henderson ◽  
Béatrice Stiat-Jeunemaitre ◽  
Richard Napier ◽  
Chris Hawes
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Plant Cells ◽  
Brefeldin A

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments

Cell Calcium ◽  
2004 ◽  
Vol 36 (6) ◽  
pp. 479-487 ◽  
Author(s):  
L. Missiaen ◽  
K. Van Acker ◽  
K. Van Baelen ◽  
L. Raeymaekers ◽  
F. Wuytack ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Hela Cells ◽  
Calcium Release

scholarly journals Dynein Supports Motility of Endoplasmic Reticulum in the FungusUstilago maydis

2002 ◽  
Vol 13 (3) ◽  
pp. 965-977 ◽  
Author(s):  
Roland Wedlich-Söldner ◽  
Irene Schulz ◽  
Anne Straube ◽  
Gero Steinberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Minor Importance ◽  
Dependent Transport

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER inU. maydis.

scholarly journals Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1798
Author(s):  
Ted Hackstadt ◽  
Abhilash I. Chiramel ◽  
Forrest H. Hoyt ◽  
Brandi N. Williamson ◽  
Cheryl A. Dooley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rna Synthesis ◽  
Light And Electron Microscopy ◽  
Brefeldin A ◽  
Viral Proteins ◽  
Protein Markers ◽  
Replication Complex ◽  
Golgi Fragmentation

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

scholarly journals Trafficking of Porin-Deficient Salmonella typhimurium Mutants inside HeLa Cells: ompR andenvZ Mutants Are Defective for the Formation ofSalmonella-Induced Filaments

1998 ◽  
Vol 66 (4) ◽  
pp. 1806-1811 ◽  
Author(s):  
Scott D. Mills ◽  
Sharon R. Ruschkowski ◽  
Murry A. Stein ◽  
B. Brett Finlay
Keyword(s):  
Salmonella Typhimurium ◽  
Hela Cells ◽  
Epithelial Cell Line ◽  
Tubular Structures ◽  
Wild Type ◽  
Human Epithelial Cell ◽  
Outer Membrane Porin ◽  
Cell Line Hela ◽  
Transposon Mutants ◽  
Line Hela

ABSTRACT Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimuriumporin-deficient mutants. In this study, isogenic transposon mutants ofS. typhimurium with insertions in ompR,envZ, ompF, ompC, ompD,osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that ompR and envZ mutants were reduced or completely inhibited for the formation ofSalmonella-induced filaments (Sifs). This result was confirmed with an ompB deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella. Genetic analysis showed that the ompR mutation could be complemented intrans by cloned ompR to restore its ability to induce Sifs. In contrast, mutations in the knownompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S. typhimurium.

scholarly journals Role of Host Cell-Derived Amino Acids in Nutrition of Intracellular Salmonella enterica

2015 ◽  
Vol 83 (12) ◽  
pp. 4466-4475 ◽  
Author(s):  
Jasmin Popp ◽  
Janina Noster ◽  
Kim Busch ◽  
Alexander Kehl ◽  
Gero zur Hellen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Host Cell ◽  
Cell Line ◽  
Hela Cells ◽  
Salmonella Enterica ◽  
Epithelial Cell Line ◽  
Intracellular Replication ◽  
Content Type ◽  
Raw264.7 Macrophages ◽  
Cell Line Hela

The facultative intracellular pathogenSalmonella entericaresides in a specific membrane-bound compartment termed theSalmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol,Salmonellais able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply ofSalmonellain the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophicSalmonellaby external amino acids was possible if bacteria were proficient in the induction ofSalmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellularSalmonellato redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.

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