MOLECULAR CHARACTERIZATION OF THE 50-KD SUBUNIT OF DYNACTIN REVEALS FUNCTION FOR THE COMPLEX IN CHROMOSOME ALIGNMENT AND SPINDLE ORGANIZATION DURING MITOSIS

Dynactin is a multi-subunit complex which has been implicated in cytop lasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and a nalyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserve d, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured ver tebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. O verexpression of p50 in COS-7 cells disrupted mitosis, causing cells t o accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically di storted. Sedimentation analysis revealed the dynactin complex to be di ssociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings repr esent clear evidence for dynactin and cytoplasmic dynein codistributio n within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dy nactin in modulating cytoplasmic dynein binding to an organelle, and i mplicate both dynactin and dynein in chromosome alignment and spindle organization.