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G E Palade

Publications and source records attributed to G E Palade.

At least 37 records · Page 2Linked to original sources
PubMed1995

Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor.

The vascular endothelial growth factor (VEGF) was originally described as vascular permeability factor due to its ability to increase microvascular permeability to plasma proteins. However, the vessel types (arteriolar, venular, and capillary) affected by VEGF and the modification of endothelial morphology in response to increased permeability induced by VEGF in vivo have not been precisely documented. By topical application or intradermal injection of recombinant human VEGF-165 we find that VEGF increases the permeability of postcapillary venules as well as muscular venules and capillaries. Surprisingly, we also find that endothelia of small venules and capillaries become fenestrated within 10 minutes of VEGF application. Fenestrations appeared in vascular beds which do not normally have fenestrated endothelium, namely the cremaster muscle and skin. Histamine, saline, and heat-inactivated VEGF do not cause fenestrations. Increased permeability is completely inhibited when VEGF is cleared by immunoprecipitation with anti-VEGF monoclonal antibodies. The VEGF effect on permeability is unlike that of any other mediator described to date since both muscular venules and capillaries are affected.

Animals↗
PubMed1994

Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide.

In a murine heart perfusion system, we were able to "turn off" the transport of derivatized albumin [dinitrophenylated albumin (DNP-albumin)] from the perfusate to the tissue, by preperfusing the system with 1 mM N-ethylmaleimide (NEM) for 5 min at 37 degrees C, followed by a 5-min perfusion of DNP-albumin in the presence of NEM. Using a postembedding immunocytochemical procedure, we showed that (i) a 30-sec to 1-min treatment of heart vasculature with 1 mM NEM reduces the transendothelial transport of DNP-albumin and nearly stops it after 5 min, and (ii) DNP-albumin is detected exclusively in plasmalemmal vesicles (PVs) while in transit across endothelial cells. Perfusion with 10 mM dithiothreitol for 1 min before NEM prevents the inhibition of vesicular transport. To quantitate the NEM effect on vesicular transport inhibition, we developed an ELISA and a dot-blot assay for measuring DNP-albumin in supernatants of perfused whole-heart homogenates. The results obtained indicate that the treatment of the heart vasculature with 1 mM NEM decreases the vesicular transport of DNP-albumin by 78-80%. Since NEM is known to inhibit the fusion of different types of vesicular carriers with their target membranes in other cell types and in in vitro reconstituted cellular systems, by alkylating a NEM-sensitive factor, we assume that the same mechanism applies in our in situ system. The decrease of vesicular transport can be explained by NEM preventing the fusion of recycling vesicles with their targets--i.e., the abluminal and luminal domains of the plasmalemma. The results open to question previous interpretations from other laboratories according to which plasmalemmal vesicles are sessile, immobile structures.

Albumins↗
PubMed1994

Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers.

From rat livers labeled in vivo for 30 min with [35S] cys-met, we have isolated two classes of vesicular carriers operating between the Golgi complex and the basolateral (sinusoidal) plasmalemma. The starting preparation is a Golgi light fraction (GLF) isolated by flotation in a discontinuous sucrose density gradient and processed through immunoisolation on magnetic beads coated with an antibody against the last 11 aa. of the pIgA-R tail. GLF and the ensuing subfractions (bound vs nonbound) were lysed, and the lysates processed through immunoprecipitation with anti-pIgA-R and anti-albumin antibodies followed by radioactivity counting, SDS-PAGE, and fluorography. The recovery of newly synthesized pIgA-R was > 90% and the distribution was 90% vs 10% in the bound vs nonbound subfractions, respectively. Albumin radioactivity was recovered to approximately 80%, with 20% and 80% in bound vs nonbound subfractions, respectively. Other proteins studied were: (a) secretory-apolipoprotein-B, prothrombin, C3 component of the complement, and caeruloplasmin; (b) membrane-transferrin receptor, EGR-receptor, asialoglycoprotein receptor, and the glucose transporter. In all the experiments we have performed, the secretory proteins distributed up to 85% in the nonbound subfraction (large secretory vacuoles), whereas the membrane proteins were segregated up to 95% in the bound subfraction (small vesicular carriers). These results suggest that in hepatocytes, membrane and secretory proteins are transported from the Golgi to the basolateral plasmalemma by separate vesicular carriers as in glandular cells capable of constitutive and regulated secretion.

