Dynactin is required for microtubule anchoring at centrosomes

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo . In living cells, perturbation of the dynein-dynactin interaction profound ly blocks mitotic spindle assembly, and inhibition or depletion of dynein o r dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein-dy nactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome-lysosome system, bu t the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules ar e reorganized into unfocused arrays. The pericentriolar components, gamma t ubulin and dynactin, are lost from centrosomes, but pericentrin localizatio n persists. Microtubule nucleation from centrosomes proceeds relatively nor mally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localiza tion. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide n ew insights into dynactin-cargo interactions.