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1.
Summary The Na+/K+ ionophore monensin is known to arrest the intracellular transport of newly synthesized proteins in the Golgi complex. In the present investigation the effect of monensin on the secretion of 3H-galactose-labeled and 3H-sialic acid-labeled thyroglobulin was studied in open thyroid follicles isolated from porcine thyroid tissue.Follicles were incubated with 3H-galactose at 20° C for 1 h; at this temperature the labeled thyroglobulin remains in the labeling compartment (Ring et al. 1987a). The follicles were then chased at 37° C for 1 h in the absence or presence of 1 M monensin. Without monensin substantial amounts of labeled thyroglobulin were secreted into the medium, whereas in the presence of the ionophore secretion was inhibited by 80%. Since we have previously shown (Ring et al. 1987 b) that monensin does not inhibit secretion of thyroglobulin present on the distal side of the monensin block we conclude that galactose is incorporated into thyroglobulin on the proximal side of this block.Secretion was also measured in follicles continuously incubated with 3H-galactose for 1 h at 37° C in the absence or presence of monensin. In these experiments secretion of labeled thyroglobulin was inhibited by about 85% in the presence of monensin. Identically designed experiments with 3H-N-acetylmannosamine, a precursor of sialic acid, gave similar results, i.e., almost complete inhibition of secretion of labeled thyroglobulin in the presence of monensin. The agreement between the results of the galactose and sialic acid experiments indicates that sialic acid, like galactose, is incorporated into thyroglobulin on the proximal side of the monensin block.Considering observations made in other cell systems the present results suggest that galactosylation and sialylation of thyroglobulin are completed within the Golgi complex. 相似文献
2.
Summary The effect of energy deprivation on the intracellular transport and secretion of thyroglobulin was studied in open follicles isolated from porcine thyroids. Follicles were pulse-labeled with 3H-leucine or 3H-galactose. Labeled thyroglobulin was secreted into the incubation medium where it was isolated by means of immunoprecipitation. Secretion was followed in chase incubations under various experimental conditions using CCCP (carbonyl-cyanide-mchlorophenylhydrazone) or DNP (dinitrophenol), both uncouplers of oxidative phosphorylation, or CN–, which inhibits respiration. CCCP (1 M) was shown to inhibit exocytosis by about 80%, DNP (0.1–5 mM) by 45–85%, and CN– (0.5–1.1 mM) by 5–55%. By combining CN– with the ionophore monensin, which blocks transport through the Golgi complex but does not essentially interfere with exocytosis, evidence was obtained that CN– also inhibits transport of thyroglobulin from the Golgi cisternae to the exocytic vesicles by 40%. Electron-miroscopic autoradiography of isolated thyroid lobes from the rat also indicated that transport of 3H-leucine label into the follicle lumen is inhibited in the presence of CCCP or CN–. Intracellular ATP content was found to be about 40% of the control level in follicles incubated with CCCP (1 uM) or CN– (0.9 mM). The results show that the transport of thyroglobulin from the Golgi complex to the exocytic vesicles as well as from the exocytic vesicles into the follicle lumen is dependent upon metabolic energy. The transport blocks are probably associated with inhibited membrane fusions and fissions.Abbreviations CCCP
carbonylcyanide-m-chlorophenylhydrazone
- FCCP
carbonylcyanide-p-trifluoromethoxyphenylhydrazone
- DNP
dinitrophenol 相似文献
3.
RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS 总被引:16,自引:12,他引:4
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A. Haddad Meredith D. Smith Annette Herscovics N. J. Nadler C. P. Leblond 《The Journal of cell biology》1971,49(3):856-882
The incorporation of fucose-3H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-3H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-3H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-3H. At 3–5 min following fucose-3H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid. 相似文献
4.
Summary Exposure of isolated pancreatic islets (mouse or rat) to low temperature (2° C) evoked a threefold increase in insulin release irrespective of the glucose concentration in the incubation medium. Cold-induced release was transient and rewarming to 37° C restored the sensitivity of B-cells to glucose stimulation. In islets cooled to 2° C, exocytotic profiles could easily be detected both by thin-section and freeze-fracture electron microscopy. As revealed by the freeze-fracture technique, the number of exocytotic profiles per membrane area was increased three-to fourfold as compared to islet cells incubated at 20° C. This was paralleled by intracellular fusion of secretory vesicles. Cold-induced insulin release was not affected by theophylline, cytochalasin B, omission of extracellular Ca++ or D600. Replacement of extracellular Na+ with choline or sucrose suppressed the increase in insulin release and in frequency of exocytotic profiles recorded after exposure to 2° C. It is suggested that a redistribution of Ca++ from intracellular stores, possibly mediated by an increase in intracellular Na+, triggers exocytosis of insulin granules upon exposure to cold. 相似文献
5.
