Uncoated Vesicles Research Articles

Endocytosis of folate labeled proteins: Ultrastructural localization in kb cells

Recently, methods for the delivery of macromolecules into cells via covalent coupling to vitamins have been described for plant and animal cells. This method utilizing vitamin receptor endocytosis concentrates macromolecules inside the cell in an active form that is nondegradative. The purpose of this study was to determine the specific location of bovine serum albumin-folic acid-colloidal gold (BSA-F-CG) conjugates within KB cells. Bovine serum albumin was covalently coupled to folic acid (BSA-F) and used to stabilize 15 nm colloidal gold (CG) particles. KB cells were incubated with BSA-F-CG or BSA-CG at either 11°C for 2 hours or 37°C for 15 minutes to allow binding, and the cells washed to remove unbound material. Some cells were fixed immediately, and others were incubated for additional time periods. BSA-CG particles nonspecifically pinocytosed by control cells were only found in large dense endosomes after 6 hours incubation. In cells incubated with BSA-F-CG, CG particles decorated the plasma membrane, and were found in uncoated pits. At 30 minutes CG particles could be found in multivesicular bodies (MVB’s), small vesicles, and dense endosomes. At 6 hours, CG particles were found in various dense endosomes, MVB’s, dense endosomes associated with the Golgi apparatus, and free in the cytoplasm. Cells that were pulsed with BSA-F-CG for 15 minutes at 37°C had CG particles on the surface and uncoated pits, small vesicles and MVB’s. After 60 and 360 minutes, BSA-F-CG was located in MVB’s, clear and dense endosomes, and MVB’s associated with the Golgi apparatus. Control cells incubated with BSA-CG had a few CG particles located in small clear endosomes at 15 minutes and large dense endosomes at 6 hours. For comparison, 5nm colloidal gold was stabilized with transferrin (TF) and incubated alone with KB cells or coincubated with 15 nm BSA-F-CG. The 5 nm TF-CG was localized in coated pits and on the rim of vesicular structures resembling CURL (Compartment of Uncoupling of Receptor and Ligand) at 15 minutes. There was no colocalization of TF-CG and BSA-F-CG at 15 minutes. TF-CG and BSA-F-CG were found separately and together in MVB’s at 1 hour. At six hours TF-CG and BSA-F-CG frequently colocalized in large dense endosomes. Folate labeled proteins endocytosed in this study shared some common compartments with transferrin, but there were several points of divergence. Transferrin-CG is taken up via coated pits whereas BSA-F-CG enters the cell via uncoated pits. At 15 minutes, internalized TF-CG is associated primarily with vesicles resembling CURL, and BSA-F-CG is found in MVB’s. At one hour, both TF-CG and BSA-F-CG may be found separately and together in MVB’s, and at 6 hours both may be found separately and together in dense endosomes. The BSA-F-CG particles free in the cytoplasm may be released during transport of the ligand, possibly by MVB’s that associate with the Golgi apparatus. The BSA-F-CG particles localized in this study may represent the normal location of endocytosed folate or their location could be an aberration of the normal pathway.

Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi.

Epimastigote forms of the pathogenic parasite Trypanosoma cruzi were used to study the endocytic process in a protozoon. These elongated unicellular organisms are highly polarized cells: endocytosis occurs only at the anterior region through the cytostome and the flagellar pocket membrane, areas of the plasma membrane where the cell cytoskeleton, formed by sub-pellicular microtubules, is absent. When the cells were incubated at 4 degrees C or 28 degrees C with gold-labeled transferrin, fixed and processed for routine transmission electron microscopy our observations show that this ligand initially binds to the cytosome and the membrane lining the flagellar pocket and is subsequently ingested through a clathrin-independent receptor-mediated endocytotic process, with formation of uncoated pits and vesicles. Ingested complexes are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the posterior end of the cell. Immunocytochemical data from Lowicryl-embedded cells demonstrated that the reservosomes are acidic compartments (pH 6.0, as shown using DAMP as a pH probe) with no acid phosphatase or typical lysosome-associated membrane proteins (LAMP 1, LAMP 2 and lgp 120), but rich in cysteine proteinase. These data suggest that the reservosome is a pre-lysosomal compartment. Since cysteine proteinase of T. cruzi contains no phosphorylated mannose residues and the cation-independent mannose 6-phosphate receptor could not be immunocytochemically detected in the reservosomes, it is possible that lysosomal enzymes in the epimastigote forms of T. cruzi are targeted to compartments related to the endocytic pathway through a mechanism different from that which occurs in other eukaryotic cells.