Animals↗
PubMed1993

Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.

An assay for endoplasmic reticulum (ER)-through-Golgi transport has been developed in streptolysin O-permeabilized murine erythroleukemia (MEL) cells. The reporter proteins are metabolically labeled native murine glycophorins, which display a distinctive shift in electrophoretic mobility after acquisition of O-linked oligosaccharides. The O-linked sugars are acquired at a site distal to a brefeldin A block, presumably in a cis Golgi compartment, and sialylation occurs in middle and/or trans Golgi compartments. In permeabilized cells supplemented with cytosolic proteins and an ATP-generating system, 20-50% of the radiolabeled precursor glycophorins can be converted to the mature, sialylated form. This maturation process is ATP- and cytosol-dependent and is blocked by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). Electron microscopy of permeabilized MEL cells shows retention of ER elements, stacked Golgi cisternae, free polysomes, and other subcellular components. In the presence of GTP[gamma S], dilated vesicles accumulate around the Golgi stacks. Antisera to the carboxyl terminus of the Golgi resident alpha subunit of Gi3 inhibit maturation of glycophorin. To our knowledge, a transport assay utilizing O-glycosylation of an endogenous protein as a monitor of ER-through-Golgi traffic in permeabilized cells has not been reported previously. Furthermore, the data provide evidence for heterotrimeric GTP-binding protein involvement in Golgi function.

Adenosine Triphosphate↗
PubMed1993

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.

Adenosine Triphosphate↗
PubMed1993

Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium.

In the capillary physiology literature, molecules and particles larger than 10 nm are assumed to leave the plasma mostly through large pores located at the level of intercellular junctions in microvessels lined with a continuous endothelium. In morphological studies of similar microvessels, outgoing particles > 10 nm were detected in endothelial plasmalemmal vesicles not in intercellular junctions. Because the probes may not be found in transit through the junctions because they may be swept away by strong currents generated by Starling forces, we have examined a large number of junctions in arteriolar, capillary, and venular segments of bipolar vascular fields of mouse diaphragms collected after perfusion with large pore probes. The results presented in this study indicate that 1) the perfused probes accumulate in the luminal introits of the junctions as filtration residues that decrease in size and frequency from arterioles to venules, and 2) large pore probes move across the endothelium exclusively through plasmalemmal vesicles.

Animals↗
PubMed1993

Thrombomodulin and thrombin localization on the vascular endothelium; their internalization and transcytosis by plasmalemmal vesicles.

We have immunolocalized thrombomodulin (TM), (an endothelial cofactor protein for thrombin-mediated protein C activation), and its ligand thrombin (TH) by light- and electron microscopy on endothelial cells in culture and in situ. Our results show that: i) TM is expressed in small clusters on the basal and the apical surface of human umbilical vein endothelial cells (HUVEC), is absent from coated pits and coated vesicles, is internalized from the cell surface via plasmalemmal vesicles, and is associated with endosomes, including a tubulovesicular network within the cytoplasm; ii) exogenous TH binds at low surface density on HUVEC and colocalizes with TM in small TM clusters; iii) on the microvascular endothelium of heart, lung, and kidney, TM is expressed at high density on the luminal plasmalemma, is associated with a fraction of the plasmalemmal vesicle population; iv) and is found internalized in multivesicular bodies in TH perfused heart samples; v) in the same samples, perfused TH is detected at low surface density: on the luminal aspect of the endothelium within small TM clusters, in the introits and within plasmalemmal vesicles, in endosomal structures, on the abluminal plasmalemma, and within the pericapillary spaces. Our findings suggest that the high level of endothelial TM expression in situ provides a safety margin for random TH generation; they also demonstrate that bound TH is transcytosed across the endothelium by plasmalemmal vesicles which might represent an additional mechanism to remove TH from the circulation.

Animals↗
PubMed1992

Common peptide epitopes in glycophorin and the endothelial sialoglycoprotein gp60.

