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M C Willingham

Publications and source records attributed to M C Willingham.

At least 217 records · Page 12Linked to original sources
PubMed1982

The morphologic pathway of exocytosis of the vesicular stomatitis virus G protein in cultured fibroblasts.

Cultured fibroblasts were infected with vesicular stomatitis virus (VSV) and the pathway of exocytosis of G protein, the transmembrane glycoprotein of VSV, was followed by immunofluorescence and electron microscopy. G protein was detected within the endoplasmic reticulum, within smooth vesicles and stacks in the Golgi region and on the cell surface. No G protein was detected in the coated regions of the Golgi. Our data are consistent with the hypothesis that coated regions of the Golgi are involved in transfer of lysosomal enzymes and other substances to lysosomes and not in exocytosis.

Animals↗
PubMed1982

Affinity labeling of the plasma membrane 3,3',5-triiodo-L-thyronine receptor in GH3 cells.

The binding of 3,3',5-triiodo-L-thyronine (T3) to GH3 rat pituitary tumor cells was studied at 15 degrees C and was shown to be saturable, reversible, and stereospecific. Least-squares analysis of the binding data showed two classes of binding sites with dissociation constants of 1.8 +/- 0.2 nM and 260 +/- 30 nM and binding capacities of (5.2 +/- 0.2) X 10(4) and (1.6 +/- 0.2) X 10(6) sites per cell, respectively. Affinity labeling of intact cells was carried out by incubation of cells with 0.3 nM N-bromoacetyl-[125I]T3 at 15 degrees C for 1 hr. Analysis of the cellular extracts by sodium dodecyl sulfate gel electrophoresis showed three labeled protein bands with apparent molecular masses of 55, 47, and 33 kilodaltons (kDal) in a ratio of 86:7:7. The labeling of only the 55-kDal protein band was selectively reduced to 50% by 20 microM unlabeled T3. Highly purified plasma membranes of GH3 cells were prepared and shown to be free of nuclei. Affinity labeling of the purified plasma membranes gave the same labeling pattern as with intact cells. Peptide mapping by Staphylococcus aureus V8 digestion of the 55-kDal protein from cells or plasma membranes gave the identical peptide fragments. Thus the 55-kDal protein labeled from intact cells is the same protein as that from purified plasma membranes. These results together with our earlier findings [Horiuchi, R., Cheng, S.-y., Willingham, M. & Pastan, I. (1982) J. Biol. Chem. 257, 3139-3144] suggest that the 55-kDal protein may be involved in mediating the uptake of T3 in GH3 cells.

Affinity Labels↗
PubMed1982

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

Acetyltransferases↗
PubMed1982

Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin.

The entry of many animal viruses into their host cells often proceeds via a specialized internalization pathway involving clathrin-coated regions of the plasma membrane. We have examined the effect of dansylcadaverine and amantadine on the entry of vesicular stomatitis virus (VSV) into mouse cells. Both compounds inhibit VSV entry as determined by fluorescence and electron microscopy, 3H-labeled VSV uptake, and VSV-dependent RNA synthesis assays. They also inhibit the uptake of alpha 2-macroglobulin, a protein that binds to specific membrane receptors and follows the same route of internalization. Dansylcadaverine is 20-fold more potent than amantadine in blocking virus and alpha 2-macroglobulin uptake. One cellular target for both of these amine-containing compounds appears to be the clustering of membrane-bound ligands or particles in clathrin-coated pits.

Amantadine↗
PubMed1982

Transit of epidermal growth factor through coated pits of the Golgi system.

Using the direct conjugate of epidermal growth factor (EGF) and horseradish peroxidase, we have followed the entry of EGF into KB (human carcinoma) cells. EGF initially was found bound diffusely to the entire cell surface at 4 degrees C; on warming to 37 degrees C, EGF was found clustered in clathrin-coated pits on the plasma membrane in 1 min or less. Within 1-2 min at 37 degrees C, EGF began to accumulate in receptosomes within the cell and remained there for up to 10 min. At 10-13 min after warming to 37 degrees C, EGF was found in thin reticular membranous elements of the Golgi system, as well as concentrated in the clathrin-coated pits present on these membranes. By 15 min after warming, EGF began to be delivered to lysosomes located near the Golgi system. These findings suggest that clathrin-coated pits in the Golgi reticular system accumulate EGF before delivery to lysosomes.

Carcinoma↗
PubMed1982

Saturable binding sites for vesicular stomatitis virus on the surface of Vero cells.

The binding of vesicular stomatitis virus (VSV) to Vero monkey cells was studied by using virus metabolically labeled with [35S]methionine. Under conditions where viral uptake did not occur (4 degrees C), apparent binding equilibrium was achieved within 12 h at a level representing 12% of the input virus. Two distinct forms of virus-cell interaction were found. At low concentrations of VSV, corresponding to multiplicities used for tissue culture studies, saturable binding was the major form of interaction. Saturation was complete at approximately 4,000 VSV virions per cell. At higher virus concentrations, nonsaturable binding prevailed. Trypsin treatment of Vero cells did not decrease the binding of VSV to the saturable binding sites. Internalization of VSV at 37 degrees C also displayed a saturable component which was directly comparable to that observed for binding. VSV binding to high-affinity, saturable sites on the plasma membrane may represent a receptor-mediated route of viral uptake.

