Biochemical and Genetic Approaches to Microtubule Function in Dictyostelium discoideum

Abstract

This chapter discusses the genetic and biochemical methods for analyzing both tubulin and the function of microtubules during the Dictyostelium life cycle. A two-dimensional (2D) gel system capable of resolving single charge differences in Dictyostelium polypeptides is developed. α- and β-tubulin is identified on high-resolution 2D gels, and microtubules is isolated in cytoskeleton preparations from amoebae and the gel system is used to screen potential microtubule mutants for electrophoretically altered microtubule proteins. Microtubules from Dictyostelium amoebae are localized by indirect immunofluorescence. The structure and nature of the microtubule system are defined in both growth and development. Conditions are established for preserving of Dictyostelium microtubules in vitro. This allowed the microtubules to be isolated and analyzed biochemically. Methods for isolating mutants with potentially altered microtubule proteins are devised. Once mutants are obtained, tubulin and the microtubule system from them are examined and the mutant phenotypes are characterized. A mutant possessing aberrations in cytoplasmic and mitotic microtubule systems is described. © 1987, Academic Press Inc.