Polyclonal anti-serum made against murine glycophorin gp3 (alpha gp) recognizes the endothelial albumin binding glycoprotein, gp60. In this study, we investigated the nature (peptide vs. carbohydrate) of the common epitope. First, a new technique was developed to remove oligosaccharides from glycoproteins that were first immobilized on filters and then subjected to beta-elimination. When greater than 90% of the glycans of gp60 were removed, alpha gp still recognized gp60 without apparent loss of affinity. Second, we used brefeldin A to accumulate unglycosylated glycophorin precursors in order to affinity-purify peptide-specific alpha gp immuno-globulins; these antibodies recognized gp60. Finally, alpha gp recognized from in vitro translations a 48 kDa putative polypeptide precursor of gp60. These different approaches indicate that gp60 and gp3 have at least one common epitope in their peptide backbones.

Animals↗
PubMed1992

Brefeldin A affects early events but does not affect late events along the exocytic pathway in pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) to Golgi complex and causes dismantling of the Golgi complex with relocation of resident Golgi proteins to the ER in some cultured cells. It is not known whether later steps in the secretory process are affected. We previously have shown that in BFA-treated rat pancreatic lobules, there is no detectable relocation of Golgi proteins to the ER and, although Golgi cisternae are rapidly dismantled, clusters of small smooth vesicles consisting of both bona fide Golgi remnants and associated vesicular carriers persist even with prolonged BFA exposure. We now report the effects of BFA on transport of proteins through the secretory pathway in exocrine pancreatic cells; we pulse-labeled pancreatic lobules with [35S]methionine and then chased for various times before adding BFA. When BFA was added at pulse, treated lobules released less than 10% of radioactive protein in comparison with controls, regardless of whether or not the lobule cultures were stimulated with carbamoylcholine. However, when lobules were pulsed and then chased for 30, 45, or 60 min before BFA addition, the amount of labeled protein released was comparable in both BFA-treated and untreated cultures. Furthermore, the kinetics and amounts of basal and carbamoylcholine-stimulated release of unlabeled alpha-amylase from storage in zymogen granules were similar in both control and BFA-treated lobules. Therefore, in the rat pancreas, BFA blocks ER to Golgi transport but does not affect later stages along the secretory pathway, including intra-Golgi transport, exit from the Golgi complex, formation and concentration of secretory granules, and exocytosis.

Animals↗
PubMed1992

Differential colchicine effects on the transport of membrane and secretory proteins in rat hepatocytes in vivo: bipolar secretion of albumin.

We carried out a comparative investigation on the effects of colchicine (25 mumoles/100 gm body wt) on the intracellular transport, processing and discharge by secretion or proteolytic processing of a membrane protein (i.e., the polymeric IgA receptor) and a secretory protein (i.e., albumin) in rat hepatocytes. The results obtained indicated the following: (a) the transport and processing of polymeric IgA receptor is strongly inhibited and delayed, but the appearance of secretory component in the bile is not arrested; (b) polymeric IgA receptor reaches the sinusoidal plasmalemma in colchicine-treated specimens, as it does in controls; (c) albumin discharge into the plasma is strongly inhibited and markedly delayed in colchicine-treated as compared with control animals; (d) the reverse applies for albumin secretion in the bile, which is increased by a large factor; (e) newly synthesized albumin secreted directly from hepatocytes in control and in colchicine-treated animals is the major source of bile albumin; and (f) colchicine affects in different ways the polymeric IgA receptor and albumin arrival at the sinusoidal front and especially at the biliary front of the hepatocyte.

Animals↗
PubMed1992

Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.

This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.

Animals↗
PubMed1992

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.

Animals↗
PubMed1991

Ultrastructural localization of albumin transport across the cerebral microvasculature during experimental meningitis in the rat.

