scholarly journals Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors.

1993 ◽  
Vol 123 (1) ◽  
pp. 89-97 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
High Efficiency ◽  
De Novo ◽  
Coated Vesicles ◽  
Transferrin Receptors ◽  
Con A ◽  
Coated Pits ◽  
Late Endosomes ◽  
Endocytic Vesicles ◽  
Endocytic Compartments ◽  
K Depletion

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin-dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)-depleted cells with Con A-gold for 15 min, approximately 75% of Con A-gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)-depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.

scholarly journals The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations.

1991 ◽  
Vol 113 (4) ◽  
pp. 731-741 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
Cell Surface ◽  
Coated Vesicles ◽  
Minor Role ◽  
Specific Marker ◽  
Con A ◽  
Early Endosomes ◽  
Gold Conjugate ◽  
Entire Cell ◽  
A Minor ◽  
Endocytic Vesicles

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.

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.

Structural heterogeneity of endocytic membranes in macrophages as revealed by the cholesterol probe, filipin

1981 ◽  
Vol 51 (1) ◽  
pp. 95-107
Author(s):  
R. Montesano ◽  
P. Vassalli ◽  
L. Orci
Keyword(s):  
Structural Heterogeneity ◽  
Cholesterol Content ◽  
Peritoneal Macrophages ◽  
Con A ◽  
Coated Pits ◽  
Mechanisms Of Formation ◽  
Smooth Membrane ◽  
Endocytic Vesicles ◽  
Cytochemical Technique ◽  
Mouse Peritoneal Macrophages

The polyene antibiotic, filipin, by specifically interacting with cholesterol, produces approximately 25-nm protuberances (filipin-sterol complexes) in freeze-fractured membranes, and the addition of filipin to aldehyde fixatives has been recently introduced as a cytochemical technique for the localization of cholesterol in cell membranes. In a previous study we showed that, in fibroblasts filipin-sterol complexes are absent from endocytic coated pits. To establish whether the absence of filipin-sterol complexes is a phenomenon restricted to coated pits or is correlated with endocytosis in general, we applied the filipin probe to cultured mouse peritoneal macrophages, in which different forms of endocytosis take place. The macrophages were incubated with bovine albumin or concanavalin A (Con A) to induce pinocytosis, and with heat-killed straphylococci or opsonized erythrocytes to induce phagocytosis, then fixed in glutaraldehyde/filipin and freeze-fractured. Filipin-sterol complexes were plentiful on the plasma membrane, on the smooth-membrane invaginations and vesicles induced by albumin, on the large endocytic vacuoles induced by Con A, and on the membrane of phagosomes but, in contrast, they were absent from coated pits and vesicles, as well as from coated segments of invagination or vesicles. These results indicate that the membranes involved in different types of endocytosis do not react in the same way with filipin and may, therefore, have a different cholesterol content. This could reflect different mechanisms of formation for the various types of endocytic vesicles.

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 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.

scholarly journals Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison.

1981 ◽  
Vol 29 (12) ◽  
pp. 1437-1441 ◽  
Author(s):  
P F Davies ◽  
L Kuczera
Keyword(s):  
Cell Surface ◽  
Vascular Endothelium ◽  
Ruthenium Red ◽  
Coated Vesicles ◽  
Membrane Glycoproteins ◽  
Coated Pits ◽  
Ruthenium Red Staining ◽  
Almost All ◽  
Endocytic Vesicles

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.

scholarly journals The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster

2005 ◽  
Vol 169 (6) ◽  
pp. 953-963 ◽  
Author(s):  
Bernhard Sommer ◽  
Adrian Oprins ◽  
Catherine Rabouille ◽  
Sean Munro
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Temperature Sensitive ◽  
Coated Pits ◽  
Vitellogenin Receptor ◽  
Golgi Membranes ◽  
Polarized Secretion ◽  
Truncation Mutant ◽  
Endocytic Vesicles ◽  
Endocytic Compartments

The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5E13) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5E13 oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless.

scholarly journals Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

1999 ◽  
Vol 10 (4) ◽  
pp. 961-974 ◽  
Author(s):  
Siv Kjersti Rodal ◽  
Grethe Skretting ◽  
Øystein Garred ◽  
Frederik Vilhardt ◽  
Bo van Deurs ◽  
...  
Keyword(s):  
Immunogold Labeling ◽  
Water Soluble ◽  
Soluble Form ◽  
Transferrin Receptors ◽  
Serum Free Medium ◽  
Free Medium ◽  
Coated Pits ◽  
Serum Free ◽  
Electron Microscopical ◽  
Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

scholarly journals Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1273-1278 ◽  
Author(s):  
D J McGookey ◽  
K Fagerberg ◽  
R G Anderson
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coated Vesicles ◽  
Low Density ◽  
Vesicle Membrane ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Transport Vesicles ◽  
Endocytic Vesicles

Filipin has been widely used as an electron microscopic probe to detect 3-beta-hydroxysterols, principally cholesterol, in cellular membranes. When it complexes with sterol, it forms globular deposits that disrupt the planar organization of the membrane. Previous studies have shown that coated pits and coated vesicles, specialized membranes involved in receptor-mediated endocytosis, do not appear to bind filipin. This has led to the suggestion that these membranes are low in cholesterol compared with the remainder of the plasma membrane. Since coated endocytic vesicles become uncoated vesicles during the transport of internalized ligands to the lysosome, we have carried out studies to determine whether or not the membranes that surround these transport vesicles are unable to bind filipin and therefore, are also low in cholesterol. Cells were incubated with ferritin-conjugated ligands that bind to low density lipoprotein (LDL) receptors in coated pits. After allowing internalization of the conjugates, we fixed the cells in either the presence or absence of filipin. This permitted us to identify all of the vesicles involved in the transport of LDL to the lysosome and to determine whether the membranes of these vesicles were able to bind filipin. We found that, coordinate with the dissociation of the clathrin coat from the endocytic vesicles, the membranes became sensitive to the formation of filipin-sterol complexes. Furthermore, all of the uncoated endocytic vesicle membranes, as well as the lysosomal membranes, bound filipin. This suggests either that coated membrane contains normal cholesterol levels, which is not easily detected with filipin, or that cholesterol rapidly moves into endocytic vesicles after the clathrin coat dissociates from the membrane.

scholarly journals A role of OCRL in clathrin-coated pit dynamics and uncoating revealed by studies of Lowe syndrome cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Ramiro Nández ◽  
Daniel M Balkin ◽  
Mirko Messa ◽  
Liang Liang ◽  
Summer Paradise ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Clinical Manifestations ◽  
Coated Vesicles ◽  
Lowe Syndrome ◽  
Syndrome Patient ◽  
Coated Pits ◽  
Intracellular Sorting ◽  
Endocytic Vesicles

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

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