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J W Slot

Publications and source records attributed to J W Slot.

At least 19 recordsLinked to original sources
PubMed1992

Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells.

The intracellular localization of human copper,zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

Aged↗
PubMed1992

Polarized distribution of glucose transporter isoforms in Caco-2 cells.

We have examined the expression and cellular location of facilitated glucose transporter proteins (GLUT1, -3, and -5) in a human colonic epithelial cell line (Caco-2) by using peptide-specific antibodies. A differential cellular distribution of these transporters was observed in differentiated (greater than 14 days postconfluence) Caco-2 cells by immunofluorescence and immunoelectron microscopy. GLUT1 was localized primarily to the basolateral membrane, whereas GLUT3 was predominantly localized to the apical membrane. GLUT5, which was detected in only approximately 40% of fully differentiated Caco-2 cells, was found primarily in the apical membrane but was also present in both basolateral and intracellular membranes. A Na(+)-independent glucose transport system in the apical membrane of Caco-2 cells has been described previously [Blais, A., Bissonnette, A. & Berteloot, A. (1987) J. Membr. Biol. 99, 113-125], and we propose that GLUT3 mediates this process. The amino acid sequence identity (57%) and structural conservation between GLUT1 and GLUT3 may make these transporters an ideal model system for examining the molecular basis for polarized sorting of membrane proteins.

Adenocarcinoma↗
PubMed1992

Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme E1 plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the 110-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-E1 monoclonal antibodies, we find that E1 is abundant both within the cytoplasm and nucleus. Within the cytoplasm, E1 was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. E1 was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, E1 was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of E1 to distinct subcellular organelles suggests that E1 may play multiple physiological roles within the cell.

Antibodies, Monoclonal↗
PubMed1992

Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi.

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle.

In muscle and adipocytes, glucose transport is regulated by the translocation of insulin regulatable glucose transporters (GLUT4) between an intracellular compartment and the cell surface. In these studies we have characterized the cellular compartments containing GLUT4 in rat skeletal muscle. Immunocytochemical studies showed that in unstimulated muscle, GLUT4 was not present in surface membranes. Tubulo-vesicular structures clustered in the trans Golgi reticulum were enriched in GLUT4. GLUT4 underwent translocation to the sarcolemma in response to combined stimulation with insulin and exercise. Using immunoisolation, the intracellular GLUT4 vesicles (IRGTV) were purified 300-fold over the cell homogenate. IRGTV from unstimulated muscle were not enriched in markers specific for the sarcolemma, transverse tubules, sarcoplasmic reticulum or mitochondria; this was confirmed using gel filtration chromatography. Insulin resulted in a 40% decrease in GLUT4 levels in IRGTV confirming that this represents the intracellular compartment of GLUT4. GLUT4 is a major component of the IRGTV, constituting at least 5% of total vesicle protein. A subset of polypeptides are also markedly enriched in the muscle IRGTV. In conclusion, these data suggest that translocation of GLUT4 from intracellular tubulo-vesicular structures is the major mechanism by which insulin and exercise regulate muscle glucose transport.

Animals↗
PubMed1992

The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus.

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

Amino Acid Sequence↗
PubMed1992

Interaction of insulin and exercise on glucose transport in muscle.

Glucose transport is the rate-limiting step for glucose utilization in muscle. In muscle and adipose tissue, glucose transport is acutely regulated by such factors as insulin and exercise. Translocation of glucose transporters (GLUT4) from an intracellular domain to the cell surface is the major mechanism for this regulation. Using immunocytochemistry, the intracellular distribution of GLUT4 under resting conditions is similar in adipocytes and myocytes. GLUT4 is concentrated in tubulovesicular structures either in the trans-Golgi region or in the cytosol, often close to the cell surface but not on the cell surface. After stimulation, cell surface GLUT4 labeling is increased by as much as 40-fold. GLUT4 is chronically regulated by altered gene expression. Neural and/or contractile activity regulates GLUT4 expression in muscle: 1) GLUT4 levels differ among muscles of different fiber type; 2) GLUT4 levels in muscle are increased with exercise training and decreased with denervation; and 3) cultured muscle cells, which lack an intact nerve supply, express very low levels of GLUT4. GLUT4 expression appears to be regulated in parallel with many oxidative enzymes in muscle, suggesting that there may be a unified developmental program that determines the overall metabolic properties of a particular muscle. Preliminary evidence suggests that impaired GLUT4 expression in muscle is not the primary defect associated with insulin resistance. Nevertheless, it is conceivable that the adaptive increase in muscle GLUT4 that is found with exercise training may have beneficial effects in insulin-resistant states such as non-insulin-dependent diabetes.

