In view of recent warnings for artifacts in fluorescence correlation spectroscopy, the diffusion coefficient of a series of labeled proteins in a wide range of molecular mass (43-670 kD) was determined and shown to be correct with respect to published values and the theory. Fluorescence correlation spectroscopy was then applied to the study of fluorescently labeled tubulin and its oligomerization in vitro induced by Mg2+ ions, paclitaxel, and a fluorescent derivative of paclitaxel (Flutax2). By applying relations derived from the theory of Oosawa, we were able to determine the association constant of the oligomers induced by Mg2+. With Flutax2 our experiments show that at nanomolar concentration, the fluorescent derivative is able to recruit tubulin dimers and to form oligomers of defined size. Flutax2 does not bind to microtubules preformed with paclitaxel, but it becomes preferentially incorporated into microtubules when Flutax2 oligomers are preformed, and microtubule formation is induced by paclitaxel addition. This shows that their incorporation into microtubules is faster than the displacement of the prebound drug. Experiments using fluorescently labeled tubulin and (unlabeled) paclitaxel confirm the induction of tubulin oligomers at limiting paclitaxel concentrations.
Copyright 2004 Biophysical Society