Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor

2000 ◽  
Vol 113 (17) ◽  
pp. 2963-2975 ◽  
Author(s):  
F. Vandenbulcke ◽  
D. Nouel ◽  
J.P. Vincent ◽  
J. Mazella ◽  
A. Beaudet
Keyword(s):  
Target Cells ◽  
Receptor Subtype ◽  
Coated Pits ◽  
Recycling Endosome ◽  
Recycling Endosomes ◽  
Receptor Complexes ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network ◽  
Acidic Organelles

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0–30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30–45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals Dynamin-dependent Transferrin Receptor Recycling by Endosome-derived Clathrin-coated Vesicles

2002 ◽  
Vol 13 (1) ◽  
pp. 169-182 ◽  
Author(s):  
Ellen M. van Dam ◽  
Willem Stoorvogel
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Coated Vesicles ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Coated Pits ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1G273D) or, as a control, dynamin-1 wild type. In dynamin-1G273D–expressing cells, 29–36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1G273D associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1G273D–expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.

Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1289-1295 ◽  
Author(s):  
V. Duprez ◽  
M. Smoljanovic ◽  
M. Lieb ◽  
A. Dautry-Varsat
Keyword(s):  
Horseradish Peroxidase ◽  
Interleukin 2 ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
High Affinity ◽  
Human T Cell ◽  
Late Endosomes ◽  
Discontinuous Sucrose Gradient ◽  
Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

scholarly journals A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

2015 ◽  
Vol 112 (12) ◽  
pp. E1443-E1452 ◽  
Author(s):  
Zhiyong Bai ◽  
Barth D. Grant
Keyword(s):  
Functional Module ◽  
Small Gtpase ◽  
Recycling Endosome ◽  
Vesicle Budding ◽  
Recycling Endosomes ◽  
Physiological Importance ◽  
Golgi Transport ◽  
Early Endosomes ◽  
Cargo Proteins ◽  
Signaling Receptors

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

scholarly journals Fusion of sequentially internalized vesicles in alveolar macrophages.

1990 ◽  
Vol 110 (4) ◽  
pp. 1013-1022 ◽  
Author(s):  
D M Ward ◽  
D P Hackenyos ◽  
J Kaplan
Keyword(s):  
Endocytic Pathway ◽  
Receptor Complexes ◽  
Intracellular Vesicle ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Changing Patterns ◽  
Intracellular Vesicle Transport ◽  
Shift Technique ◽  
Ligand Addition ◽  
Density Shift

Previously we reported that internalized ligand-receptor complexes are transported within the alveolar macrophage at a rate that is independent of the ligand and/or receptor but is dependent on the endocytic apparatus (Ward, D. M., R. S. Ajioka, and J. Kaplan. 1989. J. Biol. Chem. 264:8164-8170). To probe the mechanism of intracellular vesicle transport, we examined the ability of vesicles internalized at different times to fuse. The mixing of ligands internalized at different times was studied using the 3,3'-diaminobenzidine/horseradish peroxidase density shift technique. The ability of internalized vesicles to fuse was dependent upon their location in the endocytic pathway. When ligands were administered as tandem pulses a significant amount of mixing (20-40%) of vesicular contents was observed. The pattern of mixing was independent of the ligands employed (transferrin, mannosylated BSA, or alpha macroglobulin), the order of ligand addition, and temperature (37 degrees C or 28 degrees C). Fusion was restricted to a brief period immediately after internalization. The amount of fusion in early endosomes did not increase when cells, given tandem pulses, were chased such that the ligands further traversed the early endocytic pathway. Little fusion, also, was seen when a chase was interposed between the two ligand pulses. The temporal segregation of vesicle contents seen in early endosomes was lost within late endosomes. Extensive mixing of vesicle contents was observed in the later portion of the endocytic pathway. This portion of the pathway is defined by the absence of internalized transferrin and is composed of ligands en route to lysosomes. Incubation of cells in iso-osmotic medium in which Na+ was replaced by K+ inhibited movement of internalized ligands to the lysosome, resulting in ligand accumulation within the late endocytic pathway. The accumulation of ligand was correlated with extensive mixing of sequentially internalized ligands. Although significant amounts of ligand degradation were observed, this compartment was devoid of conventional lysosomal markers such as acid glycosidases. These results indicate changing patterns of vesicle fusion within the endocytic pathway, with a complete loss of temporal ligand segregation in a prelysosomal compartment.

scholarly journals Numb regulates the balance between Notch recycling and late-endosome targeting inDrosophilaneural progenitor cells

2016 ◽  
Vol 27 (18) ◽  
pp. 2857-2866 ◽  
Author(s):  
Seth A. Johnson ◽  
Diana Zitserman ◽  
Fabrice Roegiers
Keyword(s):  
Notch Signaling ◽  
Asymmetric Cell Division ◽  
Adaptor Protein ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Recycling Endosome ◽  
Notch Receptors ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensory Organ Precursor

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.