Animals↗
PubMed1981

Journey to the center of the cell: role of the receptosome.

Fibroblasts contain a specific internalization pathway that carries hormones as well as some proteins and viruses from the cell surface to the cell interior. Initially, the ligands bind to mobile receptors that are randomly distributed on the cell surface. Next the ligand-receptor complexes are trapped and concentrated in specialized regions of the membrane termed bristle-coated pits. From the pit a smooth-walled vesicle containing the ligand forms and carries the ligand to the cell interior. Because of its role in receptor-mediated endocytosis, this vesicle has been termed a "receptosome."

Animals↗
PubMed1981

Binding, surface mobility, internalization, and degradation of rhodamine-labeled alpha 2-macroglobulin.

We have used quantitative fluorescence methods to examine the fate of rhodamine-labeled alpha 2-macroglobulin (R-alpha 2 M) after binding to cell-surface receptors on NRK and Swiss 3T3 cells. From measurements of fluorescence intensities in NRK cells fixed after incubation with R-alpha 2M, we found that uptake was saturable and that half-maximal uptake occurred at 130 nM R-alpha 2M. Fluorescence measurements on cell extracts of NRK and Swiss 3T3 cells also showed a half-maximal uptake of R-alpha 2M near 130 nM. We estimate that NRK cells can take up 10(6) molecules of R-alpha 2M per hour via receptor-mediated endocytosis. The mobility of alpha 2-macroglobulin receptors on the surface of Swiss 3T3 cells was measured by using fluorescence photobleaching recovery. The two-dimensional effective diffusion coefficient of R-alpha 2M receptors was approximately 8 X 10(-10) cm2 s-1, a value close to that previously obtained for insulin and epidermal growth factor receptors. Degradation of R-alpha 2M by the cells was followed by using the loss of fluorescence from the 185000-dalton band in sodium dodecyl sulfate--polyacrylamide gels. Rhodamine fluorescence was detected in the gels by using a microscope fluorescence spectrophotometer. NRK cells degraded alpha 2M to low molecular weight fragments with a t 1/2 of 15 min. Swiss 3T3 cells degraded about 75% of the alpha 2M with a t 1/2 of 1 h. The remaining 25% remained as the intact 185000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.

Animals↗
PubMed1981

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts.

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.

Binding Sites↗
PubMed1981

Clathrin and coated vesicle proteins Immunological characterization.

Antisera to a purified extract of bovine brain coated vesicles have been prepared. The extract contained clathrin (greater than 90%) and polypeptides of 35,000 (less than 5%) and 38,000 (less than 5%) molecular weight. Antibodies to clathrin were affinity purified on a homogeneous clathrin-Sepharose column and demonstrated to be monospecific by sensitive immunoprecipitation and immunofluorescence experiments. These antibodies give a fluorescence pattern consistent with coated pit localization in cultured cells from diverse species indicating extensive cross-reaction, and, thus, conservation of the clathrin antigen. Antibodies to the lower molecular weight proteins (LMWP) present in the clathrin preparation were also affinity purified on a column containing these polypeptides but devoid of clathrin. These antibodies cross-reacted completely and immunoprecipitated only clathrin from 3T3 cell extracts. Although the molecular weights and isoelectric points of the LMWP presented are quite similar to tropomyosins, these immunological results and limited protease digestion data indicate that tropomyosin and the LMWP are not related. Rather, the latter may be breakdown products of clathrin.

Animals↗
PubMed1981

Binding and mobility of the cell surface receptors for 3,3',5-triiodo-L-thyronine.

A fluorescent derivative of the thyroid hormone 3,3'-triiodo-L-thyronine binds to cultured mouse fibroblasts; such binding is saturable. Video intensification fluorescence microscopy indicates that binding occurs at the plasma membrane. Diffusion coefficients, obtained by fluorescence photobleaching recovery, are consistent with binding to a protein receptor on the cell surface.

Animals↗
PubMed1981

Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of alpha2-macroglobulin.

Affinity-purified antibodies prepared against the major coat protein of brain coated vesicles, clathrin, were microinjected into cultured fibroblasts, and their intracellular distribution was followed by immunofluorescence microscopy and ultrastructural immunocytochemistry. Microinjected anticlathrin antibodies were concentrated on coated regions of the plasma membrane and the GERL apparatus. When an excess of anticlathrin antibodies was injected into the cytosol, coated pits on the plasma membrane were covered by anticlathrin antibody but still functioned to cluster an internalize alpha2-macroglobulin. These results are discussed in terms of the role of clathrin in the pathway of receptor-mediated endocytosis. Our data indicate that in cultured fibroblasts coated pits are stable elements permanently attached to the plasma membrane.

Animals↗