Endosomal system of Paramecium: coated pits to early endosomes

1992 ◽  
Vol 101 (2) ◽  
pp. 449-461 ◽  
Author(s):  
R.D. Allen ◽  
C.C. Schroeder ◽  
A.K. Fok
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Early Endosome ◽  
Coated Vesicles ◽  
Striking Similarity ◽  
Coated Pits ◽  
Early Endosomes ◽  
Membrane Components ◽  
Endosomal System

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/− 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/− 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early 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.

scholarly journals A Myosin I Is Involved in Membrane Recycling from Early Endosomes

2000 ◽  
Vol 150 (5) ◽  
pp. 1013-1026 ◽  
Author(s):  
Eva M. Neuhaus ◽  
Thierry Soldati
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Surface Proteins ◽  
Endocytic Pathway ◽  
Membrane Recycling ◽  
Myosin I ◽  
Early Endosomes ◽  
Butanedione Monoxime ◽  
Microscopy Method ◽  
Membrane Components

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin–myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA−/B−, and myoB− cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA−/B− cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.

The delivery of endocytosed cargo to lysosomes

2009 ◽  
Vol 37 (5) ◽  
pp. 1019-1021 ◽  
Author(s):  
J. Paul Luzio ◽  
Michael D.J. Parkinson ◽  
Sally R. Gray ◽  
Nicholas A. Bright
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Late Endosome ◽  
Snare Complex ◽  
Coated Pits ◽  
Free System ◽  
Endosomal Sorting ◽  
Protein Receptor ◽  
Late Endosomes ◽  
Early Endosomes

In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to 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 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 Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

2001 ◽  
Vol 12 (9) ◽  
pp. 2790-2799 ◽  
Author(s):  
Elizabeth M. Bennett ◽  
Sharron X. Lin ◽  
Mhairi C. Towler ◽  
Frederick R. Maxfield ◽  
Frances M. Brodsky
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Endocytic Trafficking ◽  
Minimal Impact ◽  
Recycling Endosomes ◽  
Receptor Mediated Endocytosis ◽  
Early Endosomes ◽  
Endosomal Membranes

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

scholarly journals A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples

2012 ◽  
Vol 447 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Gaëtan Chicanne ◽  
Sonia Severin ◽  
Cécile Boscheron ◽  
Anne-Dominique Terrisse ◽  
Marie-Pierre Gratacap ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Chromatography ◽  
Biological Samples ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Human Platelets ◽  
Bhk Cells ◽  
Early Endosomes ◽  
Important Player ◽  
Mass Level

PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

scholarly journals alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

scholarly journals Immunofluorescent localization of 100K coated vesicle proteins.

1986 ◽  
Vol 102 (1) ◽  
pp. 48-54 ◽  
Author(s):  
M S Robinson ◽  
B M Pearse
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Limited Proteolysis ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Cell Protein ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Double Labeling ◽  
Vesicle Proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.

scholarly journals A novel class of clathrin-coated vesicles budding from endosomes.

1996 ◽  
Vol 132 (1) ◽  
pp. 21-33 ◽  
Author(s):  
W Stoorvogel ◽  
V Oorschot ◽  
H J Geuze
Keyword(s):  
Plasma Membrane ◽  
Microscopic Examination ◽  
Transferrin Receptor ◽  
Brefeldin A ◽  
Coated Vesicles ◽  
Integral Membrane Proteins ◽  
Transferrin Receptors ◽  
Receptor Recycling ◽  
Coated Pits ◽  
Novel Technique

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.

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