Infectious entry pathway for canine parvovirus

We have investigated whether canine parvovirus (CPV) infection involves a low pH-dependent entry pathway. The effects of two lysosomotropic bases, NH 4Cl and chloroquine, on CPV entry were studied by immunofluorescence and ultrastructural and biochemical methods. In the presence of these reagents, input virions appear to accumulate in large vacuoles. Ultrastructural studies indicated that uptake of virions takes place predominantly in small uncoated vesicles that appear to fuse with larger vesicles. In the presence of NH 4Cl, virions accumulate in the latter structures and their uncoating appears to be prevented. Viral DNA as well as antigen synthesis were found to be significantly inhibited in the presence of these reagents. In addition, inhibition of viral DNA and antigen synthesis appeared to be most extensive when NH 4Cl was present from 30 min preinfection, whereas no significant inhibition was observed when the cells were treated after 2 hr postinfection. Thus, the results indicate that CPV requires exposure to low pH in an endosomal compartment to initiate a productive infection.

20] Reconstitution of endocytosis and recycling using perforated madin-darby canine kidney cells

Publisher Summary This chapter describes the methods to study cell surface binding, internalization, and recycling of transferrin in intact polarized epithelial cells. In general, endocytosis involves the formation of small (0.1- 0.2 μm) transport vesicles that form from the invagination and pinching off of dathrin-coated pits of the plasma membrane. While some toxins and lectins appear to be internalized in uncoated vesicles, in many cells, it is apparent that the formation of coated vesicles represents the quantitatively most important route of entry into the cell. Eukaryotic cells regulate the composition of the plasmalemma by continuously inserting and removing selected proteins and lipids on a process referred to as membrane recycling. This cycle involves the delivery of membrane by an exocytic fusion event of a vesicular carrier with the plasmalemma and the import or removal of membrane by an endocytic event or the invagination of small domains of the plasmalemma followed by budding of vesicles into the cytoplasm. The chapter presents the optimized procedure to permeahilize Madin-Darby canine kidney (MDCK) cell monolayers using nitrocellulose filters. The assays to reconstitute receptor-mediated endocytosis and recycling to permeabilized cells should help to understand the enzymology of endocytosis.

Evaluation of mucoadhesive polymers in ocular drug delivery. II. Polymer-coated vesicles.

Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P less than 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.

Contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts

In this study we have examined the mechanism underlying the contact-mediated transfer of a lysosomal enzyme from lymphocytes to fibroblasts in culture. We found that although antibody against the mannose 6-phosphate lysosomal targetting receptor (MPR) completely inhibited fibroblast uptake of the lysosomal enzyme beta-glucuronidase (Gus) from the culture medium, it had no effect on the transfer of the enzyme from normal lymphocytes. In contrast, the presence of antibody that prevented the adhesion of the lymphocytes to the fibroblasts inhibited Gus acquisition but had no effect on endocytosis. Immunogold electron microscopy of the contact site between the two types of cell showed that the transfer of Gus involved uncoated vesicles localized near the cell surface of the fibroblast at sites of contact with the lymphocytes. The acquired lymphocyte enzyme was shown to be transported to the fibroblast lysosomes.

Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of Immunoglobulin-G (IgG) in the human placenta

Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.

The effect of ricin B chain on the intracellular trafficking of an A chain immunotoxin

