DISRUPTION OF CENP ANTIGEN FUNCTION PERTURBS DYNEIN ANCHORING TO THE MITOTIC KINETOCHORE

Injection of purified autoantibodies against human centromeric protein s into HeLa cells during interphase disrupts the organization of the k inetochore and interferes with chromosomal movements during the subseq uent mitosis even though the chromosomes retain the ability to bind mi crotubules. We have investigated the hypothesis that this phenotype ar ises from effects on cytoplasmic dynein, the microtubule motor protein . In previous experiments we found that introduction of anticentromere antibodies into cell nuclei during the G(1)- or S-phases causes a pro metaphase-like arrest, while injections during G(2)-phase cause a meta phase arrest. We show here that, in both cases, the level of detectabl e cytoplasmic dynein at kinetochores is significantly decreased. In co ntrast, when injected cells were permitted to enter mitosis in the abs ence of microtubules (conditions where trilaminar kinetochores could b e detected by electron microscopy), the intensity of dynein labeling o n the kinetochores was identical to that seen in uninjected control ce lls exposed to colcemid. Therefore, the loss of dynein label on mitoti c kinetochores was correlated both with the injection of anticentromer e antibodies and with the presence of intact spindle microtubules. We suggest that the injection of anticentromere antibodies somehow weaken s the association of dynein with the kinetochore, so that when microtu bules are present, these motor molecules are pulled away from the kine tochores as they generate force. This model offers an explanation for the failure of chromosomes of injected cells to move normally in mitos is even though they have attached microtubules.