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1.
Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 and gp110. 总被引:17,自引:17,他引:0
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The processing and intracellular localization of the two predominant Epstein-Barr virus glycoproteins expressed in late lytic infection were investigated. Immune light or electron microscopy of frozen fixed sections revealed that gp110 colocalized to the endoplasmic reticulum and to the nuclear membrane with the endoplasmic reticulum-resident protein, heavy-chain-binding protein (BiP), while gp350/220 accumulated in low abundance in the endoplasmic reticulum and was present in higher abundance in cytoplasmic structures presumed to be Golgi and in plasma membranes. Consistent with endoplasmic reticulum and nuclear membrane localization, the bulk of gp110 was sensitive to endoglycosidase H, indicating high-mannose, pre-Golgi, N-linked glycosylation; while consistent with Golgi and plasma membrane localization, gp350/220 was mostly resistant to endoglycosidase H because of complex N- and O-linked glycosylation. gp350/220 was as abundant in extracellular enveloped virus as in the plasma membrane but was much less abundant or undetected in internal cytoplasmic or nuclear membranes. In contrast, gp110-specific antibodies did not label extracellular or intracellular virus. These data indicate that the major antigenic components of gp110 are not incorporated into or are occluded in virions and that gp350/220 is added to virus in cytoplasmic transit through a process of de-envelopment and re-envelopment at the plasma membrane or at post-Golgi vesicles. Consistent with cytoplasmic de-envelopment and re-envelopment at the plasma membrane was the finding of some free nucleocapsids in the cytoplasm of cells with intact nuclear membranes and nucleocapsids which appeared to bud through the plasma membrane. 相似文献
2.
Association of gelsolin with actin filaments and cell membranes of macrophages and platelets 总被引:20,自引:13,他引:7
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Recent evidence that polyphosphoinositides regulate the function of the actin-modulating protein gelsolin in vitro raises the possibility that gelsolin interacts with cell membranes. This paper reports ultrastructural immunohistochemical data revealing that gelsolin molecules localize with plasma and intracellular membranes, including rough endoplasmic reticulum, cortical vesicles and mitochondria of macrophages, and blood platelets. Anti-gelsolin gold also labeled the surface and interior of secondary lysosomes presumably representing plasma gelsolin ingested by these cells from the lung surface by endocytosis. Gelsolin molecules, visualized with colloidal gold in replicas of the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed and rapidly frozen and freeze-dried macrophages, associated with the ends of short actin filaments sitting on the cytoplasmic membrane surface. A generalized distribution of gelsolin molecules in thin sections of resting platelets rapidly became peripheral, and plasmalemma association increased following thrombin stimulation. At later times the distribution reverted to the cytoplasmic distribution of resting cells. These findings provide the first evidence for gelsolin binding to actin filament ends in cells and indicate that gelsolin functions in both cytoplasmic and membrane domains. 相似文献
3.
Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells 总被引:36,自引:27,他引:9
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An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed. 相似文献
4.
Jon Green Gareth Griffiths Daniel Louvard Paul Quinn Graham Warren 《Journal of molecular biology》1981,152(4):663-698
BHK-21 cells, infected with Semliki Forest virus, were treated with cycloheximide to stop further synthesis but not intracellular transport of the viral membrane proteins. These proteins were then localized in thin, frozen sections using specific antibodies labelled indirectly with ferritin or gold. Quantitation of the labelling on micrographs showed the movement of spike proteins from the rough endoplasmic reticulum and through the Golgi stacks. The spike proteins spent about 15 minutes in each of these intracellular organelles and their final destination was the plasma membrane. Parallel biochemical studies showed that most of the simple oligosaccharides on the viral spike proteins were modified to the complex form at the same time as these membrane proteins were passing through the Golgi stacks. Cell fractionation studies revealed the same pattern; the proteins passed from the rough endoplasmic reticulum to the plasma membrane via a vesicle fraction isolated according to its content of galactosyl transferase. Independent evidence that this fraction was derived at least in part from the Golgi complex in BHK cells was obtained by showing that it reacted specifically with an antibody raised to rat liver Golgi membranes. 相似文献
5.
Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the amyloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of membrane components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the membrane is a glycoprotein tightly inserted in other components of the compartment.Abbreviations Con A
Concanavalin A
- ER
endoplasmic reticulum
- FITC
fluorescein isothiocyanate
- NP 40
nonidet P40 相似文献
6.
The first mandibular molars of the Swiss albino mice, 1 through 4 days of age, were fixed in glutaraldehyde or Karnovsky's fixative. The tissues were postfixed in OSO4, dehydrated and embedded in Epon. The prepolarizing, polarizing and secretory odontoblasts were described. The prepolarizing cells, located in the vicinity of the cervical loop, were mesenchymal-like in morphology. The cells of the polarizing stage possessed organelles indicative of protein synthesis. The nucleus was located proximally. Aperiodic fibers were evident in the wide basement membrane. The secretory odontoblasts were long, slender, polarized cells closely adjoining one another. Each odontoblast possessed six morphologically discernible regions: (1) an infranuclear region, limited in size and containing few cellular organelles; (2) a nuclear region, housing the oval nucleus and a few associated lamellae of rough endoplasmic reticulum as well as a limited number of mitochondria; (3) a supranuclear rough endoplasmic reticulum region, possessing an abundance of these organelles as well as some mitochondria and secretory vesicles; (4) a Golgi region, occupying the middle third of the cell, housing the elements of an extensive Golgi apparatus which was surrounded by peripherally located profiles of rough endoplasmic reticulum; additionally, this region contained smooth endoplasmic reticulum, mitochondria, numerous secretory granules and vesicles and occasional intracellular collagen fibers; (5) an apical rough endoplasmic reticulum region, containing a rough endoplasmic reticulum component that was less extensive than its supranuclear counterpart; in addition, this region was the one richest in mitochondria and contained a plethora of secretory vesicles and granules; (6) the odontoblastic process, a region mostly void of organelles, containing various secretory products, some of which appeared to be in the process of being released extracellularly into the surrounding dentin matrix. 相似文献
7.