Covalent linkage of the A chain of ricin to the LICR-LOND-Fib75 monoclonal antibody produced an immunotoxin, Fib75-SS-ricin A, which demonstrated immunospecific toxicity to human bladder carcinoma cells in tissue culture (Forrester et al., 1984). The present studies have shown that ricin B chain potentiates the toxicity of the immunotoxin by two orders of magnitude and also significantly increases the rate of protein synthesis inhibition. Using immunoelectron microscopy, the receptor-mediated endocytosis and intracellular routing of the immunotoxin was studied with and without ricin B chain treatment after immunolocalisation of the conjugate. Fib75-SS-ricin A was internalised by the EJ cells predominantly in uncoated pits and vesicles and directed to the endosomes. Some degradation of the complex appeared to take place in multivesicular endosomes at early timepoints and 24 h after internalisation, most of the immunotoxin was found in lysosomes. Some ricin A chain epitopes were detected in Golgi vesicles. Cells treated with immunotoxin and ricin B chain endocytosed the complex predominantly in coated pits and coated vesicles. Using pre-embedding immunoperoxidase techniques, ricin chains were found in the whole Golgi complex and most of the conjugate escaped lysosomal degradation. Internalised immunotoxin was recycled back to the plasma membrane in an active form associated with vesicles which appeared to be derived predominantly from multivesicular endosomes. A similar mode of recycling has recently been reported (McIntosh et al., 1990) for ricin holotoxin in the same cell line. These observations may explain the potentiating effect of toxin B chains in the antibody-directed targeting of toxin A chains.

Uncoating of coated vesicles by yeast hsp70 proteins.

The ability of hsp70 isoenzymes from wild-type and mutant yeast strains to uncoat bovine brain coated vesicles was analyzed and compared with that of the brain uncoating ATPase. Results show that, among the four major cytoplasmic isoenzymes produced in wild-type yeast, almost all of the activity is associated with the SSA1 and SSA2 isoenzymes. The SSB1 and SSB2 isoenzymes have almost no uncoating activity and are not found in the clathrin-hsp70 complexes formed during the uncoating reaction. Using hsp70 mutant yeast strains we find a marked difference in uncoating activity between the SSA1 and SSA2 isoenzymes, although there is only a 3% difference between their amino acid sequences. The SSA4 isoenzyme, which is produced only under stress conditions, has an uncoating activity intermediate between SSA1 and SSA2. These results suggest that the ability of hsp70 isoenzymes to uncoat clathrin-coated vesicles is restricted to certain members of the hsp70 family and can be affected by subtle changes in amino acid sequence. We also investigated the uncoating activity of mixtures of isoenzymes and find that the isoenzyme with lower uncoating activity reduces the activity of the isoenzyme with higher uncoating activity possibly by occupying binding sites on coated vesicles.

Ultrastructure of the vegetative organisation and initial stages of silica plate deposition in the soil testate amoebaCorythion dubium

The siliceous body plates ofCorythion dubium are bound by a band of organic cement which is thickest at the lateral margins. The anterior vacuolar cytoplasm is separated by a pigment zone, which forms a dark band in the mid-body region, from the compact posterior region containing a typical vesicular nucleus surrounded by a region of dense endoplasmic reticulum. A pellicular basket of microtubules surrounds the posterior cytoplasm. The large Golgi complex lies between the nucleus and the fundus. Numerous coated and uncoated vesicles from the Golgi cisternae are seen in the peripheral cytoplasm alongside developing plates. These small siliceous plates are enclosed in silicon deposition vesicles lying in surface ruffles of the plasmalemma, often in association with a pair of microtubules. Observations are made on the formation of these vesicles and the early stages of silica deposition. A comparison is drawn between silica deposition inC. dubium and choanoflagellates where there is a similar association between silicon deposition vesicles and microtubules.

Use of combination of monoclonal antibodies directed against three distinct epitopes of a tumor‐associated antigen: Analysis of cell binding and internalization

Three monoclonal antibodies (MAbs), MOv17, MOv18 and MOv19 with tumor-restricted specificity for human ovarian carcinoma, were tested alone or in double combination with the aim of analyzing their binding and internalization behavior on different in vitro cell lines. Biochemical studies indicated that the 3 MAbs were directed against 3 epitopes of the same 38 kDa surface molecule. By immuno-electron-microscopy they exhibited a different internalization behavior since MOv17 induced evident endocytosis through coated vesicles, whereas MOv18 gave rise to occasional uncoated vesicles and MOv19 was completely unable to promote internalization of the relevant molecule. When tested 2 by 2 there was a binding synergy in one of the 9 possible combinations (125I-labelled MOv18 and unlabelled MOv19), but no change in the internalization behavior. The binding synergy, which was highly reproducible, was temperature-dependent and was also evident on glutaraldehyde-fixed cells. A metabolism involvement is therefore unlikely. This could be attributed to an easier accessibility of the CaMOv18 due to a conformational change of the molecule after MOv19 MAb binding.

