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F Cabral

Publications and source records attributed to F Cabral.

At least 55 records · Page 3Linked to original sources
PubMed1983

Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly.

In the accompanying paper (Cabral, F., 1982, J. Cell. Biol., 97:22-29) we described the isolation and properties of taxol-requiring mutants of Chinese hamster ovary cells. We now show that at least one of these mutants, Tax-18, has an impaired ability to form a spindle apparatus. Immunofluorescence studies using antibodies to tubulin demonstrate that, when incubated in the absence of taxol, Tax-18 forms only a rudimentary spindle with few and shortened microtubules associated with the spindle poles. Furthermore, midbodies were not observed, consistent with an absence of cytokinesis. Essentially normal spindles and midbodies are seen in the presence of taxol. Electron microscopic examination indicates that centrioles and kinetochores are morphologically normal in the mutant strain. Pole-to-kinetochore microtubules were seen but interpolar microtubules were not. Taxol-deprived mutant cells stained with anti-centrosome serum show an elevated centriole content, indicating that the defect in Tax-18 does not affect centriole replication or prevent progression through the cell cycle. Although Tax-18 cells do not form a complete spindle in the absence of taxol, cytoplasmic microtubule assembly occurs in association with microtubule-organizing centers, and microtubules with apparently normal morphology exist throughout the cytoplasm. Observation of chromosome movement indicates that the defect in these cells occurs after prometaphase. These studies demonstrate that the formation of spindle microtubules requires cellular conditions that are different from those required for cytoplasmic microtubule formation. They further show that a normal spindle may be necessary for cytokinesis but not for progress of the cells through the cell cycle.

Alkaloids↗
PubMed1983

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells.

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.

Animals↗
PubMed1982

Revertants of a Chinese hamster ovary cell mutant with an altered beta-tubulin: evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell.

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20:29-36, 1980). This mutant carries an altered beta-tubulin but still grows normally at 37 degrees C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3 degrees C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered beta-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant beta-tubulin polypeptide. This second alteration appears to make the mutant beta-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in beta-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered beta-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

Animals↗
PubMed1981

Purification and characterization of a transformation-dependent protein secreted by cultured murine fibroblasts.

The major excreted protein (MEP) of transformed mouse fibroblasts has been purified, and monospecific antisera against it have been prepared. Synthesis and secretion of this protein have previously been shown to be stimulated by transformation or treatment with tumor-promoting phorbol esters, but its function is still not known [Gottesman, M. M. (1978) Proc, Natl. Acad. Sci. U.S.A. 75, 2767--2771; Gottesman, M. M., & Sobel, M. E. (1980) Cell (Cambridge, Mass.) 19, 449--455]. The purified protein shows charge heterogeneity by two-dimensional gel electrophoresis; the major intracellular and extracellular species have a molecular weight of 35 000 and a pI of 6.8--7.3. The purified secreted protein contains approximately 5--10% neutral sugar by weight and binds specifically to a concanavalin A--Sepharose affinity column. Translation of messenger ribonucleic acid (mRNA) from cells actively synthesizing MEP in cell-free reticulocyte or wheat germ systems, which are reported to be unable to glycosylate translated proteins, results in a product of Mr 33 000 which is presumably devoid of neutral sugar. However, on two-dimensional electrophoresis, the MEP mRNA translation products continue to show charge heterogeneity similar to that seen in intact cells, suggesting that there may be multiple coordinately controlled mRNAs for MEP or a single mRNA species which can be translated in a variety of ways.

Animals↗
PubMed1981

Intermediate filaments from Chinese hamster ovary cells contain a single protein. Comparison with more complex systems from baby hamster kidney and mouse epidermal cells.

Intermediate filaments (IF) were prepared under identical conditions from Chinese hamster ovary (CHO), baby hamster kidney (BHK), and primary mouse epidermal cells. CHO and BHK cells each have a major IF protein (Mr = 55,000); these proteins comigrate on two-dimensional gels. BHK cells have an additional protein constituent (Mr = 54,000) which is absent from CHO. The IF preparation from the epidermal cells was considerably more complex, containing at least five proteins with molecular weights between 50,000 and 70,000, none of which has a mobility similar to the CHO protein. If assembled in vitro from crude CHO IF preparations were morphologically indistinguishable from filaments assembled from other cell types. Analysis of these in vitro polymerized filaments from CHO cells using circular dichroism and wide angle x-ray diffraction revealed that the CHO filaments were structurally similar to filaments prepared from BHK or epidermal cells. The Mr = 55,000 proteins from CHO and BHK cells were found to have identical one- and two-dimensional peptide maps, demonstrating that their amino acid sequences must be very similar. These studies indicate that CHO cells possess the simplest system thus far described for studying the assembly of intermediate filaments.

