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D Louvard

Publications and source records attributed to D Louvard.

At least 127 records · Page 7Linked to original sources
PubMed1985

Changes in the distribution of membranous organelles during mouse early development.

The unfertilized oocyte, fertilized egg and early embryo (2-cell to 16-cell) of the mouse have been examined immunocytochemically for the distribution of antigens associated with the endoplasmic reticulum, the lysosomal and acidic vesicle fraction (100 kD antigen), Golgi apparatus (135 kD antigen) and coated vesicles (clathrin). The distribution of these antigens has also been examined in isolated 8-cell and 16-cell-stage blastomeres of various ages and phenotypes. Endoplasmic reticulum is detected only weakly in the oocyte and egg, but is seen abundantly at later stages both in association with the nuclear membrane and evenly distributed throughout the cytoplasm, except in regions of cell:cell apposition from which it is excluded. Intracellular clathrin is associated with the spindle in mitotic and meiotic cells. During interphase, clathrin is distributed throughout the cell until the mid-8-cell stage when it is concentrated into the apical region of the cell under the region of membrane at which a surface pole of microvilli will form subsequently. Thus, the cytoplasmic polarization of clathrin precedes overt polarization at the surface. At mitosis, the clathrin relocates to the spindle and is distributed to both daughter cells. It resumes an apical location beneath the surface pole of microvilli in polar daughter 1/16 cells, but remains dispersed in apolar daughter 1/16 cells. Both the lysosomal and Golgi antigens are distributed throughout the cytoplasm until the early 16-cell stage. In pairs of 16-cell blastomeres both antigens aggregate in a single cluster and do so whether the surface phenotype of the blastomeres is polar or apolar. The position of this cluster is not consistently related to the point of contact with the other cell in the pair but there is a suggestion that in cells with a polar surface phenotype the polar foci of Golgi/lysosomal antigens are located between the nucleus and the surface pole at earlier time points, but shift to a position between the basolateral membrane and the nucleus at the later time point. In intact 16-cell embryos also, the aggregated Golgi/lysosomal antigens of polar cells appear to localize to the basal region. The distributions of these various organelles in embryonic cells reported here show a number of differences from those reported previously for mature, differentiated cells.

Animals↗
PubMed1984

A monoclonal anti-DNA antibody also binds to cell-surface protein(s).

A murine monoclonal anti-DNA antibody ( PME77 ) has been found to bind tightly to the plasma membrane of Raji cells. We show here that this monoclonal anti-DNA antibody reacts in a radioimmunoassay with the cell surface of a variety of mammalian cell types and that the antigenic determinant recognized by the monoclonal anti-DNA antibody at the surface of Raji cells is resistant to DNase. It belongs to polypeptides removed from the cell surface by a mild proteinase K treatment.

Animals↗
PubMed1984

Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubules reflects some type(s) of linkage(s) between them and that such linkages may play an important role in the location and intracellular transport of lysosomes inside cells.

Animals↗
PubMed1984

Ca2+-ATPase of the sarcoplasmic reticulum shares a common domain with a membrane glycoprotein associated with the cytoskeleton of microvilli.

On the basis of structural observations, it has been proposed that cytoskeletal organization of the intestinal microvilli could be related to striated muscle structure. We have prepared antibodies against an amphipathic membrane glycoprotein (140 kilodaltons) associated with microvillar cytoskeleton and investigated its occurrence in striated muscle. Frozen sections of striated muscle were prepared according to the technique of Tokuyasu and visualized by indirect immunofluorescence with antibodies to the 140-kilodalton protein. In longitudinal sections the labeling was concentrated mainly in the area of the I band. In cross sections a honeycomb pattern was observed, suggesting that the recognized antigen was probably associated with the periphery of the myofibrils. Ultrathin frozen sections prepared for electron microscopy revealed that this antigen is closely associated with the membrane of the sarcoplasmic reticulum. In muscle extracts, the antibodies to the intestinal microvillar 140-kilodalton protein recognized a protein of 100 kilodaltons that comigrates with the Ca2+-ATPase of the sarcoplasmic reticulum. They recognized a purified preparation of the Ca2+-ATPase and, more specifically, the trypsin-generated fragment A2, the NH2-terminal part of the molecule that is exposed on the cytoplasmic face of the sarcoplasmic reticulum. Although these two proteins, expressed in unrelated cells, have a different molecular size and are inserted in different types of membranes, they share a common structural domain responsible for their crossreactivity. We propose that this domain could also be responsible for a common function--namely, the bridging of actin filaments to membranes.

Animals↗
PubMed1984

Antibodies against lysosomal membranes reveal a 100,000-mol-wt protein that cross-reacts with purified H+,K+ ATPase from gastric mucosa.

