scholarly journals Involvement of ATP-dependentPseudomonasExotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

1998 ◽  
Vol 9 (2) ◽  
pp. 387-402 ◽  
Author(s):  
Mériem Alami ◽  
Marie-Pierre Taupiac ◽  
Hubert Reggio ◽  
Alain Bienvenüe ◽  
Bruno Beaumelle
Keyword(s):  
Atp Hydrolysis ◽  
Active Form ◽  
Brefeldin A ◽  
Recycling Endosomes ◽  
Confocal Immunofluorescence Microscopy ◽  
Weak Bases ◽  
Confocal Immunofluorescence ◽  
A Cell ◽  
Endocytic Vesicles ◽  
Cytosol Ph

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal125I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

scholarly journals Bilayered Clathrin Coats on Endosomal Vacuoles Are Involved in Protein Sorting toward Lysosomes

2002 ◽  
Vol 13 (4) ◽  
pp. 1313-1328 ◽  
Author(s):  
Martin Sachse ◽  
Sylvie Urbé ◽  
Viola Oorschot ◽  
Ger J. Strous ◽  
Judith Klumperman
Keyword(s):  
Kinase Inhibitor ◽  
Brefeldin A ◽  
Phosphatidylinositol 3 Kinase ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
Quantitative Immunoelectron Microscopy ◽  
Endocytic Vesicles ◽  
Coated Membrane ◽  
Proteasomal Inhibitor

In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron miscroscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 699-709 ◽  
Author(s):  
M.M. Schroeder ◽  
D.L. Gard
Keyword(s):  
Cell Cycle ◽  
Cortical Microtubule ◽  
Cortical Microtubules ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Vegetal Cortex ◽  
M Phase ◽  
Tubulin Antibodies ◽  
Microtubule Networks ◽  
Sperm Aster

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.

scholarly journals Understanding Cell Interactions Using Modular Nanoparticle Libraries

2019 ◽  
Vol 72 (8) ◽  
pp. 595 ◽  
Author(s):  
Georgina K. Such ◽  
Angus P. R. Johnston
Keyword(s):  
Immune Responses ◽  
Small Molecules ◽  
Active Form ◽  
Reticuloendothelial System ◽  
Endosomal Escape ◽  
Biological Interactions ◽  
Nanoparticle Delivery ◽  
A Cell ◽  
Nanoparticle Structure ◽  
The Right

Nanoparticle delivery systems have significant potential to facilitate the delivery of novel therapeutics, such as proteins, DNA or small molecules. However, there are multiple biological barriers that need to be overcome to deliver the cargo in an active form. These challenges include evading clearance by the reticuloendothelial system, minimising adverse immune responses, targeting specific cells and tissues, and trafficking into the right compartment of the cell. In this account, we will discuss how nanoparticle structure can be tuned to optimise biological interactions and thus improve the ability of nanoparticles to overcome these barriers. The focus of this article will be on controlling cell targeting and trafficking within a cell, e.g. endosomal escape.

scholarly journals RNA polymerase II transcription is concentrated outside replication domains throughout S-phase

1994 ◽  
Vol 107 (6) ◽  
pp. 1449-1456 ◽  
Author(s):  
D.G. Wansink ◽  
E.E. Manders ◽  
I. van der Kraan ◽  
J.A. Aten ◽  
R. van Driel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Spatial Relationship ◽  
S Phase ◽  
Double Labelling ◽  
Confocal Immunofluorescence Microscopy ◽  
Nuclear Domain ◽  
Confocal Immunofluorescence ◽  
Nuclear Domains ◽  
Transcription And Replication

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

scholarly journals Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome.

