LOCALIZATION OF CENP-E IN THE FIBROUS CORONA AND OUTER PLATE OF MAMMALIAN KINETOCHORES FROM PROMETAPHASE THROUGH ANAPHASE

We have conducted a detailed ultrastructural analysis of the distribut ion of the kinesin-related centromere protein CENP-E during mitosis in cultured human, rat kangaroo and Indian muntjac cells. Using an affin ity-purified polyclonal antibody and detection by 0.8 nm colloidal gol d particles, CENP-E was localized primarily to the fibrous corona of t he kinetochore in prometaphase and metaphase cells. Some labeling of t he kinetochore outer plate was also observed. The distribution of fibr ous corona-associated CENP-E did not change dramatically following the attachment of microtubules to the kinetochore. Thus, the normal disap pearance of this kinetochore substructure in conventional electron mic rographs of mitotic chromosomes with attached kinetochores is not due to the corona becoming stretched along the spindle microtubules as has been suggested. Examination of cells undergoing anaphase chromatid mo vement revealed the presence of CENP-E still associated with the outer surface of the kinetochore plate. At the same time, the majority of d etectable CENP-E in these cells was associated with the bundles of ant iparallel microtubules in the central spindle. CENP-E in this region o f the cell is apparently associated with the stem body matrix material . The simultaneous localization of CENP-E on centromeres and the centr al spindle during anaphase was confirmed by both wide-field microscopy of human cells and conventional fluorescence microscopy of rat kangar oo cells. Together, the observations reported here are consistent with models in which CENP-E has a role in promoting the poleward migration of sister chromatids during anaphase A.