scholarly journals Arfgef1 haploinsufficiency in mice alters neuronal endosome composition and decreases membrane surface postsynaptic GABAA receptors

2019 ◽  
Author(s):  
JiaJie Teoh ◽  
Narayan Subramanian ◽  
Maria Elena Pero ◽  
Francesca Bartolini ◽  
Ariadna Amador ◽  
...  
Keyword(s):  
Cell Body ◽  
Membrane Surface ◽  
Seizure Threshold ◽  
List Type ◽  
Recycling Endosome ◽  
Neuron Culture ◽  
Recycling Endosomes ◽  
Neuronal Inhibition ◽  
Early Endosomes ◽  
Hippocampal Neuron Culture

AbstractARFGEF1 encodes a guanine exchange factor involved in intracellular vesicle trafficking, and is a candidate gene for childhood genetic epilepsies. To model ARFGEF1 haploinsufficiency observed in a recent Lennox Gastaut Syndrome patient, we studied a frameshift mutation (Arfgef1fs) in mice. Arfgef1fs/+ pups exhibit signs of developmental delay, and Arfgef1fs/+ adults have a significantly decreased threshold to induced seizures but do not experience spontaneous seizures. Histologically, the Arfgef1fs/+ brain exhibits a disruption in the apical lining of the dentate gyrus and altered spine morphology of deep layer neurons. In primary hippocampal neuron culture, dendritic surface and synaptic but not total GABAA receptors (GABAAR) are reduced in Arfgef1fs/+ neurons with an accompanying decrease in GABAAR-containing recycling endosomes in cell body. Arfgef1fs/+ neurons also display differences in the relative ratio of Arf6+:Rab11+:TrfR+ recycling endosomes. Although the GABAAR-containing early endosomes in Arfgef1fs/+ neurons are comparable to wildtype, Arfgef1fs/+ neurons show an increase in GABAAR-containing lysosomes in dendrite and cell body. Together, the altered endosome composition and decreased neuronal surface GABAAR results suggests a mechanism whereby impaired neuronal inhibition leads to seizure susceptibility.HighlightsArfgef1fs/+ mice have lower seizure threshold but no spontaneous seizure.Arfgef1fs/+ neurons show reduced dendritic surface GABAAR.Arfgef1fs/+ neurons have decreased GABAAR-containing recycling endosome accompanied with an increase in GABAAR-containing lysosomes.

scholarly journals Rab5-mediated endosome formation is regulated at the trans-Golgi network

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Makoto Nagano ◽  
Junko Y. Toshima ◽  
Daria Elisabeth Siekhaus ◽  
Jiro Toshima
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Vesicle Transport ◽  
Nucleotide Exchange ◽  
Trafficking Pathway ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Critical Location ◽  
Golgi Network

AbstractEarly endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

scholarly journals The giant organelles in beige and Chediak-Higashi fibroblasts are derived from late endosomes and mature lysosomes.

1993 ◽  
Vol 178 (6) ◽  
pp. 1845-1856 ◽  
Author(s):  
J K Burkhardt ◽  
F A Wiebel ◽  
S Hester ◽  
Y Argon
Keyword(s):  
Cell Lines ◽  
Secretory Granules ◽  
Mutant Cell ◽  
Endocytic Pathway ◽  
Wild Type ◽  
Marker Proteins ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Acidic Organelles ◽  
Respective Wild Type

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disease affecting secretory granules and lysosomes-like organelles. In CHS fibroblasts, acidic organelles are abnormally large and clustered in the perinuclear area. We have analyzed fibroblast cell lines from a CHS patient and from the murine model for CHS, the beige mouse, to determine which lysosome-like compartments are affected. Uptake of neutral red showed that in both beige and CHS cell lines, the acidic organelles were markedly clustered in the perinuclear region of the cells. Giant organelles (> 4 microns) were observed in a fraction of the cells, and these were more dramatic in the beige fibroblasts than in the CHS fibroblasts. The total dye uptake of both mutant cell lines was similar to their respective wild type fibroblasts, suggesting that the overall volume of acidic compartments is unaffected by the disorder. Histochemistry and immunofluorescence showed that the giant organelles in both beige and CHS fibroblasts were positive for cathepsin D, lysosome-associated membrane protein (LAMP) 1, LAMP 2, and a 120-kD lysosomal glycoprotein, all marker proteins for late endosomes and lysosomes. The giant organelles were also negative for transferrin receptor and mannose-6-phosphate receptor, and most of them were also negative for rab 7. This distribution of marker proteins shows that the giant organelles in both beige and CHS are derived from late compartments of the endocytic pathway. This conclusion was confirmed using endocytic tracers. BSA was transported to the giant organelles, but only after long incubation times, and only at 37 degrees C. alpha 2-Macroglobulin was taken up and degraded at similar rates by CHS or beige cells and their respective wild type control cells. Taken together, our results indicate that the mutation in CHS specifically affects late endosomes and lysosomes, with little or no effect on early endosomes. Although the mutation clearly causes mislocalization of these organelles, it appears to have little effect on their endocytic and degradative functions.

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