Demonstration and Analysis of Tubulin Binding Sites on Centrosomes

Abstract

Microtubule nucleation on centrosomes is vital to the establishment of organized microtubule arrays in cells. Despite recent advances, little is known about the sequence of molecular events which leads to microtubule assembly on centrosomes. A putative early step in the nucleation process is interaction of free tubulin dimers with centrosomes. Here, we asked if centrosomes indeed interact in a specific manner with free 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 are incubated with rhodamine-conjugated tubulin, centrosomes emerge as conspicuous sites of tubulin accumulation while other insoluble cytoplasmic cell structures are not stained. In mitotic cells, lysed at various stages of mitosis, fluorescent tubulin stains centrosomes and other mitotic structures, such as the mitotic spindle, the midzone of the cleavage furrow, and the center part of the midbody. Fluorescent tubulin staining of centrosomes in lysed cells is not affected by addition of high concentrations of serum albumin to fluorescent tubulin solutions prior to incubation. In contrast, addition of micromolar concentrations of unlabeled tubulin, to fluorescent tubulin solutions, strongly reduces centrosomal staining. The tubulin binding capacity of centrosomes is conserved following centrosome isolation. Using quantitative methods for analysis of fluorescent tubulin binding on centrosomes, we find that centrosomes contain about 25 000 saturable tubulin binding sites. The apparent dissociation constant of tubulin−centrosome complexes is circa 5 μM. The kinetics of tubulin association with centrosomes 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 above 100 mM. Our results suggest that accumulation of tubulin dimers is one intrinsic function of centrosomes. We propose that such a function is not accounted for by the presence of γ-tubulin on centrosomes and may be an important factor in the regulation of centrosome-dependent microtubule nucleation.