Akopova I Tatur S Grygorczyk M Luchowski R Gryczynski I Gryczynski Z Borejdo J Grygorczyk R 《Purinergic signalling》2012,8(1):59-70
Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely
understood. In alveolar A549 cells much of the experimental data is consistent with Ca2+-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways
have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy
to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine
or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent
field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence,
as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic
stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic
event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent
exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence
assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton
(nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a
major pathway of hypotonic stress-induced ATP secretion from A549 cells. 相似文献
6.
Dr. M. Nilsson R. Ekholm G. Fayet S. Maroux L. E. Ericson 《Cell and tissue research》1987,250(2):295-303
Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council 相似文献
7.
Lisette Waits Stephanie Dunkle F. E. Wilkinson P. Moreau Keri Safranski T. Reust Dorothy M. Morré D. J. Morré 《Protoplasma》1990,154(1):8-15
Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [14C]acetate. Time- and temperature-dependent transfer of [14C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [14C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [3H]squalene which was converted into sterol or with [14C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS
dictyosome-like structure(s)
- PBS
phosphatebuffered saline
- HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- BSA
bovine serum albumin 相似文献
8.
《The Journal of cell biology》1983,97(3):607-617
Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species. 相似文献
9.
The effect of monensin on the secretion of thyroglobulin was studied in open follicles isolated from pig thyroid tissue; in this system, thyroglobulin is secreted into the incubation medium. When monensin was present during a 4-h chase incubation after pulse-labelling with 3H-leucine, the secretion of labelled thyroglobulin was reduced by about 85%; in electron-microscopic autoradiographs of rat thyroid lobes labelled and chase-incubated under similar conditions the relative number of grains over follicle lumina was strongly reduced when monensin was present during the chase. These observations are in agreement with the consensus that monensin arrests transport of secretory proteins in the Golgi complex. In other experiments, pulse-labelled follicles were chase-incubated for 1.5 h whereby labelled thyroglobulin was transported from the RER to exocytic vesicles. Monensin present during a subsequent chase of 0.5 h caused only a moderate decrease of labelled thyroglobulin secretion. TSH present during the second chase-stimulated secretion in both control and monensin-exposed follicles. TSH also caused a drastic reduction of exocytic vesicles in rat thyroid lobes, and the number of vesicles remaining in the cells was the same in controls and lobes exposed to the ionophore. The observations are interpreted to show that monensin does not inhibit the basal or TSH-stimulated transport of thyroglobulin from the site of monensin-induced arrest in the Golgi complex to the apical cell surface or the exocytosis of thyroglobulin. 相似文献
10.
Cheryl Peters Franz Edward M. Croze D.James Morr Gerhard Schreiber 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,678(3):395-402
Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [3H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed. 相似文献
11.
Summary Liver slices incubated at 1 °C underwent swelling of both cellular and intercellular compartments, as judged by electronmicroscopy. The ultrastructure showed marked changes, including disorganization of the cytocavitary network and plasma membrane and alterations of mitochondria. Restoration of metabolically favorable conditions (oxygenated medium at 38 °C) caused a nearly complete recovery of ultrastructure closely associated with extrusion of water; measurements of inulin space and electronmicroscopy both indicate a recovery of cell volume, with intercellular spaces remaining somewhat expended. The fluid lost was a roughly isotonic solution of Na+ and Cl–, while K+ was reaccumulated in exchange for Na+. Cyanide prevented recovery. Ouabain and oligomycin each partially prevented fluid extrusion, but had little effect on ultrastructural recovery except to induce intracellular vesicles containing particles of thorium dioxide derived from sinusoidal spaces. The vesicles were, however, markedly different in form with each inhibitor. There are, thus ouabain-sensitive and-insensitive components of volume regulation; the former appears to depend on the coupled transport of Na+ and K+ and the latter, we suggest, on a secretion of Na+ and Cl– into vesicles which release their contents into the bile canaliculi by an oligomycin-sensitive mechanism. Mitochondria showed conformational changes between orthodox and condensed forms, but these could not be directly related to tissue energy states; the numbers of mitochondrial dense granules bore a closer relation to tissue ATP. 相似文献
12.
