scholarly journals Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.

1983 ◽  
Vol 97 (2) ◽  
pp. 508-521 ◽  
Author(s):  
C R Hopkins ◽  
I S Trowbridge
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Gold Colloid ◽  
A431 Cells ◽  
Coated Pits ◽  
Gold Complexes ◽  
Thin Sections ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Peripheral Cytoplasm

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

scholarly journals Local clustering of transferrin receptors promotes clathrin-coated pit initiation

2010 ◽  
Vol 191 (7) ◽  
pp. 1381-1393 ◽  
Author(s):  
Allen P. Liu ◽  
François Aguet ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Major Pathway ◽  
Receptor Ligand ◽  
Local Clustering ◽  
Pit Initiation

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor–ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

scholarly journals Rapid endocytosis of the transferrin receptor in the absence of bound transferrin.

1985 ◽  
Vol 100 (2) ◽  
pp. 633-637 ◽  
Author(s):  
C Watts
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Protein A ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Prolonged Incubation ◽  
Surface Receptors ◽  
Recycling Pathway

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.

scholarly journals Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells.

1986 ◽  
Vol 102 (2) ◽  
pp. 500-509 ◽  
Author(s):  
K Miller ◽  
J Beardmore ◽  
H Kanety ◽  
J Schlessinger ◽  
C R Hopkins
Keyword(s):  
Acid Phosphatase ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Epidermoid Carcinoma ◽  
Egf Receptors ◽  
A431 Cells ◽  
Thin Sections ◽  
Receptor Complexes ◽  
Epidermal Growth

We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.

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.

Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold

1987 ◽  
Vol 87 (4) ◽  
pp. 495-506
Author(s):  
J. Beardmore ◽  
K.E. Howell ◽  
K. Miller ◽  
C.R. Hopkins
Keyword(s):  
Transferrin Receptor ◽  
Colloidal Gold ◽  
A431 Cells ◽  
Receptor Complexes ◽  
Fold Enrichment ◽  
Versus Protein ◽  
Continuous Sucrose Gradient ◽  
Modification Procedure ◽  
Enzyme Markers ◽  
Human Epidermoid Carcinoma

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.

scholarly journals Endocytosis occurs independently of annexin VI in human A431 cells

1994 ◽  
Vol 124 (3) ◽  
pp. 301-306 ◽  
Author(s):  
E Smythe ◽  
PD Smith ◽  
SM Jacob ◽  
J Theobald ◽  
SE Moss
Keyword(s):  
Transferrin Receptor ◽  
Squamous Carcinoma ◽  
Cell Types ◽  
Northern Blotting ◽  
Endocytic Pathway ◽  
A431 Cells ◽  
Coated Pits ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Annexin Vi

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild-type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.

scholarly journals In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella.

1994 ◽  
Vol 125 (6) ◽  
pp. 1265-1274 ◽  
Author(s):  
C R Hopkins ◽  
A Gibson ◽  
M Shipman ◽  
D K Strickland ◽  
I S Trowbridge
Keyword(s):  
Plasma Membrane ◽  
Chick Embryo ◽  
Cell Surface ◽  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Intracellular Processing ◽  
Recycling Pathway ◽  
Embryo Fibroblasts ◽  
Migrating Cells

By following the intracellular processing of recycling transferrin receptors and the selective sorting of a-2 macroglobulin in chick embryo fibroblasts, we have shown that the concentration of 60 nm diam tubules which surrounds the centrioles represents a distal compartment on the recycling pathway. In migrating cells transferrin receptor tracers can be loaded into this compartment and then chased to the cell surface. When they emerge the recycling transferrin receptors are distributed over the surface of the leading lamella.

Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils

1989 ◽  
Vol 94 (1) ◽  
pp. 155-168
Author(s):  
A.H. Janeczek ◽  
W.A. Marasco ◽  
P.J. van Alten ◽  
R.J. Walter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Active Phase ◽  
Coated Pits ◽  
Surface Receptors ◽  
Dense Granules ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Autoradiographic Analysis ◽  
Formylpeptide Receptor

Formylpeptide chemoattractant binds specifically to leukocyte cell surface receptors and during chemotactic activation, is internalized and processed by these cells. Using electron microscope autoradiographic techniques, we have examined the binding, uptake and disposition of fNle-Leu-Phe-Nle-[125I]Tyr-Lys by rabbit peritoneal neutrophils. Cells were incubated with ligand for 15 min at 4 degrees C, rinsed and then further incubated in buffer at 4 degrees C, 15 degrees C, 24 degrees C, or 37 degrees C for 0–40 min. For all cells incubated at 4 degrees C, grains were predominantly localized on the plasma membrane. Initially, this formylpeptide was seen in small clusters or microaggregates that were not associated with coated pits. Upon further incubation at 37 degrees C for 1 min, formylpeptide clustering on the plasma membrane increased and extensive internalization of formylpeptide was observed. Endocytosis of formylpeptide-receptor complexes clearly involved uncoated membrane pits and vesicles but did not appear to involve coated pits or coated vesicles. In the following 3 min, peptide proceeded in waves through compartments composed of small uncoated endocytic vesicles, then large vesicles, and then into dense granules. After 4 min at 37 degrees C, the most active phase of intracellular processing subsided. The percentage of grains in the cytoplasm, granule and small vesicle compartments very gradually increased during the remainder of the 40 min incubation. Formylpeptide neither bound at 4 degrees C nor accumulated at 37 degrees C on the cell surface in proximity to the underlying Golgi/centrosome region of the cell. At 24 degrees C, processing was slowed but formylpeptide-receptor complexes proceeded through a similar series of compartments. The t1/2 for formylpeptide uptake at 37 degrees C was 15–20 s, whereas at 24 degrees C the t 1/2 was approximately 10 min. No uptake was observed at 15 degrees C. The distinctive characteristics of formylpeptide binding and receptor-complex uptake seen here may be essential in initiating and maintaining continued chemotactic responsiveness.

scholarly journals Transcriptional regulation by iron of the gene for the transferrin receptor.

1986 ◽  
Vol 6 (1) ◽  
pp. 236-240 ◽  
Author(s):  
K Rao ◽  
J B Harford ◽  
T Rouault ◽  
A McClelland ◽  
F H Ruddle ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
K562 Cells ◽  
Iron Chelator ◽  
Transcriptional Level ◽  
Transferrin Receptors ◽  
Intracellular Iron ◽  
Human Transferrin Receptor ◽  
Diferric Transferrin ◽  
Permeable Iron

Treatment of K562 cells with desferrioxamine, a permeable iron chelator, led to an increase in the number of transferrin receptors. Increasing intracellular iron levels by treatment of cells with either human diferric transferrin or hemin lowered the level of the transferrin receptors. By using a cDNA clone of the human transferrin receptor, we showed that the changes in the levels of the receptor by iron were accompanied by alterations in the levels of the mRNA for the receptor. The rapidity of these changes indicated that the mRNA had a very short half-life. By using an in vitro transcriptional assay with isolated nuclei, we obtained evidence that this regulation occurred at the transcriptional level.

scholarly journals Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

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