Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in African trypanosomes

1995 ◽  
Vol 108 (11) ◽  
pp. 3611-3621 ◽  
Author(s):  
M.J. Brickman ◽  
J.M. Cook ◽  
A.E. Balber
Keyword(s):  
Intracellular Transport ◽  
Fluid Phase ◽  
Morphological Alteration ◽  
Flagellar Pocket ◽  
African Trypanosomes ◽  
Endocytic Compartment ◽  
A Value ◽  
Membrane Bound ◽  
Collecting Tubules ◽  
Endocytic Vesicles

We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of fluid phase bovine serum albumen gold complexes and membrane bound concanavalin A through endosomal compartments of bloodstream forms of Trypanosoma brucei rhodesiense. Both markers were rapidly endocytosed from the flagellar pocket. Within 20 minutes at 37 degrees C the markers reached a large, vesicular, perinuclear compartment that stained heavily with the CB1 monoclonal antibody. Neither marker left the flagellar pocket and entered cells at 4 degrees C. When cells were incubated at 12 degrees C, both markers entered the cell and were transported to collecting tubules, a tubular endosomal compartment that receives endocytosed material from coated endocytic vesicles. However, no material was transported from collecting tubules to the late, perinuclear compartment at 12 degrees C. The morphology of collecting tubule membranes was specifically altered at 12 degrees C; tubules became shorter and were arrayed near the flagellar pocket. The morphological alteration and the block in transport of endocytic markers to the perinuclear compartment seen at 12 degrees C were reversed 10 minutes after cells were returned to 37 degrees C. We also used flow cytofluorimetric measurements of pH dependent fluorescence quenching to measure the pH of the terminal endocytic compartment. Fluoresceinated lectins accumulated in a terminal compartment with a pH of 6.0-6.1, a value considerably higher than that of mammalian lysosomes. Fluorescence from fluoresceinated lectins in this terminal endocytic compartment was dequenched when bloodstream forms were incubated in the presence of chloroquine.

scholarly journals Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

1994 ◽  
Vol 302 (2) ◽  
pp. 451-454 ◽  
Author(s):  
M H Lafrance ◽  
C Vézina ◽  
Q Wang ◽  
G Boileau ◽  
P Crine ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Cell Extracts ◽  
Glycosylation Sites ◽  
Sugar Addition ◽  
Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

scholarly journals Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein.

1986 ◽  
Vol 103 (1) ◽  
pp. 255-263 ◽  
Author(s):  
J D Bangs ◽  
N W Andrews ◽  
G W Hart ◽  
P T Englund
Keyword(s):  
Cell Surface ◽  
Phospholipase C ◽  
Intracellular Transport ◽  
Signal Sequence ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Hydrophobic Peptide ◽  
Membrane Bound ◽  
A Site ◽  
Kinetics Of

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.

scholarly journals Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

2009 ◽  
Vol 106 (41) ◽  
pp. 17425-17430 ◽  
Author(s):  
C. Gadelha ◽  
S. Rothery ◽  
M. Morphew ◽  
J. R. McIntosh ◽  
N. J. Severs ◽  
...  
Keyword(s):  
Membrane Domains ◽  
Flagellar Pocket ◽  
African Trypanosomes

scholarly journals Transfected plasmacytoma cells do not transport the membrane form of IgM to the cell surface.

1988 ◽  
Vol 167 (2) ◽  
pp. 652-657 ◽  
Author(s):  
J Hombach ◽  
F Sablitzky ◽  
K Rajewsky ◽  
M Reth
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Intracellular Transport ◽  
Expression Vectors ◽  
Myeloma Cells ◽  
B Lymphoma ◽  
Cell Lineages ◽  
B Lymphoma Cells ◽  
Golgi Compartment ◽  
Membrane Bound

Expression vectors coding for membrane-bound IgM antibodies were introduced into myeloma and B lymphoma cells. Only the lymphoma but not the myeloma cells were able to express the antibodies on the cell surface, although in both cases, complete antibodies were assembled intracellularly. In myeloma cells, the Ig molecules did not reach the Golgi compartment. Thus, the intracellular transport of membrane-bound antibodies is controlled in the B cell lineages in a developmentally ordered fashion.

scholarly journals Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies.

1996 ◽  
Vol 133 (6) ◽  
pp. 1205-1215 ◽  
Author(s):  
H Krämer ◽  
M Phistry
Keyword(s):  
Amino Acids ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Specific Pattern ◽  
Multivesicular Bodies ◽  
Endocytic Compartment ◽  
Coiled Coil Domain ◽  
Type Pattern ◽  
Cell Type Specific ◽  
Endocytic Vesicles

