MICROINJECTION OF MITOTIC CELLS WITH THE 3F3 2 ANTI-PHOSPHOEPITOPE ANTIBODY DELAYS THE ONSET OF ANAPHASE/

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosome have aligned at the metaphase plate. We provide evidence i ndicating that a kinetochore phosphoepitope plays a role in this check point pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes bef ore their congression to the metaphase plate. Once chromosomes are ali gned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a co ncentration-dependent delay in the onset of anaphase. Injected antibod y inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope a t kinetochores. Microinjection of the antibody eliminated the asymmetr ic expression of the phosphoepitope normally seen on sister kinetochor es of chromosomes during their movement to the metaphase plate. Chromo some movement to the metaphase plate appeared unaffected in cells inje cted with the antibody suggesting that asymmetric expression of the ph osphoepitope on sister kinetochores is not required for chromosome con gression to the metaphase plate. In antibody-injected cells? the epito pe remained expressed at kinetochores throughout the prolonged metapha se, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with age nts that perturb microtubules, the 3F3/2 phosphoepitope quickly reappe ared at kinetochores. Immunoelectron microscopy revealed that the 3F3/ 2 epitope is concentrated in the middle electron-lucent layer of the t rilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubul e attachment or is a signaling component of the checkpoint pathway reg ulating the metaphase to anaphase transition.