CENP-U cooperates with Hec1 to orchestrate kinetochore-microtubule attachment.
ABSTRACT Mitosis is an orchestration of dynamic interaction between chromosomes and spindle microtubules by which genomic materials are equally distributed into two daughter cells. Previous studies showed that CENP-U is a constitutive centromere component essential for proper chromosome segregation. However, the precise molecular mechanism has remained elusive. Here, we identified CENP-U as a novel interacting partner of Hec1, an evolutionarily conserved kinetochore core component essential for chromosome plasticity. Suppression of CENP-U by shRNA resulted in mitotic defects with an impaired kinetochore-microtubule attachment. Interestingly, CENP-U not only binds microtubules directly but also displays a cooperative microtubule binding activity with Hec1 in vitro. Furthermore, we showed that CENP-U is a substrate of Aurora-B. Importantly, phosphorylation of CENP-U leads to reduced kinetochore-microtubule interaction, which contributes to the error-correcting function of Aurora-B. Taken together, our results indicate that CENP-U is a novel microtubule binding protein and plays an important role in kinetochore-microtubule attachment through its interaction with Hec1.
- SourceAvailable from: Takashi Sado[Show abstract] [Hide abstract]
ABSTRACT: CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.Chromosome Research 01/2014; · 2.69 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.The Journal of cell biology. 08/2014; 206(4):509-24.
- [Show abstract] [Hide abstract]
ABSTRACT: Breast cancer is a heterogeneous disease that progresses to the critical hallmark of metastasis. In the present study, we show that the High Mobility Group A1 (HMGA1) protein plays a fundamental role in this process in basal-like breast cancer subtype. HMGA1 knockdown induces the mesenchymal to epithelial transition and dramatically decreases stemness and self-renewal. Notably, HMGA1 depletion in basal-like breast cancer cell lines reduced migration and invasion in vitro and the formation of metastases in vivo. Mechanistically, HMGA1 activated stemness and key migration-associated genes which were linked to the Wnt/beta-catenin, Notch and Pin1/mutant p53 signalling pathways. Moreover, we identified a specific HMGA1 gene expression signature that was activated in a large subset of human primary breast tumours and was associated with poor prognosis. Taken together, these data provide new insights into the role of HMGA1 in the acquisition of aggressive features in breast cancer.Oncotarget 07/2013; · 6.63 Impact Factor