Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells

Abstract

The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis. We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.

Mechanical factors activateß‐catenin‐dependent oncogene expression in APC1638N/+mouse colon

“…In addition, it has been shown in vivo that mechanical irritation of the epithelium can lead to an increased mutation rate (Takahashi et al, 2000). This suggests that cells of the APC deficient colon, which can already experience chromosomal missegregation due to dominant negative effects of truncated APC protein or insufficient full length APC during mitosis (Fodde et al, 2001;Tighe et al, 2004;Aoki et al, 2007;Draviam et al, 2006), may be exposed to an alternative route to loss of heterozygosity when subjected to mechanical stress, in addition to the effects demonstrated here on tumor gene expression in the haploinsufficient tissue.…”

MCAK associates with EB1

“…We therefore propose that EB1 binding at this site holds MCAK in an inactive conformation. Given recent progress in our understanding of the roles played by EB1 and MCAK in mitosis (Kline-Smith et al, 2004;Draviam et al, 2006;Knowlton et al, 2006;Ohi et al, 2007), the possibility that these proteins could directly influence one another's respective activities at MT plus ends in mitotic cells is intriguing.…”

Cyclin A/cdk2 Regulates Adenomatous Polyposis Coli-dependent Mitotic Spindle Anchoring

“…The mitotic arrest imposed by cyclin A/cdk2 inhibition is a consequence of activation of the mitotic spindle assembly checkpoint. The effects of APC depletion on initiating the spindle checkpoint are controversial, with some studies reporting mitotic arrest (8,31) and another reporting no effect (4). There is also some controversy about the exact role of APC in the spindle assembly checkpoint with some studies indicating no effect on the checkpoint (8, 31), whereas others FIGURE 7.…”

“…The majority of APC mutations occurs in a region from codons 1,000 to 1,500 called the mutation cluster region (MCR) and result in truncations of the C-terminal half of the protein, which includes the ␤-catenin, microtubule, and EB1 binding sites of APC (1,2). Depletion of either APC or EB1 produce almost identical mitotic defects, indicating their interaction is critical to normal spindle formation (4,8). However, expression of various truncation mutants across the MCR revealed interesting differences the spindle defects observed, suggesting that this role of APC in spindle function is not solely due to interaction with EB1 (4).…”