Modulation of CENP-E organization at kinetochores by spindle microtubule attachment

Abstract

CENP-E is a protein of the kinesin superfamily that appears as small paired globules at kinetochores of chromosomes in mammalian cells during prometaphase and metaphase of mitosis [Yen et al., 1992: Nature 359:536–539]. In the present study we found that a significant number of chromosomes during early prometaphase in HeLa cells (approximately 30%) were stained with a CENP-E antibody in the form of large C-shaped “collars” that partially encircled the chromosomes. The C-shaped CENP-E collars were present only transiently and were completely replaced by small paired globular forms prior to metaphase. Most chromosomes had persistent CENP-E collars in cells blocked at mitosis with a vinblastine concentration sufficient to prevent all microtubule formation. Attachment of newly formed microtubules to the kinetochores after removal of vinblastine resulted in loss of the collars and replacement with small paired globules. Similarly, a higher proportion of chromosomes isolated from vinblastine-treated cells contained CENP-E collars (73%), and the “capture” (i.e., attachment) of microtubules by the chromosomes resulted in conversion of the collars into small paired globules in vitro. Thus, the CENP-E collars form prior to microtubule attachment and disappear after attachment of the chromosomes to the spindle. The CENP-E collars may facilitate capture of microtubules by chromosomes during prometaphase.