The size and the bilayer thickness of detergent-resistant membranes isolated from rat brain neuronal membranes using Triton X-100 or Brij 96 in buffers with or without the cations, K+/Mg2+ at a temperature of either 4 °C or 37 °C were determined by dynamic light scattering and small-angle neutron scattering. Regardless of the precise conditions used, isolated membrane preparations consisted of vesicles of ∼ 100 to 200 nm diameter as determined by dynamic light scattering methods, equating to an area of the lipid based membrane microdomain size of 200 to 400 nm diameter. By means of small angle neutron scattering it was established that the average thickness of the bilayers of the complete population of detergent-resistant membranes was similar to that of the parental membrane at between 4.6 and 5.0 nm. Detergent-resistant membranes prepared using buffers containing K+/Mg2+ uniquely formed unilamellar vesicles while membranes prepared in the absence of K+/Mg2+ formed a mixture of uni- and oligolamellar structures indicating that the arrangement of the membrane differs from that observed in the presence of cations. Furthermore, the detergent-resistant membranes prepared at 37 °C were slightly thicker than those prepared at 4 °C, consistent with the presence of a greater proportion of lipids with longer, more saturated fatty acid chains associated with the Lo (liquid-ordered) phase. It was concluded that the preparation of detergent-resistant membranes at 37 °C using buffer containing cations abundant in the cytoplasm might more accurately reflect the composition of lipid rafts present in the plasma membrane under physiological conditions. 相似文献
13.
Judy M. Strum Janice Wicken John R. Stanbury Morris J. Karnovsky 《The Journal of cell biology》1971,51(1):162-175
Iodination within the thyroid follicle is intimately associated with a thyroid peroxidase. In order to locate the in vivo site of iodination, the initial cytochemical appearance of this enzyme has been determined in fetal rat thyroid and its presence correlated with the onset of iodinated thyroglobulin synthesis. Peroxidase first appears in follicular cells during the 18th day of gestation. It is seen first in the perinuclear cisternae, the cisternae of the endoplasmic reticulum, and within the inner few Golgi lamellae. These organelles presumably represent sites of peroxidase synthesis. During the 19th and 20th days of gestation, there is a tremendous increase in peroxidase activity. In addition to the stained sites described, there are now many peroxidase-positive apical vesicles in the follicular cells. Newly forming follicles stain most conspicuously for peroxidase, the reaction product being heavily concentrated at the external surfaces of apical microvilli and in the adjacent colloid. Iodinated thyroglobulin becomes biochemically detectable in thyroids during the 19th day of gestation and increases greatly during the 20th day. The parallel rise in peroxidase staining that just precedes, and overlaps, the rise in iodinated thyroglobulin, suggests that apical vesicles and the apical cell membrane are the major sites of iodination within the thyroid follicle. 相似文献
14.
Experiments have been carried out on Chinese hamster fibroblasts. Cultures at the log-stage of growth were incubated at 15 or 25° C for 24 hrs. In two groups of experiments the cells were labeled with H3-TdR for 6 hrs at the respective temperature, washed and further incubated at 37° C. In each group of experiments cultures labeled with H3-TdR at 37° C for 20 min were used as a control. It was found: 1. the delay in the onset of cell passage through the mitotic cycle at 37° in cultures exposed to 15 or 25° C was about equal to 1,5-1 hr resp. and cells proceeded through the life cycle without blockages at any phase of the cycle; 2. the patterns of chromosome reproduction during the second half of the S-phase were the same after labeling at 15, 25 and 37° C. — In the third group the cells were labelled with HP-TdR for 10–60 min and 6 hrs resp. at 25° C. The patterns of reproduction of chromosome pairs 1–4 and small metacentrics were found to be the same in cells labeled briefly and those labeled for 6 hrs. After brief labeling asynchronous reduplication of different segments in many chromosomes became evident. It was masked because of heavy labeling after 6 hrs treatment. 相似文献
15.
B Rousset S Selmi C Alquier P Bourgeat B Orelle C Audebet R Rabilloud F Bernier-Valentin Y Munari-Silem 《Biochimie》1989,71(2):247-262
Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. In conclusion: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
16.
D. J. Morré C. Penel Dorothy M. Morré Anna S. Sandelius P. Moreau B. Andersson 《Protoplasma》1991,160(2-3):49-64
Summary The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [14C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step. 相似文献
17.
