Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin ontocentrosomes

Centrosome assembly is important for mitotic spindle formation and if defec tive may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule n ucleation, pericentrin and gamma tubulin, is inhibited in the absence of mi crotubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cy toplasmic dynein by microinjection of dynein antibodies or by overexpressio n of the dynamitin subunit of the dynein binding complex dynactin. Consiste nt with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin h ave a diminished capacity to nucleate microtubules. In living cells express ing a green fluorescent protein-pericentrin fusion protein, green fluoresce nt protein particles containing endogenous pericentrin and gamma tubulin mo ve along microtubules at speeds of dynein and dock at centrosomes. In Xenop us extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtub ules and inhibited by dynein antibodies. From these studies we conclude tha t pericentrin and gamma tubulin are novel dynein cargoes that can be transp orted to centrosomes on microtubules and whose assembly contributes to micr otubule nucleation.