CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

Abstract

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate.Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function.The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

CENP-meta, an Essential Kinetochore Kinesin Required for the Maintenance of Metaphase Chromosome Alignment in Drosophila

“…Consistent with this view, microinjection of cells with antibodies specific for CENP-E caused a delay in anaphase onset (Yen et al 1991) and a misalignment of chromosomes (Schaar et al 1997). Similarly, both immunodepletion and inhibitory antibody addition experiments with Xenopus cell-free extracts caused failure in chromosome alignment at metaphase (Wood et al 1997), while antisense oligonucleotide meditated suppression of CENP-E accumulation in mammalian cultured cells yields chronically mono-oriented chromosomes with spindles flattened along the plane of the substrate (Yao et al 2000).…”

Prime movers: the mechanochemistry of mitotic kinesins

“…The initial capture of kinetochores by the microtubule lattice and their subsequent transport to microtubule plus ends can be mediated by dynein 96 and the kinesin-7 family motor KIF10 [97][98][99] (see Fig. 1b, chromosome v; FIG.…”

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

“…3F3/2 epitopes are located within the middle, electron-lucent kinetochore layer and have also been implicated in the checkpoint that controls anaphase onset (Campbell and Gorbsky 1995). The attachment and proper positioning of chromosomes on the spindle likely involves cytoplasmic dynein (Echeverri et al 1996) andCENP-E (1997;Schaar et al 1997;Wood et al 1997), both of which are located in the outer plate and corona. The possible functions of other kinetochore components, including ZW10 (e.g., Starr et al 1997), CENP-F (also known as mitosin; Liao et al 1995) and MCAK (Wordeman and Mitchison 1995), remain to be determined.…”