Animals↗
PubMed1981

In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells.

This paper presents evidence that the intermediate filament (IF) subunits of muscle cells (skeletin or desmin) and fibroblastic cells (decamin or vimentin) separately form homopolymer IF in vitro and, when mixed, prefer to form copolymer IF in vitro. Because they coexist in cells, they may also form copolymers in vivo. The IFs of baby hamster kidney fibroblasts (BHK-21) consist of a major subunit, decamin, and two minor subunits which, on the basis of two-dimensional gel and peptide mapping criteria, are identical to the alpha and beta subunits of smooth muscle desmin. The subunits differ only in their degrees of phosphorylation: alpha desmin contained 2 mol/mol of O-phosphoserine whereas beta desmin contained none. The decamin and desmin subunits assembled into homopolymer IF in vitro in high yield from purified denatured subunits under identical conditions of pH and ionic strength. However, homopolymer decamin IF disassembled into soluble protofilaments in solutions of ionic strength less than 0.05 mol/liter whereas homopolymer desmin IF disassembled at ionic strength less than 0.03 mol/liter. When decamin and desmin were mixed together as denatured subunits or as soluble protofilaments, the IF assembled in vitro had solubility properties intermediate between those of the homopolymer IFs, indicating that the two subunits had formed copolymer IF. The stoichiometry of copolymerization as determined in mixtures in which one subunit was present in excess was suggestive of the formation of three-chain units. The possibility of nonspecific aggregation was eliminated by isolation of stable three-chain alpha-helix-enriched particles from such IF. When tracer amounts of [35S]methionine-labeled decamin were mixed with desmin, labeled IFs were obtained under conditions in which homopolymer decamin IFs were soluble. These in vitro findings may be of physiological significance because native BHK-21 IF also had solubility properties similar to those of the copolymer IF.

Animals↗
PubMed1981

Isolation of a taxol-resistant Chinese hamster ovary cell mutant that has an alteration in alpha-tubulin.

Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules [Schiff, P. B., Fant, J. & Horwitz, S. B. (1979) Nature (London) 277, 665-667]. Taxol-resistant cells were selected from a population of UV-mutagen-treated Chinese hamster ovary cells by a single-step procedure. These mutants have normal morphologies and growth rates but are 2- to 3-fold more resistant to the toxic effects of the drug than the wild-type parent. One out of 20 mutants screened by two-dimensional electrophoresis for chemical alterations in tubulin had an "extra" spot with a more acidic isoelectric point that alpha-tubulin. This extra spot was shown to be an electrophoretic variant alpha-tubulin by its copurification with tubulin in crude microtubule-containing preparations and by one-dimensional peptide mapping. The alpha-tubulin mutant was found to be temperature sensitive for growth, and this property was used as the basis for the selection of revertants. Seventeen temperature-resistant revertants of the alpha-tubulin mutant were selected for their ability to grow at 40 degrees C and three of these revertants were found to have simultaneously lost their taxol resistance and the electrophoretic variant alpha-tubulin. These results provide evidence that an alteration in alpha-tubulin can confer taxon resistance on a mammalian cell line and suggest that alpha-tubulin is essential for cell viability.

Alkaloids↗
PubMed1981

Induction of specific protein synthesis by phorbol esters in mouse epidermal cell culture.

The induction of specific protein synthesis by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was studied in cultured mouse epidermal cells by two-dimensional gel electrophoresis after pulse-labeling with [35S]methionine. While overall protein synthesis was moderately inhibited by TPA, the synthesis of five specific proteins was increased. Three of these proteins (M.W. 25,000, M.W. 55,000, and M.W. 70,000) were either not synthesized or synthesized at low rates in untreated cells, while two proteins (M.W. 35,000 and M.W. 50,000) were also synthesized in controls but to a lesser extent than in TPA-treated cells. Increased synthesis of the M.W. 50,000 protein could be seen as early as 1 hr after TPA exposure, while maximum induction of all proteins was observed at 6 hr. By 24 hr, synthesis rates of these proteins had returned to near basal levels even if TPA exposure was continued. The amino acid analog canavanine (at levels which inhibited protein synthesis to the same extent as does TPA) or nonpromoting analogs of TPA did not induce these proteins. Pulse-chase studies indicated that these proteins were not degradation products which may have resulted from TPA exposure and that the M.W. 25,000 and M.W. 35,000 protein appear to be rapidly turned over. 32PO4 labeling indicated that only the M.W. 55,000 protein was significantly phosphorylated and that TPA did not induce a qualitative change in the pattern of phosphorylation of epidermal proteins. Definitive identification of these proteins has not been made, but the specific stimulation of their synthesis supports a model of tumor promoter action in which promoters induce a specific program of changes in macromolecular synthesis in the epidermis.

