DEMONSTRATION AND ANALYSIS OF TUBULIN BINDING-SITES ON CENTROSOMES

Microtubule nucleation on centrosomes is vital to the establishment of organized microtubule arrays in cells. Despite recent advances, littl e is known about the sequence of molecular events which leads to micro tubule assembly on centrosomes. A putative early step in the nucleatio n process is interaction of free tubulin dimers with centrosomes. Here , we asked if centrosomes indeed interact in a specific manner with fr ee tubulin dimers. Using lysed cells, we show that centrosomes have a specific capacity to accumulate free tubulin molecules as compared to most other cytoplasmic cell structures. When interphasic lysed cells a re incubated with rhodamine-conjugated tubulin, centrosomes emerge as conspicuous sites of tubulin accumulation while other insoluble cytopl asmic cell structures are not stained. In mitotic cells, lysed at vari ous stages of mitosis, fluorescent tubulin stains centrosomes and othe r mitotic structures, such as the mitotic spindle, the midzone of the cleavage furrow, and the center part of the midbody. Fluorescent tubul in staining of centrosomes in lysed cells is not affected by addition of high concentrations of serum albumin to fluorescent tubulin solutio ns prior to incubation. In contrast, addition of micromolar concentrat ions of unlabeled tubulin, to fluorescent tubulin solutions, strongly reduces centrosomal staining. The tubulin binding capacity of centroso mes is conserved following centrosome isolation. Using quantitative me thods for analysis of fluorescent tubulin binding on centrosomes, we f ind that centrosomes contain about 25 000 saturable tubulin binding si tes. The apparent dissociation constant of tubulin-centrosome complexe s is circa 5 mu M. The kinetics of tubulin association with centrosome s are slow, with a half-saturation time of about 3 min and a very slow dissociation rate. Tubulin binding to centrosomes is inhibited at low temperatures, at pH above neutrality, and at NaCl concentrations abov e 100 mM. Our results suggest that accumulation of tubulin dimers is o ne intrinsic function of centrosomes. We propose that such a function is not accounted for by the presence of gamma-tubulin on centrosomes a nd may be an important factor in the regulation of centrosome-dependen t microtubule nucleation.