CENP-C IS REQUIRED FOR MAINTAINING PROPER KINETOCHORE SIZE AND FOR A TIMELY TRANSITION TO ANAPHASE

The human autoantigen CENP-C has been demonstrated by immunoelectron m icroscopy to be a component of the inner kinetochore plate. Here we ha ve used antibodies raised against various portions of CENP-C to probe its function in mitosis. We show that nuclear microinjection of anti-C ENP-C antibodies during interphase causes a transient arrest at the fo llowing metaphase. Injection of the same antibodies after the initiati on of prophase, however, does not disrupt mitosis. Correspondingly, in direct immunofluorescence using affinity-purified human anti-CENP-C an tibodies reveals that levels of CENP-C staining are reduced at centrom eres in cells that were injected during interphase, but appear unaffec ted in cells which were injected during mitosis. Thus, we suggest that the injected antibodies cause metaphase arrest by reducing the amount of CENP-C at centromeres. Examination of kinetochores in metaphase-ar rested cells by electron microscopy reveals that the number of trilami nar structures is reduced. More surprisingly, the few remaining kineto chores in these cells retain a normal trilaminar morphology but are si gnificantly reduced in diameter. In cells arrested for extended period s, these small kinetochores become disrupted and apparently no longer bind microtubules. These observations are consistent with an involveme nt of CENP-C in kinetochore assembly, and suggest that CENP-C plays a critical role in both establishing and/or maintaining proper kinetocho re size and stabilizing microtubule attachments. These findings also s upport the idea that proper assembly of kinetochores may be monitored by the cell cycle checkpoint preceding the transition to anaphase.