Biochemical and genetic approaches to microtubule function in Dictyostelium discoideum

Methods have been developed for analyzing tubulin and microtubules from the cellular slime mold D. discoideum. alpha- and beta-tubulin have been identified on high-resolution 2D gels, and microtubules have been isolated in cytoskeleton preparations from amoebae (White et al., 1983). These studies have revealed properties unique to Dictyostelium tubulin. Amoebal microtubules can be visualized by indirect immunofluorescence, which has aided in the identification of inhibitors which specifically depolymerize microtubules and block amoebae in mitosis. The mitotic inhibitors CIPC, NOC, and TBZ have been used to select resistant mutants which are currently the subjects of biochemical, morphological, and genetic analysis (Katz et al., 1982; White, 1983). One mitotic inhibitor-resistant mutant, CIPC 6, was found to be temperature-sensitive for growth at 27 degrees C as well as CIPC-resistant. At the restrictive temperature amoebae from this mutant are deficient in the passage through mitosis. After incubation for 12 hours at the restrictive temperature, 20% of the CIPC 6 amoebae displayed condensed chromosomes, compared to 2% at the permissive temperature, as determined by Giemsa staining. Examination of the microtubules of this mutant by indirect immunofluorescence showed abnormal spindle microtubule formation at the restrictive temperature, which is the likely cause of the mitotic arrest (White, 1983). Cytoplasmic microtubules were also disrupted in nonmitotic amoebae of CIPC 6 at 27 degrees C. This temperature-sensitive loss of microtubule function suggested the possibility that tubulin from CIPC 6 might be altered. When tubulin from CIPC 6 was examined on 2D gels, no reproducible electrophoretic change was observed from that of the wild type. Through further characterization of mitotic inhibitor-resistant mutants like CIPC 6, more mitotic or microtubule mutants will be identified. Among these mutants, some should contain electrophoretically altered tubulin, microtubule-associated proteins, or components of the amoebal cytoskeleton. Possessing Dictyostelium mutants with known biochemical alterations in cytoskeletal proteins should reveal significant information regarding the function of these proteins in eukaryotic growth and development.