Specific antibodies against lysosomal membranes were prepared by using techniques previously described (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol., 92:92-107) for obtaining organelle-specific antibodies. The purified antibodies stained an acidic vacuolar compartment as shown by double-labeling experiments with acridine orange and indirect immunofluorescence. Characterization of the antibodies by immunoreplica methods revealed one major protein of approximately 100,000 mol wt. The antibodies cross-reacted with purified H+,K+ ATPase from pig gastric mucosa, the enzyme responsible for HCl secretion, but not with ATPases transporting other ions. They may therefore recognize a component of the proton pump involved in the acidification of lysosomes. As was expected, secondary lysosomes contained immunoreactive antigen, as determined by the fine-structural localization of reaction product for peroxidase or immunogold probes in several cell types. The antigen was also found in vacuoles containing phagocytosed bacteria in macrophages so it is present in at least some of the compartments of an endocytic pathway. In liver, the antigen was present in small amounts on the plasma membrane and in large amounts in some coated vesicles (near the sinusoidal surface of hepatocytes), putative endosomes, two cisternae on the cis side of the Golgi complex, adjacent vesicles and vacuoles, and pericanalicular dense bodies. In summary, the antigen seems to be present in those compartments that have recently been demonstrated to be acidified by an ATP-driven pump.

Adenosine Triphosphatases↗
PubMed1984

[A new specificity for anti-DNA antibodies].

A monoclonal antibody directed against double stranded DNA obtained by fusion between myeloma cells and spleen cells from auto-immune B/W Mice, binds to protein(s) at the plasma membrane of human B lymphoblastoid cell lines Raji.

Animals↗
PubMed1984

[Asymmetrical distribution of clathrin in the Golgi apparatus of polypeptide-secreting cells].

Using an antibody revealed by the protein A-gold technique, we have studied the distribution of clathrin antigenic sites in the Golgi area of pancreatic B-cells. Golgi compartments showing an immunolabelling comprised extensive segments of cisternae, typical coated vesicles, dilated extremities of cisternae with condensing secretory material, and newly formed secretory granules. Most of the labelled membranes were observed at the trans Golgi pole while little immunoreactivity was found on the cis pole.

Clathrin↗
PubMed1983

Characterization of ouabain-resistant mutants of a canine kidney cell line, MDCK.

Madin-Darby canine kidney (MDCK) cells were mutagenized and variants resistant to 10, 160, and 2000 times the ouabain lethal dose for wild type cells selected. The phenotypes were stable in the absence of selection. The frequencies with which variants were recovered were consistent with genetic alterations being responsible for drug resistance. It was shown that 50% of the (Na+, K+)-ATPase activity present in mutant cells had a higher Kd for ouabain than normal while 50% remained wild type for ouabain binding. Wild type MDCK cells were measured to have 2 X 10(6) ouabain binding sites per cell with a Kd for the drug of 0.6-1.0 X 10(-7) M. The novel (Na+, K+)-ATPase activities in the mutants demonstrated Kd values for ouabain of 10(-5) M, 3 X 10(-4) M, or 3 X 10(-3) M for the different mutant classes tested. The rate of synthesis of the (Na+, K+)-ATPase as well as the total amount of enzyme per unit of cell protein was unaltered in the mutants. Comparison of the alpha subunit of the enzyme, known to contain the ouabain-binding site, by sodium dodecyl sulfate-gel electrophoresis did not reveal any difference in the size of this subunit in mutant versus wild type cells.

Animals↗
PubMed1983

Characterization of membrane glycoproteins involved in attachment of microfilaments to the microvillar membrane.

We have characterized a novel integral membrane glycoprotein, from intestinal microvilli, with a relative molecular mass (Mr) of 140 000 as measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This glycoprotein has been purified to homogeneity, and specific antibodies have been prepared to localize it immunocytochemically and to determine its topological organization with respect to the membrane bilayer. This protein's major features are: (1) in contrast to other major glycoproteins of the microvillus membrane it cannot be quantitatively extracted by detergents; (2) treatment of the core residue (an insoluble fraction which remains after Triton X-100 treatment of purified microvilli) with low-ionic-strength buffer in the presence of chelating agents promotes partial solubilization of the amphipathic glycoprotein; (3) controlled proteolysis with papain, using right-side-out sealed vesicles derived from microvilli, results in solubilization of the protein. Once solubilized by papain, the protein co-migrates (on SDS-PAGE) with the protein obtained by dialysis but, unlike the low-ionic-strength form, it does not bind detergents or exhibit hydrophilic properties. These observations are consistent with the protein having a small hydrophobic domain that anchors it to the microvillar membrane. Most of these features were reported some years ago for aminopeptidase, but this newly described protein has a distinct behaviour with respect to its association with microfilaments. We have demonstrated that the 110K protein, a major cytoskeletal protein of the lateral bridges, will bind to the glycoprotein in vitro. These observations suggest that this 140K polypeptide is a transmembrane protein and may provide attachment sites in vivo for cytoskeletal proteins.

Cytoskeleton↗
PubMed1983

Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli.

