CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells.
CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduces tension across the centromere, increases the incidence of spindle pole fragmentation, and results in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes show typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibit normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule-binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.
McEwen
B F
BF
Division Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA. bruce.mcewen@wadsworth.org
Chan
G K
GK
Zubrowski
B
B
Savoian
M S
MS
Sauer
M T
MT
Yen
T J
TJ
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D020090:Chromosome Segregation; D002875:Chromosomes / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism / Q000648:ultrastructure; D008677:Metaphase; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008938:Mitosis; D009038:Motion; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure
D002868:Chromosomal Proteins, Non-Histone; C068688:centromere protein E