Binding and Internalization of Biotinylated Interleukin-2 in Human Lymphocytes

The binding, internalization, and fate of interleukin-2 (IL-2) were studied in phytohemagglutinin (PHA)-activated human lymphocytes using biotinylated recombinant IL-2 (rIL-2). Streptavidin adsorbed to 18-nm colloidal gold beads (Au18-streptavidin) and streptavidin covalently bound to horseradish peroxidase (HRP-streptavidin) were used to follow the movement of biotinylated rIL-2 within cells over a 4-hour period. Results obtained from either probe were similar. Biotinylated rIL-2 was taken up in coated pits, transferred to a series of small uncoated vesicles and tubules in the peripheral cytoplasm of the cell, then concentrated and sequestered in uncoated vesicles, multivesicular bodies (MVB), and dense bodies (DB) in the peripheral and juxtanuclear cytoplasm of the cell. Occasionally, MVB containing Au18-streptavidin, or HRP-streptavidin, appear to have fused with the plasma membrane of the cell. No labeling of the Golgi cisternae, nuclear envelope, or nucleus was observed. Results from a competitive receptor binding assay and a cell proliferation assay indicate that both the affinity of rIL-2 for high affinity rIL-2 receptors and the proliferative activity of rIL-2 were negligibly affected by the biotinylation procedure. These studies suggest that in activated lymphocytes, IL-2 is bound to receptors on the cell surface, gathered in coated pits, internalized by receptor-mediated endocytosis, concentrated in the endosomal compartments, and delivered to lysosomes for degradation.

Binding and internalization of biotinylated interleukin-2 in human lymphocytes

The binding, internalization, and fate of interleukin-2 (IL-2) were studied in phytohemagglutinin (PHA)-activated human lymphocytes using biotinylated recombinant IL-2 (rIL-2). Streptavidin adsorbed to 18-nm colloidal gold beads (Au18-streptavidin) and streptavidin covalently bound to horseradish peroxidase (HRP-streptavidin) were used to follow the movement of biotinylated rIL-2 within cells over a 4-hour period. Results obtained from either probe were similar. Biotinylated rIL-2 was taken up in coated pits, transferred to a series of small uncoated vesicles and tubules in the peripheral cytoplasm of the cell, then concentrated and sequestered in uncoated vesicles, multivesicular bodies (MVB), and dense bodies (DB) in the peripheral and juxtanuclear cytoplasm of the cell. Occasionally, MVB containing Au18-streptavidin, or HRP-streptavidin, appear to have fused with the plasma membrane of the cell. No labeling of the Golgi cisternae, nuclear envelope, or nucleus was observed. Results from a competitive receptor binding assay and a cell proliferation assay indicate that both the affinity of rIL-2 for high affinity rIL-2 receptors and the proliferative activity of rIL- 2 were negligibly affected by the biotinylation procedure. These studies suggest that in activated lymphocytes, IL-2 is bound to receptors on the cell surface, gathered in coated pits, internalized by receptor-mediated endocytosis, concentrated in the endosomal compartments, and delivered to lysosomes for degradation.

Morphologic Demonstration of Receptor-Mediated Endocytosis of Epidermal Growth Factor by Isolated Bile Duct Epithelial Cells

It was recently shown that intrahepatic bile duct epithelial cells in situ or after isolation from rat liver have coated pits and vesicles, suggesting that they participate in receptor-mediated endocytosis. Therefore, using a morphologic approach and epidermal growth factor coupled to horseradish peroxidase or colloidal gold as probes, we studied freshly isolated or short-term cultured intrahepatic bile duct epithelial cells prepared from normal rat liver to determine if they participate in receptor-mediated endocytosis. Immunoelectron microscopy using a monoclonal antibody against the epidermal growth factor receptor was also used to examine for the presence of the growth factor receptor on the cells. Immediately after isolation, the cells did not internalize either epidermal growth factor-horseradish peroxidase or epidermal growth factor-colloidal gold; no growth factor receptor could be shown on these cells by immunocytochemistry, either. In contrast, cells cultured for 24 h bound and internalized both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold at 37 degrees C and showed growth factor receptors diffusely distributed on the plasma membrane. When cultured cells exposed to epidermal growth factor-colloidal gold were fixed with glutaraldehyde containing saponin and tannic acid, colloidal gold particles were observed in coated pits and in coated and uncoated vesicles. Preincubation of cultured cells with native epidermal growth factor completely blocked the internalization of both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold. When rat liver was stained in situ for epidermal growth factor receptor, reaction product was observed by immunoelectron microscopy exclusively on the basal surface of the plasma membrane of the intrahepatic bile duct epithelial cells. These results indicate that bile duct epithelial cells internalize epidermal growth factor by endocytosis via coated pits containing receptors localized in situ exclusively to the basal domain of their plasma membranes. The data demonstrate for the first time that intrahepatic bile duct epithelial cells participate in receptor-mediated endocytosis and raise the possibility that they are a target for epidermal growth factor.

