Quantitative electron microscopic autoradiographic studies on internalization of 125I-labelled epidermal growth factor in term human placenta

1986 ◽  
Vol 84 (1) ◽  
pp. 41-52
Author(s):  
N. Chegini ◽  
C.V. Rao
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Placental Tissue ◽  
Electron Microscopic ◽  
Membrane Area ◽  
Capillary Endothelial Cells ◽  
Intracellular Organelles ◽  
Epidermal Growth ◽  
Silver Grains

The electron microscopic autoradiographic studies described here revealed the presence of specific silver grains over nuclei, lysosomal vesicles, rough endoplasmic reticulum and Golgi apparatus after incubation of placental tissue for 2 h at 38 degrees C with 1 nM-[125I]EGF. Three-step mask analysis, which corrects for radiation spread, showed that the relative grain density was the highest in nuclei, followed by lysosomal vesicles, then Golgi and rough endoplasmic reticulum, equally. The nuclear grain density, however, was lower than that in microvillus plasma membranes. There were very few grains in basolateral plasma membranes, none in the basement membrane area and a considerable number in capillary endothelial cells. The present results demonstrating the association of internalized [125I]EGF with a variety of intracellular organelles raise the possibility of EGF acting on the intracellular sites in addition to cell surface sites.

The presence of epidermal growth factor binding sites in the intracellular organelles of term human placenta

1986 ◽  
Vol 84 (1) ◽  
pp. 19-40
Author(s):  
N. Ramani ◽  
N. Chegini ◽  
C.V. Rao ◽  
P.G. Woost ◽  
G.S. Schultz
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Rough Endoplasmic Reticulum ◽  
Human Placenta ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Intracellular Organelles ◽  
Epidermal Growth

Highly purified lysosomes, rough and smooth endoplasmic reticulum, and Golgi apparatus, as well as microvillus plasma membranes, bound 125I-labelled epidermal growth factor ([125I]EGF) with similar affinity. Scatchard plots for all the organelles were curvilinear. The apparent number of available binding sites per mg protein of intracellular organelles was 27–71% of that found in microvillus plasma membranes. The bound and free [125I]EGF were not degraded by any of the organelles. Binding and dissociation of [125I]EGF in all organelles were dependent on the time and temperature of incubation. The specificity of [125I]EGF binding was similar in all organelles. The optimal pH for binding to lysosomes was 6.0, in contrast to 7.0 for all the other organelles. Exposure of different organelles to enzymes and protein-modifying reagents resulted in numerous binding differences between the intracellular organelles and microvillus plasma membranes. Covalent affinity labelling with [125I]EGF revealed two major proteins of 155 and 140(X10(3)) Mr in all the organelles. The 155 X 10(3) Mr protein was labelled predominantly in all organelles except rough endoplasmic reticulum, where both proteins were equally labelled. Addition of proteolytic inhibitors during isolation of organelles did not alter the pattern of [125I]EGF-labelled binding proteins found in the organelles. EGF also stimulated phosphorylation of the 155 and 140(X10(3)) Mr proteins in all the organelles. The 155 X 10(3) Mr protein was phosphorylated more than the 140 X 10(3) Mr protein in microvillus plasma membranes and smooth endoplasmic reticulum, whereas the 140 X 10(3) Mr protein was phosphorylated more than the 155 X 10(3) Mr protein in lysosomes and both proteins were equally phosphorylated in rough endoplasmic reticulum. Several organelles also contained minor [125I]EGF-binding proteins that did not show phosphorylation response and proteins that showed phosphorylation response but did not bind [125I]EGF. Thus, the present study demonstrates by a number of different criteria, that several intracellular organelles of term human placenta also contain EGF-binding and kinase activities.

Altered labelling of the cell surface and intracellular organelles with [3H]mannose in enucleated amoebae

1984 ◽  
Vol 68 (1) ◽  
pp. 83-94
Author(s):  
C.J. Flickinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Cell Organelles ◽  
Intracellular Organelles ◽  
And Control ◽  
Kinetics Of ◽  
Heavy Labelling

The production, transport, and disposition of material labelled with [3H]mannose were studied in microsurgically enucleated and control amoebae. Cells were injected with the precursor and samples were prepared for electron-microscope radioautography at intervals, up to 24 h later. Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus at early intervals after injection. Later, labelling of groups of small vesicles increased, and the percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and cell surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains over the rough endoplasmic reticulum varied less with time in enucleated than in control cells. Second, the distribution of radioactivity was altered. A greater percentage of grains was associated with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of surface material was slower in the absence of the nucleus. It is suggested that this is related to the decreased motility of enucleate cells.

scholarly journals The effect of epidermal growth factor (EGF) on the differentiation of the rough endoplasmic reticulum in fetal mouse small intestine in organ culture.

