Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment

Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation. test and two-way ANOVA as per the experimental requirement. Recombinant DNA and transfections CENPB (1C158aa) cDNA fragment was PCR amplified from a PLK1 plasmid in which the C-terminal PBD domain was replaced with the first 158 amino acids of CENPB (pQCXIN-Flag-Plk1deltaC-CENPB(1C158)) (a gift from Mark Burkard) and cloned into full length pEGFP-hBLM and pEGFP-hBLM(Q672) plasmids at AgeI site to generate N-terminally tagged CENPB (1C158aa) fusion proteins. Transfections of DNA plasmids were performed using FuGene HD (Promega) according to the manufacturers Rhoa guidelines. All plasmids and their sequences are available upon request. Forward primer: (CENPB-For1) 5-TAAGCAACCGGTATGGGCCCCAAGAGGCGACAG-3; Reverse primer: (CENPB-linker-Rev1)5-TAAGCAACCGGTCTAGCACTTGCGCCCCCAGCACTTGCTCCACCGGCCGGACTG GCAGGCGCCGC-3 Reporting summary Further information on research design is available in the?Nature Research Reporting Summary linked to this article. Supplementary information Supplementary Information(6.4M, pdf) Peer Review File(512K, pdf) Supplementary Movie 1(1.7M, mov) Supplementary Movie 2(171K, mov) Supplementary Movie 3(373K, mov) Supplementary Movie 4(1.0M, mov) Supplementary Movie 5(995K, mov) Supplementary Movie 6(437K, mov) Reporting Summary(89K, pdf) source data(2.1M, xlsx) Acknowledgements We would like to thank the people in the Genome Centre for their great support. We thank Robert Lera and Mark Burkard for providing the RPE1 derivative cells and the CENPB cDNA, Marcel van Vugt for HAP1 and HAP1 BLM-knockout cells, and Phillip North for GM08505 Bloom Syndrome fibroblasts. We thank Jessica Hudson for BLM siRNA oligos. We also thank Kim Nasmyth, Jon Baxter and Mark Burkard for helpful PK11007 comments on the study. This work is supported by Sir Henry Dale Fellowship (Ref. 104178/Z/14/Z) from Wellcome Trust and the Royal Society, and by the Genome Damage and Stability Centre. K.L.C. is the recipient of Sir Henry Dale Fellowship.?Funding for open access charge: Charity Open Access Fund (COAF). Author contributions O.A.J. and K.L.C designed and performed the experiments with help from A.T., T.O. and A.H. O.A.J, A.T., T.O. and K.L.C. analysed the data. K.L.C. wrote the paper with inputs from all authors. Data availability The authors declare that the data supporting the findings of this study are available within the PK11007 paper and its Supplementary?information files. Raw imaging data are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Footnotes Peer review information: would PK11007 like to thank anonymous reviewers for their contribution to the peer review of this work. Peer review reports are available. Publishers note: Springer Nature remains neutral with PK11007 regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Ankana Tiwari, Tomisin Olukoga. Supplementary information Supplementary Information accompanies this paper at 10.1038/s41467-019-10938-y..