1989 ◽  
Vol 109 (6) ◽  
pp. 3303-3314 ◽  
Author(s):  
K W Dunn ◽  
T E McGraw ◽  
F R Maxfield
Keyword(s):  
Digital Image Analysis ◽  
High Efficiency ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Recycling Endosomes ◽  
Double Label ◽  
Fluorescent Ligands ◽  
Endocytic Vesicles ◽  
Endocytic Compartments ◽  
Very High

To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min. We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2-4 min further incubation in the absence of external ligands, LDL-containing compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with Tf. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.

scholarly journals Inhibition of Transferrin Recycling and Endosome Tubulation by Phospholipase A2Antagonists

2001 ◽  
Vol 276 (50) ◽  
pp. 47361-47370 ◽  
Author(s):  
Paul de Figueiredo ◽  
Anne Doody ◽  
Renée S. Polizotto ◽  
Daniel Drecktrah ◽  
Salli Wood ◽  
...  
Keyword(s):  
Cell Surface ◽  
Molecular Mechanism ◽  
Golgi Complex ◽  
Broad Spectrum ◽  
Tubule Formation ◽  
Recycling Endosomes ◽  
Low Concentrations ◽  
A Cell ◽  
Recycling Pathway ◽  
Lines Of Evidence

We report here that a broad spectrum of phospholipase A2(PLA2) antagonists produce a concentration-dependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (∼1 μm), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (∼10 μm), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA2antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA2antagonists. And third, endosome tubule formation in a cell-free, cytosol-dependent reconstitution system was equally sensitive PLA2antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA2and that PLA2-dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA2activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.

Subcellular movement and expression of HSP27, αB-crystallin, and HSP70 after two bouts of eccentric exercise in humans

2009 ◽  
Vol 107 (2) ◽  
pp. 570-582 ◽  
Author(s):  
G. Paulsen ◽  
F. Lauritzen ◽  
M. L. Bayer ◽  
J. M. Kalhovde ◽  
I. Ugelstad ◽  
...  
Keyword(s):  
Eccentric Exercise ◽  
Ultrastructural Level ◽  
Protective Role ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Small Hsps ◽  
Generating Capacity ◽  
Αb Crystallin ◽  
Shock Proteins ◽  
Repeated Bout

The aims of this study were to investigate the sarcomeric accumulation and expression of heat shock proteins (HSPs) after two bouts of maximal eccentric exercise. Twenty-four subjects performed two bouts of 70 maximal voluntary eccentric actions using the elbow flexors in one arm. The bouts were separated by 3 wk. The changes in concentric (60°/s) and isometric (90°) force-generating capacity were monitored for 9 days after each bout, and biopsies were taken 1 and 48 h and 4 and 7 days after bout 1 and 1 and 48 h after bout 2. The content of HSP27, αB-crystallin, HSP70, and desmin in the cytosolic and cytoskeleton/myofibrillar fractions of homogenized muscle samples was determined by immunoassays, and the cellular and subcellular localization of the HSPs in the myofibrillar structure was analyzed by conventional and confocal immunofluorescence microscopy and quantitative electron microscopy. The force-generating capacity was reduced by ∼50% and did not recover completely during the 3 wk following bout 1. After bout 2, the subjects recovered within 4 days. The HSP levels increased in the cytosolic fraction after bout 1, especially HSP70 (∼300% 2–7 days after exercise). Increased levels of HSP27, αB-crystallin, and HSP70 were found in the cytoskeletal/myofibrillar fraction after both bouts, despite reduced damage after bout 2. At the ultrastructural level, HSP27 and αB-crystallin accumulated in Z-disks, in intermediate desmin-like structures (αB-crystallin), and in areas of myofibrillar disruption. In conclusion, HSP27 and αB-crystallin accumulated in myofibrillar structures, especially in the Z-disks and the intermediate structures (desmin). The function of the small HSPs is possibly to stabilize and protect the myofibrillar structures during and after unaccustomed eccentric exercise. The large amount of HSP27, αB-crystallin, and HSP70 in the cytoskeletal/myofibrillar fraction after a repeated bout of exercise suggests a protective role as part of the repeated-bout effect.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX

2001 ◽  
Vol 358 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Philippe ULSEMER ◽  
Catherine STRASSEL ◽  
Marie-Jeanne BAAS ◽  
Jean SALAMERO ◽  
Sylvette CHASSEROT-GOLAZ ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Ovary Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Vessel Injury ◽  
Von Willebrand

The multisubunit leucine-rich glycoprotein (GP) Ib–IX–V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard–Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse–chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbα undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbα and surface expression of the complex. Biosynthetic studies were then performed on a Bernard–Soulier variant based on previous description of abnormal GPIbα size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbα. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments (‘ERGIC’) and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

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