CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

Abstract

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAbl77) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, -250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAbl77 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAbl77 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.

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).…”

“…CENP-E was first identified as a protein that is present on the kinetochores of mitotic cells during chromosome movement (Compton et al 1991; Yen et al 1991). The sequence of mammalian CENP-E revealed significant sequence similarity between the NH 2 -terminal domain of the 312-kD CENP-E protein and the motor domain of members of the kinesin superfamily (Yen et al 1992).…”

CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can Be Achieved via Redundant Mechanisms in Mammalian Cells

“…Injected rabbit and rat antibodies were detected with Cy-5-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA). Endogenous CENP-E was detected with a mouse monoclonal antibody (mAb) 177 or rat polyclonal antibody that was generated and affinity-purified as described previously for rabbit HX-1 anti-CENP-E antibodies (Yen et al, 1991;Schaar et al, 1997). Rabbit anti-hBUB1 and rabbit anti-hBUBR1 were previously described Jablonski et al, 1998).…”

“…CENP-E is a kinesin-like protein that binds to kinetochores during mitosis (Yen et al, 1991(Yen et al, ,1992Cooke et al, 1997;Yao et al, 1997). Depletion of CENP-E from HeLa cell kinetochores via antibody microinjection or expression of dominant negative mutants results in mitotic arrest with unaligned chromosomes Chan et al, 1998).…”

Skeletor, a Novel Chromosomal Protein That Redistributes during Mitosis Provides Evidence for the Formation of a Spindle Matrix

“…In female meiotic cells, in the absence of centrosomes, microtubules nucleated around chromosomes and then self-organized into a bipolar spindle (Gard 1992; Theurkauf and Hawley 1992). Various nuclear molecules have been directly shown to undergo a mitosis-specific redistribution pattern and associate with microtubules and centrosomes, including the centromere proteins (CENPs) (Pankov et al 1990; Yen et al 1991; Casiano et al 1993), the inner centromere proteins (INCENPs) (Cooke et al 1987; Earnshaw and Cooke 1991; Mackay et al 1993), and the nuclear mitotic apparatus (NuMA) proteins (Lydersen and Pettijohn 1980; Compton et al 1992; Yang and Snyder 1992). Furthermore, the discovery of microtubule flux at both ends of spindle microtubules (Mitchison 1989; Sawin and Mitchison 1991, Sawin and Mitchison 1994), in conjunction with the demonstration that chromosomes whose kinetochore fibers have been severed by UV-microbeam irradiation still moved poleward (Forer et al 1997), suggest that current models for how chromosomes move to the poles are incomplete and that additional structural components of the spindle apparatus may exist (for review see Pickett-Heaps et al 1997).…”