Incorporation of spin-labelled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. As abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.

Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.

The retinal microvasculature of spontaneously diabetic BB rats: Structure and luminal surface properties

Endothelial cell permeability and luminal surface anionic sites were studied in the retinal microvasculature of spontaneously diabetic rats. Horseradish peroxidase (HRP) was used as a tracer of pinocytotic transport, and cationized ferritin (CF) was used as a marker of luminal surface anionic sites. Diabetic and control rats were injected with HRP, and their retinas were fixed. Retinal tissue sections were then incubated in CF, reacted to visualize HRP, and prepared for quantitative electron microscopic analysis. In both control and diabetic rats treated with serotonin and histamine antagonists to prevent HRP-induced vascular changes, the endothelium formed a barrier to the tracer. Pinocytotic uptake was relatively low in most vessels. Reaction product was restricted to pinocytotic vesicles, tubular cisternae, and multivesicular bodies. HRP uptake appeared high in some of the deep capillaries of the diabetic retinas as compared with that of the controls, but the difference was not statistically significant. HRP-induced transendothelial permeability was observed in both control and diabetic rats when serotonin and histamine antagonist pretreatment was omitted. CF studies showed anionic sites in four luminal surface microdomains in control and diabetic endothelial cells. CF-binding, anionic sites were present on the plasma membrane, on all coated vesicles, on some uncoated vesicles, and on most diaphragms of uncoated vesicles. Plasma membrane binding was sparse and patchy in some diabetic vessels, especially in the deep vessels of rats that were not treated with the serotonin and histamine antagonists. However, statistical analysis showed similar numbers of plasma membrane binding sites in diabetic and control rats pretreated with serotonin and histamine antagonists. Our data suggest that the retinal microvasculature in diabetic rats remains normal in terms of permeability and luminal membrane anionic charge. (Preliminary report was presented at the Society for Neuroscience; M. E. C. Fitzgerald and R. B. Caldwell, 1987, Soc. Neurosci. Abstr., 13, 773.)

Endocytosis of simian virus 40 into the endoplasmic reticulum.

The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.

Differentiation of trophoblast of the baboon blastocyst.

The structure of trophoblast of the baboon blastocyst undergoes a number of maturational changes from the early blastocyst to the late blastocyst stage. The striking expansion of the blastocyst that occurs during the preimplantation period is accompanied by the development of an extensive endocytic apparatus. Cationized ferritin labels coated depressions and vesicles near the apical cell surface, numerous uncoated tubules and larger apical vesicles, and multivesicular bodies within trophoblast cells. Basally and laterally the labeled components are primarily small uncoated vesicles and tubules. Small, discrete clusters of ferritin particles were seen within the basolateral compartment between trophoblast and its basal lamina and beneath trophoblast cells that do not have a basal lamina. the results indicate that ingested materials may be directed in two pathways, one involving breakdown within the lysosomal system and one involving transcytosis. The zona pellucida is a trilaminar structure consisting of a fibrillar outer layer that often contains spermatozoa, an intermediate zone, and an inner layer containing columns of dense zonal material. Loss of the zona occurs after expansion of the blastocyst and development of the endocytic organelles. During the late blastocyst stage, syncytial trophoblast differentiates at the margin of the polar trophoblast. Because blastocysts were flushed from the uterus, it could not be determined whether azonal blastocysts had been adherent to the uterine surface prior to collection.

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes.

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLPF) were found to interact with isolated oligodendrocytes from bovine brain at 4 degrees C as well as at 37 degrees C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t = 3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSAF) led to a weak cell labeling.