FLUORESCENT TAXOIDS AS PROBES OF THE MICROTUBULE CYTOSKELETON

Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FL UTAX) and N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-li ganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alpha beta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affin ity. FLUTAX-induced microtubule elongation is markedly Mg2+-dependent, encompassing the binding of one Mg2+ ion more per tubulin dimer polym erized than in the case of taxol. A small perturbation of the absorpti on spectrum of bound FLUTAX is consistent with a cationic microenviron ment relative to the solution. The fluorescence anisotropy of FLUTAX i ncreases by an order of magnitude upon binding to microtubules and tim e-resolved measurements Indicate that the fluorescein moiety remains c onsiderably mobile on a protein surface. The rare of labeling suggests that this is the outer microtubule wall. Alternatively, the microtubu le lumen would be functional. FLUTAX- and ROTAX-induced microtubules, radial structures, and organized microtubule bundles are readily obser ved under the fluorescence microscope. Rapid and accurate visualizatio n of native (or very mildly fixed) cytoplasmic and spindle microtubule s of a variety of permeabilized cells is simply obtained with micromol ar FLUTAX, with an advantage over immunofluorescence. In addition, FLU TAX labels the centrosomes of PtK2 cells more intensely than antibodie s to alpha- or beta-tubulin, and io-localizing with antibodies to gamm a-tubulin. Two brightly fluorescent spots, probably separating or dupl icating centrioles, can be resolved in the centrosomes of interphase c ells. This finding indicates that centrosomes may well be additional t argets of action of taxoids. FLUTAX strongly labels microtubules near the spindle poles, as well as microtubules at the telophase spindle eq uator and the central part of the midbody in cytokinesis (instead of t he dark zone frequently observed with immunofluorescence), suggesting a predominant interaction of FLUTAX with sites at which tubulin is new ly polymerized. Nanomolar concentrations of FLUTAX also permit specifi c imaging of centrosomes, half-spindles and midbodies in growing U937 cells. (C) 1998 Wiley-Liss, Inc.