Fluorescent taxoid probes for microtubule research

Abstract

The use of the antitumor drug taxol as an experimental microtubule-stabilizing agent is widespread. Fluorescent taxol conjugates, although less employed, are very useful tools for several purposes in microtubule research. These include easily visualizing microtubule cytoskeletons in a variety of cells as well as in vitro assembled microtubules, studying the molecular recognition processes of taxoids by microtubules and investigating new microtubule-stabilizing agents. This chapter describes both the methods for working with fluorescent taxol conjugates and several applications employing the active fluorescent taxoids Flutax-1, Flutax-2, Hexaflutax, Rotax, and FChitax-3. These methods include visualizing microtubules in native and mildly fixed cytoskeletons from cultured cells, ciliate and flagellate protozoans and in living tumor cells, purification of tubulin from tumor cell lines and measurement of its taxoid binding capacity. The applications discussed include a homogeneous assay to screen for compounds binding the taxol site, the determination of the pathway of taxol entry into microtubules and the design of high affinity microtubule-stabilizing agents.