Designer microenvironments for the analysis of cell and tissue function.
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Animals↗
Biomedical subjects
C H Streuli
Publications and source records attributed to C H Streuli.
Expression of extracellular matrix components is regulated by substratum.
Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.
Animals↗
Recombination resulting in unusual features in the polyomavirus genome isolated from a murine tumor cell line.
Polyomavirus-induced tumor formation in the adult natural mouse host has been investigated. Tumors were produced in nude mice with the transformation-defective mutant strain NG18 after a long latency period by apparent activation of a cryptic endogenous transforming viral function. A tumor cell line, designated ScB, was established and characterized. Cells from this morphologically distinct line were unusual in that they grew in soft agar but did not form foci. They were highly tumorigenic. They had a 3.1-kilobase major viral transcript that hybridized to probes derived from regions encoding both the T antigens and the structural proteins. ScB cells expressed polyomavirus small T antigen, a slightly altered middle T antigen, and a truncated large T antigen but no capsid proteins. Middle T antigen preserved its interactions with host proteins of 60 and 37 kilodaltons and with c-src. Analysis of cDNA and genomic clones indicated that the stable viral insert in the ScB genome contained multiple copies of the viral B-enhancer. The genome contained two intragenic inversions which created novel early- to late-strand switches. A simple model for the generation of one inversion is proposed that involves the juxtaposition of two stem-loop structures at an illegitimate recombination site; the location of the inverted segment within the integrated sequence permits use of the viral late polyadenylation signal in early-region transcripts, as confirmed by DNA sequence. A repetitive sequence may facilitate recombination at the other inversion site. Both the biological consequences of the observed rearrangements and the structure of the integrated viral DNA suggest that the recombination events are nonrandom.
Animals↗
Myristylated polyomavirus VP2: role in the life cycle of the virus.
The double-stranded genome of the small DNA tumor virus, polyomavirus, is enclosed in a capsid composed of a major protein, VP1, which associates as pentameric capsomeres into an icosahedral structure, and two minor proteins, VP2 and VP3, whose functions and positions within the structure are unknown. The N-terminal glycine of the VP2 coat protein has been shown to be cotranslationally acylated with myristic acid. To study the function of this modification and the role of VP2 in the life cycle of polyomavirus, the N-terminal glycine, critical to the myristylation consensus sequence, has been altered to a glutamic acid or a valine residue by site-directed oligonucleotide mutagenesis. The glycine----glutamic acid mutant DNA has been further studied. When transfected into cells permissive for the polyomavirus full lytic life cycle, this mutant DNA replicated at levels comparable to those of wild-type viral DNA, and small amounts of nonrevertant (mutant) virus could be harvested from the cultures. The virus particles viewed by electron microscopy appeared slightly distorted, but the ratio of full to empty particles was similar to that produced in a wild-type viral infection. Mutant virus was capable of reinfecting permissive cells but with a considerably reduced efficiency.
Animals↗
Subcellular localisation of the middle and large T-antigens of polyoma virus.
The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.
Animals↗
Efficient oligodeoxyribonucleotide-directed deletion mutagenesis using pEMBL vectors: removal of early region introns from polyoma virus mutants.
We have used oligodeoxyribonucleotide-directed deletion mutagenesis to remove early region introns from polyoma virus mutants. To this end we compared single priming, double priming, and gapped duplex approaches using either priming at 37 degrees C or at the critical temperature. The gapped duplex approach, coupled with priming at the critical temperature, resulted in up to 70% yield of the desired product. In conjunction with the use of the pEMBL vector system this method was simplified to yield specific deletions from cloned large DNA fragments with high efficiency. The resulting mutant plasmids could be used directly for biological assays without retransformation or recloning. RNA and protein analyses showed that removal of the large T- or middle T-antigen introns from polyoma early region mutants dl23 and dl8 was specific and resulted in DNA competent for the synthesis of only one T antigen.
