Abstract

The isolation and characterization of microtubule mutants using drug-resistance selections has provided insights into the role of microtubules in mitosis, the mechanism of drug-resistance in mammalian cells, and regulatory mechanisms in microtubule assembly. Recently, methods have been devised which enhance our ability to isolate mutants; and ideas on how to use mutants to study microtubule function, assembly, and regulation have been refined. Using this information, we propose to isolate many new mutants to determine the range of mutant phenotypes and to test developing ideas on the mechanism of drug action and cellular mechanisms of resistance to their effects. Considerable effort will be devoted toward the development of new selections for obtaining mutants in microtubule assembly and in the expression of microtubule associated proteins (MAPs). We already have one mutant of the first type and believe we may have one of the second. At the same time it will be necessary to develop tools to fully characterize these mutants. Included in these studies will be the isolation and characterization of the spindle apparatus from CHO cells, the identification of MAPs, and the generation of antibodies against various MAP and spindle components. This work should complement and extend our current knowledge of microtubule physiology and provide a basis for understanding drug action and cellular mechanisms of resistance.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029955-07
Application #
3277603
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1982-02-01
Project End
1990-01-31
Budget Start
1988-02-01
Budget End
1989-01-31
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225

  Publications

Minotti, A M; Barlow, S B; Cabral, F (1991) Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin. J Biol Chem 266:3987-94
Barlow, S B; Cabral, F (1991) Alterations in microtubule assembly caused by the microtubule-active drug LY195448. Cell Motil Cytoskeleton 19:9-17
Minotti, A M; Loeb, L M; Cook, R et al. (1990) Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography. Anal Biochem 184:28-34
Sawada, T; Cabral, F (1989) Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells. J Biol Chem 264:3013-20
Schibler, M J; Barlow, S B; Cabral, F (1989) Elimination of permeability mutants from selections for drug resistance in mammalian cells. FASEB J 3:163-8
Cabral, F; Barlow, S B (1989) Mechanisms by which mammalian cells acquire resistance to drugs that affect microtubule assembly. FASEB J 3:1593-9
Boggs, B A; Minotti, A M; Loeb, L M et al. (1988) Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus. J Biol Chem 263:14566-73
Brady, R C; Schibler, M J; Dedman, J R et al. (1987) Biochemical characterization of isolated CHO cell mitotic spindles: identification of calmodulin-binding proteins. J Cell Sci 87 ( Pt 5):643-9
Boggs, B; Cabral, F (1987) Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells. Mol Cell Biol 7:2700-7
Brady, R C; Cabral, F; Dedman, J R (1986) Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells. J Cell Biol 103:1855-61

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