Summary This study investigates by electron microscopy the transformational process of the endosomal compartment of the Drosophila nephrocyte, the garland cell, which occurs during endocytotic processing of internalized material. The endosomal compartment of the garland cell consists of a prominent tubular/vacuolar complex in the cortical cytoplasm. When internalization of coated pits is blocked at 29°C using the endocytosis mutant, shibire
ts, the tubules gradually disappear after 7 min at 29°C. By 12 min at 29°C, the vauoles also disappear. Thus, the endosomal compartment appears to constantly undergo a transformational process that necessitates continuous replenishment by coated vesicles. The data suggest that the tubular component of the endosomal compartment gradually transforms into vacuoles by the expansion of the tubular membrane. The vacuoles then transform by invaginating into themselves, creating flattened cisternae. The electron-lucent substance in the lumina of the vacuoles appears to be extruded into the cytoplasm through the invaginating membrane. No shuttle vehicles such as vesicles or tubules could be identified that might have been involved in the transporting of endocytosed materials and membrane from the endosomal compartment to lysosomes or back to the plasma membrane. 相似文献
18.
《Biology of the cell / under the auspices of the European Cell Biology Organization》1990,68(1-3):183-193
This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations. 相似文献
19.
COLCHICINE-INDUCED INHIBITION OF LIPOPROTEIN AND PROTEIN SECRETION INTO THE SERUM AND LACK OF INTERFERENCE WITH SECRETION OF BILIARY PHOSPHOLIPIDS AND CHOLESTEROL BY RAT LIVER IN VIVO 总被引:18,自引:6,他引:12
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Rats were injected with colchicine and the secretion of triglycerides into the serum was studied for 90 min after injection of [14C]palmitic acid and Triton WR 1339. The release of labeled and chemically determined triglyceride was reduced to about 20–30% of control values. The effect of colchicine on serum triglyceride levels was not dependent on the presence of Triton and was similar in males and females and in fed and fasted rats. The effect was dose dependent and was reversible 6–7 h after injection of 0.05 mg/100 g body weight. Colchicine inhibited also the release of labeled proteins into the serum but did not affect the amount of [3H]leucine incorporated into liver proteins. Within 4 h of colchicine treatment there was an 80% fall in serum very low density lipoproteins (VLDL), a 30% fall in serum high density lipoproteins (HDL), and no change in the d > 1.21 protein level, but reduction in the appearance of labeled proteins was encountered in all serum fractions. Colchicine had no effect on the rate of bile flow and on the secretion of phospholipids and cholesterol into the bile. In the hepatocyte there was accumulation of Golgi-derived secretory vesicles, containing nascent VLDL particles; these vesicles were seen also in the vicinity of the sinusoidal cell surface, but the space of Disse contained few or no VLDL particles. There was an apparent reduction in microtubules and some increase in microfilaments. It is suggested that microtubules affect the secretion of lipoproteins and proteins into the serum by maintaining the organization of the plasma membrane required for its fusion with secretory vesicles. The lack of effect of colchicine on biliary lipid secretion indicates that the latter is not dependent on vesicular transport. 相似文献
20.
Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface.
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The route of transport of Semliki Forest virus (SFV) membrane glycoproteins to the plasma membrane was studied using immunoperoxidase electron microscopy. SFV glycoproteins were localized in cultured BHK-21 fibroblasts infected with a temperature-sensitive mutant ts-1 of SFV, which shows a temperature-dependent, reversible defect in the transport of membrane glycoproteins to the cell surface. At 39 degrees C (restrictive temperature) the viral proteins were retained in the endoplasmic reticulum and the nuclear membrane. After shift of the infected cultures to 28 degrees C (permissive temperature) the proteins were synchronously transported to the Golgi complex. In the Golgi complex the labeled proteins were first (at 2.5 min) detected in large Golgi-associated vacuoles (GAV). Subsequently, i.e., at 5-30 min, the viral glycoproteins appeared in the cisternal stack: at 5 min the label was found in one or two of the proximal cisternae whereas at 15 or 30 min also the more distal cisternae were partially or uniformly labeled. At all time points examined after the temperature-shift, peroxidase label was found in 50 nm vesicles which were frequently coated. At 30 min, in addition to the 50 nm vesicles, larger 80 nm vesicles, which often had a cytoplasmic coat were labeled in the Golgi region. These results identify two major size classes of both coated and smooth vesicles which appear to function in the transport of the viral membrane proteins from the endoplasmic reticulum via distinct GAV and the stacked Golgi cisternae to the plasma membrane. 相似文献