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Biomedical subjects

C R Hopkins

Publications and source records attributed to C R Hopkins.

At least 55 records · Page 3Linked to original sources
PubMed1986

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

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.

Antibodies, Monoclonal↗
PubMed1985

The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells.

Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin receptor, the distribution of transferrin receptors on the surfaces of human epidermoid carcinoma A431 cells has been mapped at high resolution. On prefixed cells and cells incubated at 5 degrees C, the receptors are predominantly within and around clathrin-coated microdomains near the free cell margin. By preincubating the cells with saturating concentrations of free antibody at 5 degrees C and warming them to 37 degrees C in the presence of the gold complexes, the appearance of new receptors in the membrane has been followed. The majority first appear near the free cell margin and then move centripetally. At first, they are monodisperse, but as they move toward the site of internalization they form loose aggregates. Within the immediate vicinity of the clathrin-coated microdomains the migrating receptors form closely packed, ordered aggregates. These observations indicate recycling transferrin receptors move to their site of internalization without cross-linking.

Antibodies, Monoclonal↗
PubMed1985

Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake.

The distribution of cell surface receptors for transferrin-iron and epidermal growth factor (EGF) on the surface of cultured epithelioid (A431) cells has been identified by immunocytochemical electron microscopy. The patterns of movement displayed by these two receptor populations as they transfer to their sites of internalization on the cell surface are different. The movement of recycling transferrin receptors over the surface is ligand-independent whereas EGF receptors are more stable residents and remain monodisperse until they bind ligand. Prior to uptake transferrin receptors cluster, predominantly within existing clathrin-coated pits while the aggregates formed by EGF ligand-receptor complexes induce new membrane invaginations. These results are discussed in relation to receptor populations concerned with constitutive, high capacity uptake processes and receptors involved in signal transduction.

Antibodies↗
PubMed1983

Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells.

Using transferrin peroxidase (Tfn-HRP) and a transferrin receptor-specific antibody complexed to colloidal gold (ATR) we have identified the intracellular compartments concerned with processing internalized transferrin-receptor complexes. We have identified major membrane-bound systems in the peripheral cytoplasm and in the juxtanuclear area, from which components of these complexes are returned to the cell surface. Time course studies indicate that the peripheral system is concerned with a "short circuit," recycling ligand and receptor complexes back to the upper surface of the cell. The juxtanuclear compartment is part of a longer circuit that routes some receptors to the basal surface and others, along with ligand, to the lysosome.

Carcinoma, Squamous Cell↗
PubMed1983

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

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.

Biological Transport, Active↗
PubMed1982

Localization of fibronectin, laminin-entactin, and entactin in Reichert's membrane by immunoelectron microscopy.

Immunoelectron microscopy using protein A-colloidal gold complexes of different sizes was used to study the relative distribution of extracellular matrix glycoproteins within Reichert's membrane (RM) of 13.5-day mouse embryos. Labelling for fibronectin was distributed asymmetrically; the highest concentration occurring in the outermost layer adjacent to the trophoblast cells and negligible labelling in the inner matrix, beneath the parietal endoderm cells. Within the main body of the membrane, fibronectin was concentrated in discrete electron-opaque deposits. Antibodies raised against the native complex between laminin and entactin , and against entactin alone labelled the RM more uniformly.

Animals↗
PubMed1982

Studies on the kinetics of dopamine-regulated prolactin secretion.

The regulation of prolactin (PRL) release by dopamine has been studied using short-term dissociated rat pituitary cell cultures. Concentrations of dopamine between 10(-12) and 10(-10) M stimulate PRL release, whereas concentrations between 10(-9) and 10(-7) M inhibit. By studying the kinetics of PRL release it was found that (1) the rate of PRL release increases only after a post-stimulus delay of 20 min, and (2) the response is phasic, stimulatory intervals being separated by intervals in which the rate of PRL release falls below the level of basal release. Inhibition of PRL release by dopamine occurs within 10 min after adding dopamine and is maintained tonically for a least 2 h. Cells treated with non-specific secretogogue (57 mM K+) during the inhibitory phases of dopamine-regulated release, are stimulated to secrete significant amounts of hormone.

Animals↗
PubMed1981

Regulation of gonadotropin secretion in the anterior pituitary.

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.

Animals↗
PubMed1981

Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes.

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.

Animals↗
PubMed1981

Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A-coated gold in conjunction with biotin and fc-bearing ligands.

Procedures for preparing gold colloid particles stabilized with either avidin or protein A are described. Methods of using these general utility tracers for localizing biotinylated and fc bearing immunoglobulins are outlined and, as examples of the way in which these methods can be applied, procedures for identifying epidermal growth factor receptors and surface fibronectin on ovarian granulosa cells are described.

Animals↗
PubMed1981

Early events following the binding of epidermal growth factor to surface receptors on ovarian granulosa cells.

Receptors for epidermal growth factor (EGF) have been localized on the surface of ovarian granulosa cells using a colloidal gold avidin complex in conjunction with biotinylated EGF. On cells incubated at low temperature the receptors are predominantly monodisperse and have a random distribution. When cells are warmed to 37 degrees C the receptors aggregate into clusters.

Animals↗
PubMed1979

Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections.

ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cells was identified as containing numerous round or ovoid 170--300 nm secretory granules.

Adrenocorticotropic Hormone↗