Effect of gibberellic Acid and actinomycin d on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells 总被引:6,自引:2,他引:4
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Analysis of structural changes in barley aleurone cells during germination or following incubation of isolated layers in gibberellic acid with or without actinomycin D revealed extensive development of rough endoplasmic reticulum. Following the assembly of stacked rough endoplasmic reticulum, vesiculation occurred mainly in basal regions of the cell, resulting in a polar distribution of rough endoplasmic reticulum vesicles. It is postulated that these vesicles are involved in protein secretion, because smooth vesicles, derived from the rough endoplasmic reticulum, apparently become appressed to the plasma membrane. The increased α-amylase in the ambient medium and in cell homogenates correlated directly with formation and subsequent vesiculation of the rough endoplasmic reticulum. Furthermore, when cells were treated with actinomycin D and gibberellic acid, α-amylase synthesis was inhibited by 45% and secretion by 63%. These cells were characterized cytologically by large areas of disarrayed segments of fragmented rough endoplasmic reticulum, corresponding to a high intracellular level of α-amylase. In addition, small lipid bodies common to the segmented regions of rough endoplasmic reticulum were surrounded by fine fibrous material, short segments of rough endoplasmic reticulum, and free ribosomes, suggesting that actinomycin D had interfered with development and organization of rough endoplasmic reticulum. 相似文献
8.
Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver. 总被引:6,自引:0,他引:6
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Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled. 相似文献
9.
《The Journal of cell biology》1982,92(1):92-107
Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue. There were may unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles. These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane. The anit-RER antibodies labeled this structure alone at the light and electron microscopic levels. They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000. Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental condition . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation. 相似文献
10.
F Capron B Coltoff-Schiller A B Johnson G M Fleischner S Goldfischer 《The journal of histochemistry and cytochemistry》1979,27(5):961-966
Ligandin (glutathione-s-transferase) and Z protein are soluble hepatocellular proteins that are involved in the transfer of organic ions, including bilirubin and some hormones and carcinogens from the plasma to the liver. The intracellular distribution of ligandin and Z protein was studied by applying the peroxidase-antiperoxidase procedure of L. A. Sternberger (Immunocytochemistry, Prentice Hall Inc., 1974) to paraffin sections and free-floating 10-micrometers frozen sections that were processed for both light and electron microscopy. Ligandin and Z protein were localized to the cytosol of hepatocytes in association with smooth endoplasmic reticulum (SER), but no reaction product was present between cisternae of rough endoplasmic reticulum. Penetration of reagents was enhanced in 10-micrometers frozen sections and the preservation of subcellular structures was equivalent to thicker, unfrozen sections. 相似文献
11.
Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicle-associated nucleation. 相似文献
12.
Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Br?kelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Summary Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization. 相似文献
14.
Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization. 相似文献
15.
To investigate the impaired cell surface expression of human major histocompatibility antigen (HLA) in transfected L cells, we examined their intracellular localization by immunocytochemistry. HLA class II molecules produced in transfected L cells were mainly detected in the intracellular vesicles and in the nuclear envelope as granular precipitates. The results suggest that the intracellular transport of the newly synthesized molecules in transfected L cells is impaired at some point along the pathway from the rough endoplasmic reticulum (RER) to the medial-, trans-Golgi apparatus. 相似文献
16.
Summary Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicleassociated nucleation. 相似文献
17.
Synopsis The effects of starvation on the intracellular distribution of acid glycerophosphate phosphatase have been studied in the cells of the proximal convoluted tubule of mouse kidney. Severe cytological and cytochemical changes begin in the cell two hours after withdrawal of food. They consist primarily in the appearance of numerous acid phosphatase-positive cytoplasmic vesicles arising from a hypertrophy of the rough endoplasmic reticulum. This is followed by the appearance of numerous acid phosphatase-positive Golgi zones and by degenerative changes of the mitochondria. After eighteen hours the cells also contain a large number of droplets of nonosmiophilic material, probably neutral fats, sited mainly in the area of the basement membrane, some cytosegresomes and several residual bodies sited below the brush border in the apical region of the cells. Since there appears to be a lack of correlation between the number and time of appearance of the cytosegresomes, the time of appearance and intensity of the reaction for nonlysosomal acid phosphatase, and the entity of cellular damage, this has led the authors to suggest the possibility that, beside the lysosomal autophagy described by other workers, there may occur in these experimental circumstances also a direct digestion of subcellular organelles by the acid phosphatasepositive vesicles of the rough endoplasmic reticulum, and that this mechanism may explain the formation of lipid droplets. 相似文献
18.
Subcellular localization of B apoprotein of plasma lipoproteins in rat liver 总被引:23,自引:9,他引:14
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Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte. 相似文献
19.
Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles 总被引:29,自引:15,他引:14
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《The Journal of cell biology》1983,97(6):1815-1822
Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8- nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae. 相似文献
20.
M. A. Epstein 《The Journal of cell biology》1957,3(6):851-858