Short Communication Assembly of Microtubules in a Cytoplasmic Extract of Tobacco BY-2 Miniprotoplasts in the Absence of Microtubule-Stabilizing Agents

The assembly of microtubules occurred even in the absence of microtubule-stabilizing agents in a cytoplasmic extract obtained from evacuolated protoplasts (miniprotoplasts) of tobacco BY-2 cells. The assembled microtubules were arranged side by side forming big bundles and two types of bridge-like structure were observed between adjacent microtubules. Key words: Cross-bridge (microtubule) — Microfilament — Microtubule bundle — Miniprotoplast — Self-assembly — Tobacco BY-2 cells. An experimental system in which tubulin could assem-ble in vitro into microtubules and in which molecules that regulate the polymerization of tubulin could be identified would greatly facilitate studies on the regulation of micro-tubule dynamics in living cells. Since the conditions for the assembly of microtubules in extracts of brain tissue were established (Weisenberg 1972, Shelanski et al. 1973), similar procedures for the polymerization of tubulin in vitro have been employed for a variety of other tissues and cultured cell lines from various animal species in attempts to study the dynamics of formation of microtubules and to isolate tubulin and microtubule-associated proteins (Vallee and Bloom 1984, Olmsted 1986, Wiche et al. 1991). How-ever, efforts to adapt this method to the isolation of tubulin from higher plants have been hampered by the low level of tubulin in plant cell extracts and by the presence in such ex-tracts of low-molecular-weight inhibitors of the polymeriza-tion of tubulin and endogenous peptidases (Fosket et al. 1981). The polymerization of microtubules from purified or partially purified plant tubulin has been achieved in the presence of microtubule-stabilizing agents (Morejohn and Fosket 1982, Mizuno 1985), but no reports have been published, to our knowledge, on the assembly of microtu-bules in crude extracts to which no microtubule-stabilizing agents have been added. In the present study, we succeeded in polymerizing microtubules in a cytoplasmic extract ob-tained from evacuolated protoplasts of tobacco BY-2 cell