Animals↗
PubMed1980

CHO mutants resistant to colchicine, colcemid or griseofulvin have an altered beta-tubulin.

Single-step mutants of Chinese hamster ovary (CHO) cells have been isolated which are resistant to killing by the anti-mitotic drugs colchicine, colcemid or griseofulvin. Two-dimensional gel analysis showed that two mutants resistant to griseofulvin, one resistant to colcemid and one resistant to colchicine carry an alteration in the beta-tubulin subunit. Most of the remaining isolates are believed to be permeability mutants on the basis of their cross resistance to drugs which do not interefere with microtubular polymerization or function (Ling and Thompson, 1974; Bech-Hansen, Till and Ling, 1976). A reduced amount of the wild-type beta-tubulin protein remained in each of the beta-tubulin mutants, but a beta-tubulin protein with a more basic isoelectric point also appeared. Messenger RNAs coding for both wild-type and variant beta-tubulins were found in at least one mutant as assayed by in vitro translation in a reticulocyte lysate. This indicates that the altered tubulin does not arise as the result of a posttranslational modification.

Animals↗
PubMed1980

Ultrastructural localization to 10 nm filaments of an insoluble 58K protein in cultured fibroblasts.

Using a simple procedure, we have isolated a Triton X-100 insoluble protein (58,000 mol wt) from cultured Chinese hamster ovary (CHO) cells, a fibroblastic cell line. The isolated protein, judged homogeneous by two-dimensional gel electrophoresis, was used to elicit antibodies in rabbits. The antiserum obtained is specific for the 58K protein as determined by immunoprecipitation from detergent solubilized 35S-labeled CHO cells and by antibody labeling of SDS solubilized Swiss 3T3 cell extracts separated on a 10% polyacrylamide gel. Indirect immunofluorescence localization with this antiserum in fixed permeabilized Swiss 3T3 cells, whose flat morphology makes them superior for localization studies, reveals filaments that radiate from the perinuclear region to the cell periphery. These filaments were identified as 10 nm filaments by electron microscopic localization using the EGS and ferritin bridge procedures. We conclude that the 58K protein is a major constituent of fibroblast 10 nm filaments. Other intracellular structures were not labeled by this technique.

Animals↗
PubMed1978

Identification of cytochrome c oxidase subunits in nuclear yeast mutants lacking the functional enzyme.

Yeast mutants specifically lacking cytochrome c oxidase activity were screened for cytochrome c oxidase subunits by one- and two-dimensional electrophoresis, electrophoresis in exponential gradient gels, and immunoprecipitation with antisera against one or more of the cytoplasmically made subunits of the enzyme. Two cytochrome c oxidase-less nuclear mutants previously described from this laboratory each lack one or more mitochondrially synthesized cytochrome c oxidase subunits while possessing all four cytoplasmically synthesized subunits of that enzyme. The subunits remaining in these mutants were not assembled with each other; the cytoplasmically made subunits IV and VI could be released from the mitochondria by sonic oscillation, in contrast to the situation in wild type cells. No electrophoretically detectable alterations were found in any of the cytochrome c oxidase subunits present in the mutants. Nuclear mutations may thus cause both a loss as well as a defective assembly of mitochondrially made cytochrome c oxidase subunits.

Cell Nucleus↗
PubMed1978

The determination of similarities in amino acid composition among proteins separated by two-dimensional gel electrophoresis.

A simple and rapid procedure has been developed to determine similarities in amino acid composition among cellular proteins separated by two-dimensional gel electrophoresis. Cells in tissue culture are simultaneously labeled with two different amino acids each tagged with a different radioisotope. The proteins are then separated on two-dimensional gels and their location on the gels determined by Coomassie-blue staining or autoradiography. Elution of the protein from the appropriate region of the gel followed by liquid scintillation counting yields an isotope ratio which reflects the ratio of the two amino acids in the protein. Examples of the use of this technique in analyzing mutant proteins, proteins altered by carbamylation, and cell proteins with similar amino acid composition (e.g., actin and tubulin) are given.

Amino Acids↗