Transmembrane ligands can be internalized across cell boundaries into receptor-expressing cells. In the developing Drosophila eye imaginal disc, the bride of sevenless transmembrane protein (boss) is expressed on the surface of R8 cells. After internalization into neighboring R7 cells, the boss protein accumulates in multivesicular bodies. In a search for genes that affect this cell-type-specific pattern of boss endocytosis, we found that mutations in the hook gene inhibit the accumulation of boss in multivesicular bodies of R7 cells. In addition, hook flies exhibit pleiotropic phenotypes including abnormal bristle morphology and eye degeneration. The wild-type-pattern of boss endocytosis was restored in hook mutants by a genomic rescue fragment containing the hook gene or by a hook cDNA expressed in R7 cells under control of a sevenless (sev) enhancer. The hook gene encodes a novel cytoplasmic protein of 679 amino acids with a central coiled-coil domain of some 200 amino acids. Truncated, epitope-tagged hook proteins coimmunoprecipitated the full-length protein, indicating dimerization mediated by the coiled-coil domain. The hook protein localizes to vesicular structures that are part of the endocytic compartment. The requirement of the hook protein in R7 cells for the accumulation of boss protein in multivesicular bodies, and the localization of the hook protein to endocytic vesicles indicate that the hook gene encodes a novel component of the endocytic compartment that plays an important role in the endocytosis of transmembrane ligands or their transport to multivesicular bodies.

scholarly journals Geochronological and Geochemical Study of Zircon from Tourmaline-Muscovite Granites of the Archaean Kolmozero–Voronya Greenstone Belt: Insights into Sources of the Rare-Metal Pegmatites

Minerals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 760
Author(s):  
Nikolay M. Kudryashov ◽  
Oksana V. Udoratina ◽  
Matthew A. Coble ◽  
Ekaterina N. Steshenko
Keyword(s):  
Greenstone Belt ◽  
Fluid Phase ◽  
Rare Metal ◽  
Zircon Crystal ◽  
A Value ◽  
Geochemical Study ◽  
Zircon Crystallization ◽  
Geochronological Study ◽  
Ree Patterns ◽  
Phase Fluid

In order to determine the U-Pb crystallization age of zircon from the tourmaline-muscovite granites of the Kolmozero–Voronya greenstone belt located in the northeastern Fennoscandian Shield (Kola Peninsula), an isotope-geochronological study of the zircon grains was performed using a SHRIMP-RG microprobe. The belt is represented by the Archaean volcano-sedimentary rocks (2.9–2.8 Ga). Deposits of rare-metal pegmatites (Li and Cs with associated Nb, Ta, and Be) occur within the belt and on its margins. The age of the pegmatites within the belt was estimated at 2.7–2.6 Ga. Until now, there has been no generally accepted view on the genetic relation of the pegmatites with granite. Various authors have suggested that the pegmatites could potentially be associated with many type of granitoids within the region, i.e., plagiogranites, tonalites, amphibole-biotite granodiorites, microcline granites, alkaline granites, or muscovite-tourmaline granites. Zircon crystals from the muscovite-tourmaline granites are heterogeneous; they have less altered cores and strongly altered rims. The zircon cores are slightly enriched in U at a value of 173–1030 ppm, Th/U = 0.1–0.4. The zircons’ rims are heavily enriched in U at a value of 700–3300 ppm, Th/U = 0.03–0.08, indicating metasomatic processes. Zircon characteristics show that it crystallized from a melt enriched in a fluid phase. Fluid activity lasted after zircon crystallization as reflected in the irregular composition of the mineral and its rare earth element (REE) patterns that are typical of a metasomatic zircon. The computed zircon crystallization temperature in the tourmaline-muscovite granites is in the range of 650–850 °C. The discordant age calculated for five analyzed points of the zircon crystal cores is 2802 ± 13 Ma. The discordant age for four analyzed points of the zircon crystal rims is found to be 2728 ± 14 Ma. On the basis of the obtained isotope-geochronological data, we conclude that the tourmaline-muscovite granites located in the immediate vicinity of rare-metal pegmatite veins are the most probable source of matter for the pegmatites.

Effects of colchicine on endocytosis of horseradish peroxidase by rat peritoneal macrophages

1980 ◽  
Vol 45 (1) ◽  
pp. 59-71 ◽  
Author(s):  
A. Piasek ◽  
J. Thyberg
Keyword(s):  
Horseradish Peroxidase ◽  
Digestive Enzymes ◽  
Cytochalasin B ◽  
Fluid Phase ◽  
Peritoneal Macrophages ◽  
The Other ◽  
Fluid Phase Endocytosis ◽  
Cytoplasmic Microtubules ◽  
Endocytic Vesicles ◽  
Cytoplasmic Complex

Horseradish peroxidase (HRP) was used as an exogenous marker to study the effects of microtubule-disruptive drugs on endocytosis in cultures of thioglycollate-elicited rat peritoneal macrophages. Colchicine and vinblastine, but not lumicolchicine or cytochalasin B, reduced HRP uptake by about 30–40%. However, as determined by stereological measurements, the size of the HRP-containing compartment within the cells remained unaltered. In both control cells and cells treated with colchicine or vinblastine the HRP-reactive vesicles were preferentially located close to the dictyosomes (stacks of cisternae) despite the fact that the Golgi complex was disorganized in the treated cells. These results suggest that intact cytoplasmic complex was disorganized in the treated cells. These results suggest that intact cytoplasmic microtubules are required to maintain a normal rate of fluid phase endocytosis in macrophages. On the other hand, it seems as if microtubules are not necessary for the translocation of newly formed endocytic vesicles/lysosomes to the dictyosomes, from which they probably are supplied with digestive enzymes.

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