An integral membrane glycoprotein of pig intestinal microvilli which exists in two polypeptide forms [mol. wt. 140 K and 200 K as measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)] was purified to homogeneity and characterized. The 200-K form is probably a precursor of the 140-K species. We have localized the glycoprotein by electron microscope immunochemistry using specific antibodies and determined its topological organization with respect to the membrane bilayer. Triton X-100 treatments which solubilize most other microvillar membrane glycoproteins from purified, closed, right-side out vesicles do not efficiently extract this protein. The protein can be partially solubilized from the detergent-insoluble residue, either by treatment with proteases (trypsin or papain) or by exposure to low ionic strength buffer in the presence of chelating agents and detergents. Once solubilized by papain or trypsin, the protein co-migrates on SDS-PAGE with the protein obtained by low ionic strength extraction. However, the form of the protein released by papain does not bind detergents and exhibits hydrophilic properties. Our observations are consistent with the 140-K protein having a small hydrophobic domain that anchors it to the microvillar membrane. The 140-K glycoprotein binds in vitro to a 110-K protein of the core cytoskeleton residue. These observations suggest that the 140-K glycoprotein may be a transmembrane protein which may in vivo provide attachment sites for direct or indirect association with polypeptides of the microvillus cytoskeleton.

Actin Cytoskeleton↗
PubMed1983

A monoclonal antibody to the heavy chain of clathrin.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti-clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re-build another coated pit.

Animals↗
PubMed1983

Uvomorulin: a nonintegral membrane protein of early mouse embryo.

A monoclonal antibody has allowed the characterization of various forms of uvomorulin, a glycoprotein involved in the process of compaction of mouse morula. In addition to various degradation products, uvomorulin exists as a 120-kilodalton exocellular molecule stable at the cell surface. A short-lived 135-kilodalton precursor of uvomorulin has been detected after 10-min pulse labeling. Uvomorulin-like molecules are found on various tissues at various stages of development of the mouse.

Animals↗
PubMed1983

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

Animals↗
PubMed1983

Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence.

The G protein of vesicular stomatitis virus was implanted in the apical plasma membrane of Madin-Darby canine kidney cells by low pH-dependent fusion of the viral envelope with the cellular membrane. The amount of fusion as determined by removal of unfused virions, either by tryptic digestion or by EDTA treatment at 0 degree C, was 22-24% of the cell-bound virus radioactivity. Upon incubation of cells after implantation, the amount of G protein as detected by immunofluorescence diminished on the apical membrane and appeared within 30 min on the basolateral membrane. At the same time some G protein fluorescence was also seen in intracellular vacuoles. The observations by immunofluorescence were confirmed and extended by electron microscopy. Using immunoperoxidase localization, G protein was seen to move into irregularly shaped vacuoles (endosomes) and multivesicular bodies and to appear on the basolateral plasma membrane. These results suggest that the apical and basolateral domains of Madin-Darby canine kidney cells are connected by an intracellular route.

Animals↗
PubMed1982

A monoclonal antibody against a 135-K Golgi membrane protein.

A monoclonal antibody ( 53FC3 ) has been produced against a Golgi membrane protein with a mol. wt. of 135 000 which was originally identified using a polyclonal antiserum. Treatment of isolated, intact Golgi vesicles with protease caused a decrease in mol. wt. of 5000-10 000, whereas in the presence of Triton X-100, the protein was completely degraded. This shows that the protein spans the bilayer and that most of its mass is on the luminal side of Golgi membranes. Using two immunoelectron microscopic techniques, the protein was found in one or two cisternae on one side of the Golgi stack which, in normal rat kidney cells, had 4-6 cisternae. As an illustration of the use to which this monoclonal antibody can be put we present a light microscopic study of the disassembly and reassembly of the Golgi complex during mitosis.

Animals↗
PubMed1982

Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound.

We have used the technique of experimental wounding of confluent monolayers of normal fibroblasts to induce essentially unidirectional and synchronous cell movement at the edge of the wound. The intracellular location of the Golgi apparatus and the microtubule-organizing center was determined by double indirect immunofluorescence microscopy, using antibodies specific for the membranes of the Golgi apparatus and antibodies specific for tubulin, respectively. In cells at the wound edge, the immunolabeled Golgi apparatus and microtubule-organizing center were in close proximity to one another and located predominantly forward of the cell nucleus facing the wound. In the same cultures in cells removed from the wound, the two organelles were also coordinately located; however, they were randomly oriented with respect to the wound edge. This reorientation of the two organelles in cells at the wound edge was evident within minutes after wounding and persisted as cell extension subsequently occurred into the wound. These results suggest that both the Golgi apparatus and the microtubule-organizing center may participate in directing cell movement. The possible mechanisms involved are discussed in the light of previous hypotheses and experimental evidence concerning cell motility.

Animals↗
PubMed1982

Antibodies to the Golgi complex and the rough endoplasmic reticulum.

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.

Animals↗