Injury to the blood brain barrier (BBB) is a fundamental sequela of bacterial meningitis, yet the precise mechanism facilitating exudation of albumin across the endothelium of the cerebral microvasculature remains conjectural. After intracisternal inoculation of Escherichia coli (0111:B4) lipopolysaccharide in rats to elicit a reversible meningitis and BBB injury, we utilized in situ tracer perfusion and immunolabeling procedures to identify by transmission electron microscopy the precise topography and microvascular exit pathway(s) of bovine serum albumin (BSA). Results revealed that during meningitis there was: (a) an inducible increase in immunodetectable monomeric BSA binding to the luminal membrane of all microvascular segments in the pia-arachnoid and superficial brain cortex; (b) similar uptake of both colloidal Au-BSA (as well as monomeric BSA) by plasmalemmal vesicles but no detectable transcytosis to the abluminal side; and (c) predominant exit of both perfused Au-BSA and immunodetectable monomeric BSA through open intercellular junctions of venules in the pia-arachnoid. This was corroborated in separate experiments documenting focal pial venular leaks of in situ perfused 0.01% colloidal carbon black during experimental meningitis. These results provide precise localization of BBB injury in meningitis to meningeal venules, confirm a paracellular exit pathway of albumin via open intercellular junctions, and suggest an injury mechanism amenable to specific therapeutic intervention.

Animals↗
PubMed1991

Effects of brefeldin A on the processing of viral envelope glycoproteins in murine erythroleukemia cells.

This paper documents the effects of brefeldin A (BFA) on the processing and transport of viral envelope glycoproteins in a retrovirus-transformed murine erythroleukemia (MEL) cell line. BFA is a fungal metabolite that disrupts intracellular membrane traffic at the endoplasmic reticulum (ER)-Golgi complex junction. In MEL cells, BFA inhibited the processing of the newly synthesized precursor, gPr90env, of the murine leukemia virus envelope protein, gp70, and curtailed the budding of virions into the culture medium by blocking the transport of this protein out of the ER. The block resulted in the intracellular accumulation of gPr90env and two putative products of its processing (78 and 66 kDa). The results of endoglycosidase (endo) H and D digestion of the viral glycoproteins in the presence and absence of BFA indicated that (i) there was no glycoprotein processing during the first approximately 2 h of the BFA block; (ii) active Golgi enzymes relocated to the ER in approximately 2 h during BFA treatment, resulting in the production of partially endo H-resistant forms of the spleen focus-forming virus glycoprotein, gp55 (in controls, this glycoprotein was generally retained in the ER as an endo H-sensitive entity); and (iii) proteolytic processing of gPr90env to gp70 occurred prior to the acquisition of endo H resistance and at approximately the same time as endo D sensitivity (i.e. in a cis Golgi compartment). In control cells, the spleen focus-forming virus glycoprotein, gp55, underwent turnover with a half-life of approximately 5 h. In contrast, its turnover was considerably slower during BFA treatment (t 1/2 = approximately 20 h), suggesting that transport of gp55 out of the ER was required for its degradation or that BFA afforded it protection from proteolysis within the ER.

Animals↗
PubMed1991

Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocytes after bile duct ligation.

In rat hepatocytes, transcytotic vesicular carriers transport the mature 120 x 10(3) Mr form of the polymeric IgA receptor (pIgA-R), with or without its ligand, pIgA, from the sinusoidal to the biliary plasmalemma, where the ectodomain of the receptor is cleaved to produce an 80 x 10(3) Mr fragment that is secreted into the bile. Here we show that cholestasis induced by bile duct ligation results in the accumulation of transcytotic carriers, identified by the 120 x 10(3) Mr pIgA-R and pIgA, in the pericanalicular cytoplasm of hepatocytes. To determine the extent of pIgA-R accumulation, hepatic total microsomes (TM) were prepared from control and cholestatic rats. Solubilized TM proteins were separated by SDS-PAGE and receptor forms were detected by immunoblotting and autoradiography. Quantitative densitometry of these autoradiograms showed that after duct ligation the 120 x 10(3) Mr receptor accumulated to a level approximately threefold higher than the control. Concomitantly, immunologically related, novel 124, 90 and 80 x 10(3) Mr proteins (cholestatic antigens) became detectable. Immunoblot analyses of biliary and serum proteins showed that cholestasis resulted in: (1) a marked decrease in the concentrations of the 80 x 10(3) Mr receptor and pIgA in the bile, whereas albumin concentrations remained at control levels; and (2) a marked increase in the concentration of the 80 x 10(3) Mr receptor in the serum. Positive sites for pIgA-R were localized to the pericanalicular cytoplasm of hepatocytes by indirect immunofluorescence on semithin frozen sections in cholestatic hepatocytes. The sites were more numerous and the positive signal stronger than in controls. One day post-ligation, pIgA-positive sites were located to the same pericanalicular cytoplasm of hepatocytes; by three days, however, most pIgA appeared in sinusoidal endothelia and Kupffer cells. To validate the vesicular character of the receptor-positive sites, sham-operated and cholestatic livers were processed for either transmission electron microscopy (TEM) or immunogold localization of receptors on thin frozen sections. TEM verified the accumulation of pericanalicular vesicles in cholestatic hepatocytes. Immunogold tests localized pIgA-R to pleiomorphic, pericanalicular vesicles, which were increased in number, size and concentration of antigenic sites in cholestatic hepatocytes. These findings indicate that bile duct ligation provides a method for manipulating the in vivo transcytotic pathway and for accumulating previously unstudied transcytotic carriers in hepatocytes.