Animals↗
PubMed1991

Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.

The insulin-regulated glucose transporter GLUT4 was immunolocalized in rat cardiac muscle under conditions of basal and stimulated glucose uptake, achieved by fasting and a combined exercise/insulin stimulus, respectively. In basal myocytes there was very little (less than 1%) GLUT4 in the different domains of the plasma membrane (sarcolemma, intercalated disk, and transverse tubular system). GLUT4 was localized in small tubulo-vesicular elements that occur predominantly near the sarcolemma and the transverse tubular system and in the trans-Golgi region. Upon stimulation approximately 42% of GLUT4 was found in the plasma membrane. Each domain of the plasma membrane contributed equally to this effect. GLUT4-positive, clathrin-coated pits were also present at each cell surface domain. The remainder of the labeling was in tubulo-vesicular elements at the same sites as in basal cells and in the intercalated disk areas. The localization of GLUT4 in cardiac myocytes is essentially the same as in brown adipocytes, skeletal muscle, and white adipocytes. We conclude that increased glucose transport in muscle and fat is accounted for by translocation of GLUT4 from the intracellular tubulo-vesicular elements to the plasma membrane. The labeling of coated pits indicates that in stimulated myocytes, as in adipocytes, GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and that stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation.

Animals↗
PubMed1991

Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.

Cytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC. Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing. In this study, the subcellular localization of perforin, granzymes, and known endosomal and lysosomal marker proteins was determined in human and murine CTL, by immunogold labeling of ultrathin cryosections followed by electron microscopy. Perforin and granzymes can be detected in rough endoplasmic reticulum, Golgi complex, trans-Golgi reticulum, and in all cytotoxic granules. Within the granules, they have a similar distribution and are localized not only in the so-called dense core but also over the region containing small internal vesicles. This finding implies that perforin and granzymes can be released in membrane-enveloped and/or -associated form into the intercellular cleft formed upon CTL-TC interaction. On the basis of the present evidence, additional release of these molecules in soluble form cannot be excluded. The lysosomal membrane glycoproteins lamp-1, lamp-2, and CD63, are abundantly present on the granule-delimiting outer membrane, which becomes incorporated into the CTL plasma membrane during lethal hit delivery. In contrast, the cation-dependent mannose 6-phosphate receptor, known to be present in endosomes and absent from lysosomes, is found only in a minority of the granules. Together with our previous findings that the granules are acidic and connected to the endocytic pathway, these observations define CTL granules as secretory lysosomes.

Animals↗
PubMed1991

Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells.

Antigen processing requires intracellular antigen catabolism to generate immunogenic peptides that bind to class II MHC molecules (MHC-II) for presentation to T-cells. We now provide direct evidence that these peptides are produced within dense lysosomes, as opposed to earlier endocytic compartments. The protein antigen hen egg lysozyme was targeted to endosomes or lysosomes by encapsulating it in liposomes of different membrane composition. Acid-sensitive liposomes released their contents in early endosomes, whereas acid-resistant liposomes sequestered their contents from potential endosomal processing events and released their contents only after delivery to lysosomes. Antigen encapsulated in acid-resistant liposomes was processed in a chloroquine-sensitive manner and presented more efficiently than soluble antigen or antigen encapsulated in acid-sensitive liposomes. Thus, peptides may be recycled from lysosomes, transported to endosomes to bind MHC-II, and then expressed at the cell surface.

Animals↗
PubMed1991

Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.

Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.

Adipose Tissue, Brown↗
PubMed1991

Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type.

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.

Adipose Tissue↗
PubMed1991

Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution.

Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freeze-substitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK I cells, even though they were connected by tight junctions. The labeling density on the apical plasma membrane was 1.4-1.6 times higher than basolateral. Roughly two-thirds of the gold particles were found intracellularly. The Golgi complex was labeled for Forssman as were endosomes, identified by endocytosed albumin-gold, and lysosomes, defined by double labeling for cathepsin D. In most cases, the nuclear envelope was Forssman positive, but the labeling density was 10-fold less than on the plasma membrane. Mitochondria and peroxisomes, the latter identified by catalase, remained free of label, consistent with the notion that they do not receive transport vesicles carrying glycosphingolipids. The present method of lipid immunolabeling holds great potential for the localization of other antigenic lipids.

Animals↗
PubMed1990

Characterization of human factor VIII and interaction with von Willebrand factor. An electron microscopic study.

Blood coagulation factor VIII is a large glycoprotein that circulates in plasma at relative low concentration (0.1 microgram/ml). It consists of a heterogeneous mixture of a series heavy-chain peptides (90-200 kDa), each associated with a light chain of 80 kDa. To gain insight into the physical properties of the protein, we have characterized purified human factor VIII by electron microscopy and rotary shadowing. Electron microscopy of rotary shadowed factor VIII molecules showed predominantly a single globular domain structure, with a somewhat asymmetric shape, while two-domain structures were also encountered. The overall dimensions of the globular domains ranged from 4 x 6 nm to 8 x 12 nm. EDTA treatment of factor VIII reduced the overall dimensions (2.5 x 5 nm to 6 x 10 nm) while treatment with thrombin reduced the dimensions to a small extent. In complexes with von Willebrand factor, factor VIII appeared localized at the globular domains of von Willebrand factor multimers. In addition, incubation of factor VIII with Staphylococcus aureus V8 protease fragments SpII and SpIII revealed only binding to the globular domains of SpIII. In this study, the first morphological characterization of human factor VIII is presented, together with its direct localization on von Willebrand factor multimers.

Blotting, Western↗
PubMed1990

No evidence for expression of the insulin-regulatable glucose transporter in endothelial cells.

A major effect of insulin is to increase glucose transport in muscle and fat. A family of genes encoding distinct mammalian glucose transporters has recently been elucidated. One of these, the insulin-regulatable glucose transporter (IRGT), is primarily expressed in muscle and fat, tissues that exhibit insulin-dependent glucose transport. Insulin promotes glucose transport in these tissues by stimulating movement of the glucose transporter from an intracellular location to the plasma membrane. Recent studies, however, suggest that an additional effect of insulin in these tissues may be the facilitation of glucose transport, presumably across capillary endothelium. This hypothesis is based on the localization of the IRGT in endothelial cells specific to muscle and adipose tissue. We report here, however, on morphological and biochemical studies using several different IRGT-specific antibodies in which we could not reproduce these results.

Adipose Tissue, Brown↗
PubMed1990

Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing.

Antigen presentation requires intracellular processing of native antigens to produce immunogenic peptides that bind to major histocompatibility complex class II (MHC-II) molecules. In functional studies of antigen processing by elicited peritoneal macrophages, MHC-II-peptide complexes were formed intracellularly. Immunogenic peptides were not released to bind surface MHC-II molecules. Ultrastructural studies employing immunogold staining in ultrathin cryosections of these macrophages showed large amounts of MHC-II molecules in intracellular sac-like vacuoles in the peripheral cytoplasm; most of these were negative for the lamp 1 lysosomal/endosomal membrane protein and cathepsin D. MHC-II molecules were also present in endosomes containing cathepsin D and lamp 1 as well as previously internalized gold-transferrin. The intracellular pool of MHC-II molecules was only slightly decreased by treatment with cycloheximide for 3 hr, indicating that it consisted mainly of endocytosed, recycling molecules, as opposed to nascent ones. These ultrastructural studies support the notion that there is endocytosis of MHC-II molecules into endocytic compartments, consistent with our earlier biochemical data. Furthermore, we have defined the distinct endocytic compartments that must mediate important functions in antigen processing, including the formation of MHC-II-peptide complexes.

Animals↗
PubMed1989

Molecules relevant for T cell-target cell interaction are present in cytolytic granules of human T lymphocytes.

An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.

Clone Cells↗