1981 ◽  
Vol 29 (6) ◽  
pp. 765-770 ◽  
Author(s):  
J F Beaulieu ◽  
R Calvert
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Organ Culture ◽  
Rough Endoplasmic Reticulum ◽  
Fetal Mouse ◽  
Absorptive Cells ◽  
Mouse Small Intestine ◽  
Epidermal Growth ◽  
Mouse Fetuses

The differentiation of the rough endoplasmic reticulum (RER) of mouse duodenal absorptive cells located at the tip of the villi at 17 days of gestation was compared to that of absorptive cells in duodenal explants of 15-day-old mouse fetuses cultured for 72 hr 1) with Trowell T8 medium (without insulin) alone or supplemented 2) with epidermal growth factor (EGF; 100 ng/ml) or 3) with 25% bovine amniotic fluid (BAF). Glucose-6-phosphatase activity (G6Pase) was localized cytochemically to ensure a better identification of the RER. The intersections of a double lattice falling over and outside the RER were counted and the percentage of intersections over the RER was estimated. With this method, the extent of the RER is not statistically different when the absorptive cells in utero are compared to those of explants cultured with EGF. However, the extent of the RER in the absorptive cells cultured with Trowell T8 medium alone or supplemented with BAF is 50% lower than in the former two groups. It is concluded that EGF promotes the maturation of duodenal absorptive cells in organ culture.

scholarly journals Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

1975 ◽  
Vol 23 (6) ◽  
pp. 439-451 ◽  
Author(s):  
H Miyayama ◽  
R Solomon ◽  
M Sasaki ◽  
C W Lin ◽  
W H Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Epithelial Cells ◽  
Normal Mouse ◽  
Plasma Membranes ◽  
Mouse Kidney ◽  
Lead Salt ◽  
Acetate Buffer ◽  
Electron Microscopic

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.

scholarly journals Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle.

1995 ◽  
Vol 129 (2) ◽  
pp. 459-471 ◽  
Author(s):  
N Benlimame ◽  
D Simard ◽  
I R Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Mdck Cells ◽  
Electron Microscopic ◽  
Autocrine Motility Factor ◽  
Lysosomal Fraction ◽  
Motility Factor ◽  
Variable Diameter ◽  
Triton X

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule-associated tubular organelles.

scholarly journals Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

1984 ◽  
Vol 219 (2) ◽  
pp. 679-685 ◽  
Author(s):  
A E Richardson ◽  
R L Dormer
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Ionophore A23187 ◽  
Uptake System ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Pancreatic Acini ◽  
Dense Fraction

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction (SF2) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction (SF1) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum (SF2) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.

scholarly journals Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

1977 ◽  
Vol 74 (3) ◽  
pp. 992-1015 ◽  
Author(s):  
J Paiement ◽  
CP Leblond
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Follicular Cells ◽  
Lysosomal Degradation ◽  
High Concentration ◽  
Different Types ◽  
Apical Vesicles ◽  
Rat Thyroid ◽  
Silver Grains ◽  
Distinct Distribution

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.

scholarly journals IN VIVO INCORPORATION OF 3H-GALACTOSE BY THYROID FOLLICULAR CELLS OF THE RAT, AS SHOWN BY ELECTRON MICROSCOPIC RADIOAUTOGRAPHY

1972 ◽  
Vol 20 (3) ◽  
pp. 220-224 ◽  
Author(s):  
A. HADDAD
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Side Chains ◽  
Follicular Cells ◽  
Electron Microscopic ◽  
Thyroid Follicular Cells ◽  
Apical Vesicles ◽  
Electron Microscopic Radioautography

Radioactive galactose was injected intravenously into rats and localized in thyroid follicular cells by electron microscopic radioautography at intervals ranging from 2.5 to 30 min after injection. The galactose label was mostly present in the Golgi apparatus at 2.5 min, with some of it in the adjacent rough endoplasmic reticulum. By 30 min, the label was found in apical vesicles and colloid. It was concluded that galactose is added to the carbohydrate side chains of incomplete thyroglobulin molecules during their travel through the cisternae of the endoplasmic reticulum into the Golgi apparatus; the uptake begins as this organelle is approached, but predominates within it. The thyroglobulin molecule which has thus been labeled is transported by the apical vesicles to the colloid.

scholarly journals Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

1996 ◽  
Vol 16 (5) ◽  
pp. 1946-1954 ◽  
Author(s):  
L V Lotti ◽  
L Lanfrancone ◽  
E Migliaccio ◽  
C Zompetta ◽  
G Pelicci ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Rough Endoplasmic Reticulum ◽  
Receptor Tyrosine Kinase ◽  
Receptor Activation ◽  
Kinase Activation ◽  
Epidermal Growth

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.

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