Antigens, Viral, Tumor↗
Variants of BALB/c 3T3 cells lacking complex gangliosides retain a fibronectin matrix and spread normally on fibronectin-coated substrates.
Evidence has accumulated that di- and trisialogangliosides are involved in the interaction of cells with fibronectin. We have therefore tested the ability of variants of BALB/c 3T3 deficient in such gangliosides to organize a fibronectin matrix and to spread on fibronectin-coated substrates. Whereas BALB/c 3T3 cells contained gangliosides GM3, GM1, and GD1a, direct chemical analysis showed that five out of six variants isolated contained no detectable GD1a. By the overlaying of thin layer chromatograms of cellular gangliosides with 125I-cholera toxin, these variants were also found to lack ganglioside GM1. In contrast, the sialogalactoprotein profile of these cells, analyzed using an 125I-ricin/SDS polyacrylamide gel overlay technique, was similar to that of the parent cell line. All variants organized an extensive fibronectin matrix comparable to that of BALB/c 3T3, as shown using either immunofluorescence or lactoperoxidase-catalyzed iodination. The variants could also spread on fibronectin-coated substrates and adopt a morphology similar to that of BALB/c 3T3 cells, with little or no difference in the concentration of fibronectin required for 50% cell spreading. Cell spreading of the variants was accompanied by the formation of focal contacts and microfilament bundles, in a manner closely resembling that seen with BALB/c 3T3 cells. Treatment of BALB/c 3T3 cells with neuraminidase, which converts much of the cellular GD1a to GM1, did not affect cell spreading on fibronectin. The results clearly demonstrate that complex gangliosides are not essential for retention of a fibronectin matrix or for spreading on fibronectin-coated substrates.
Actin Cytoskeleton↗
Isolation of variants of BALB/c 3T3 cells defective in complex ganglioside biosynthesis.
In an attempt to clarify the relationship between altered glycosphingolipid metabolism and other aspects of the transformed phenotype, we have isolated variants of BALB/c 3T3 cells (clone A31) which are defective in synthesis of the more complex gangliosides (GM2, GM1 and GD1a). The selection protocol was based on the specificity of cholera toxin for ganglioside GM1, and the ability to lyse cells which bound toxin using anti-toxin and complement. Following treatment of cells with ethane methane sulfonate (EMS), populations resistant to lysis were obtained after 5-6 rounds of selection, despite a low and rather variable killing efficiency (75-95%). Five out of six clones isolated from such populations showed reduced toxin-binding capacity and loss of gangliosides more complex than GM3, as determined by metabolic labelling with [1-14C] palmitate. An identical phenotype was displayed by a variant isolated from a non-mutagenized population of cells. The phenotype remained stable for several months in culture and for over at least 40 cell doublings. Ganglioside nomenclature is according to Svennerholm [24].
Animals↗
Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.
Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].
Animals↗
The use of cholera toxin as a probe to study the organisation of ganglioside GM1 in membranes.
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Animals↗
The cholera toxin receptor ganglioside GM remains associated with triton X-100 cytoskeletons of BALB/c-3T3 cells.
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Animals↗
Myristic acid is coupled to a structural protein of polyoma virus and SV40.
In the lytic cycle of papova viruses, both uncoating of the viral genome after infection and assembly of functional virions take place in the cell nucleus. The mechanisms by which newly internalized virions are targeted to the nucleus and viral DNA encapsidated into particles are poorly understood. Although the major capsid protein VP1 is involved in endocytosis, and largely defines virion structure, the functions of the minor proteins VP2 and VP3 have remained obscure. Here we show that VP2 from both polyoma virus and simian virus 40 (SV40) is covalently linked to myristic acid; this is the first report of a myristylated protein in the nucleus and of a fatty acid being important in the structure of a nonenveloped virus. We consider the implications of this unusual modification on encapsidation and suggest that VP2 may be a scaffolding protein for virion assembly.
Amino Acid Sequence↗