MODIFICATION OF TUBULIN WITH THE FLUOROCHROME 5-(4,6-DICHLOROTRIAZIN-2-YL)AMINO FLUORESCEIN AND THE INTERACTION OF THE FLUORESCENT PROTEIN WITH THE ISOLATED MEIOTIC APPARATUS

¹ Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755 and The Marine Biological Laboratory, Woods Hole, Massachusetts 02543

Microtubules (MTs) assembled in vitro have been labeled with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)amino fluorescein (DTAF) using a modification of the procedure of Keith et al. (1981). When the fluorescent MTs were analyzed by polyacrylamide gel electrophoresis under reducing conditions both the major components of the MTs, the tubulin dimers and microtubule-associated proteins (MAPs), appeared distinctly fluorescent and thus were modified covalently with the fluorochrome. The fluorescent MTs were cold labile and reassembled with kinetics identical to control microtubule proteins. In addition, fluorescent tubulin dimers, purified from the mixture of labeled MT proteins, assembled with both an initial rate and final extent of assembly indistinguishable from a control, unlabeled sample when both were examined at the same protein concentration. On occasion, however, the fluorescent protein has been observed to demonstrate a one to two minute lag, an anomaly that is a function of total protein concentration. When the reassembled fluorescent MTs were examined by negative stain electron microscopy, morphologically normal polymers were observed. Since fluorescent tubulin dimers are potentially valuable probes for the examination of MT growth polarity, the ability of the fluorescent protein to assemble in association with nucleation centers in vitro was examined. For these experiments, meiotic spindles from the surf clam Spisula solidissima were isolated and used to nucleate the assembly of fluorescent MTs in vitro. The results of such experiments revealed linear, fluorescent arrays within the spindle and asters. The results of the experiments reported here demonstrate that tubulin labeled with DTAF retained its ability to self-assemble and to assemble in association with microtubule organizing centers (MTOCs) in vitro. Moreover, the polymer formed from the DTAF labeled proteins was distinctly fluorescent and could be observed and recorded using conventional fluorescence optics.