Animals↗
PubMed1991

Differential and specific labeling of epithelial and vascular endothelial cells of the rat lung by Lycopersicon esculentum and Griffonia simplicifolia I lectins.

In the rat lung, we found that the Lycopersicon esculentum (LEA) lectin specifically binds to the epithelium of bronchioles and alveoli whereas Griffonia simplicifolia I (GS-I) binds to the endothelium of alveolar capillaries. The differential binding affinity of these lectins was examined on semithin (approximately 0.5 microns) and thin (less than 0.1 (microns) frozen sections of rat lung lavaged to remove alveolar macrophages. On semithin frozen sections, LEA bound to epithelial cells lining bronchioles and the alveoli (type I, but not type II epithelial cells). On thin frozen sections, biotinylated Lycopersicon esculentum (bLEA)-streptavidin-gold conjugates were confined primarily to the luminal plasmalemma of type I cells. bGS-I-streptavidin-Texas Red was detected on the endothelial cells of alveolar capillaries and postcapillary venules but not on those of larger venules, veins or arterioles. By electron microscopy, GS-I-streptavidin-gold complexes were localized primarily to the luminal plasmalemma of thick and thin regions of the capillary endothelium. Neither lectin labeled type II alveolar cells, but both lectins labeled macrophages in the interstitia and in incompletely lavaged alveoli.

Animals↗
PubMed1991

Effects of Brefeldin A on the Golgi complex, endoplasmic reticulum and viral envelope glycoproteins in murine erythroleukemia cells.

This report concerns the effects of Brefeldin A (BFA): i) on the Golgi complex and the ER of retrovirus-transformed murine erythroleukemia (MEL) cells and, ii) on the viral proteins these cells express. Golgi complexes were extensively disorganized by BFA. Within 5 min, most stacked cisternae were converted to vesicles scattered throughout the centrosphere region. By 30 min, the Golgi complexes were completely disassembled. Only clusters of small vesicles ("Golgi remnants") persisted in the vicinity of the centrioles and microtubule-organizing centers. Some of these small vesicles had a simple coat structure on their membranes. Over the next 1 to 2 h of BFA treatment, the number of vesicles in the Golgi area decreased concomitantly with the expansion of a predominantly smooth membrane portion of the ER, consisting of a network of dilated tubules in continuity with regular RER cisternae, annulate lamellae and the nuclear envelope. By electron microscopy, viral glycoproteins appeared to accumulate on the membranes of this network, and immature virions were found to bud preferentially into its cisternal space. Viral accumulations increased with time under BFA. The rest of the RER appeared normal, apparently unaffected by the drug. Preferential virion budding suggests that this expanding network is a chemically differentiated part of the ER. By immunofluorescence, antibodies to viral envelope proteins gave a punctate staining at the surface of control cells, presumably in the areas of virion budding, whereas relatively large intracellular masses of antigens were found in BFA-treated cells. We assume that these masses represent the differentiated parts of the ER. Taken together, these findings suggest that BFA blocks intracellular transport of newly synthesized cellular and viral proteins immediately distal to the distinct compartment of the ER in which virion budding preferentially occurs. BFA effects are rapidly and fully reversible. Within 1 min of the removal of the drug, stacks of Golgi cisternae began to reappear in the vicinity of the centrioles, and by 30 min, Golgi complexes regained their normal structural appearance.

Animals↗