Miyamoto Tatsuo, Akutsu Silvia Natsuko, Fukumitsu Akihiro, Morino Hiroyuki, Masatsuna Yoshinori, Hosoba Kosuke, Kawakami Hideshi, Yamamoto Takashi, Shimizu Kenji, Ohashi Hirofumi, Matsuura Shinya
Department of Genetics and Cell Biology.
Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
Hum Mol Genet. 2017 Nov 15;26(22):4429-4440. doi: 10.1093/hmg/ddx330.
Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by congenital reduction of head circumference. Here, we identified compound heterozygous mutations c.731 C > T (p.Ser 244 Leu) and c.2413 G > T (p.Glu 805 X) in the WDR62/MCPH2 gene, which encodes the mitotic centrosomal protein WDR62, in two siblings in a Japanese family with microcephaly using whole-exome sequencing. However, the molecular and cellular pathology of microcephaly caused by WDR62/MCPH2 mutation remains unclear. To clarify the physiological role of WDR62, we used the CRISPR/Cas9 system and single-stranded oligonucleotides as a point-mutation-targeting donor to generate human cell lines with knock-in of WDR62/MCPH2 c.731 C > T (p.Ser 244 Leu) missense mutation. In normal metaphase, the mitotic spindle forms parallel to the substratum to ensure symmetric cell division, while WDR62/MCPH2-mutated cells exhibited a randomized spindle orientation caused by the impaired astral microtubule assembly. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for the maintenance of spindle orientation through astral microtubule development. In this study, we demonstrated that WDR62 is a PLK1 substrate that is phosphorylated at Ser 897, and that this phosphorylation at the spindle poles promotes astral microtubule assembly to stabilize spindle orientation. Our findings provide insights into the role of the PLK1-WDR62 pathway in the maintenance of proper spindle orientation.
原发性小头畸形（MCPH）是一种常染色体隐性疾病，其特征为先天性头围减小。在此，我们通过全外显子组测序，在一个患有小头畸形的日本家庭的两名同胞中，鉴定出WDR62/MCPH2基因中的复合杂合突变c.731 C > T（p.Ser 244 Leu）和c.2413 G > T（p.Glu 805 X），该基因编码有丝分裂中心体蛋白WDR62。然而，由WDR62/MCPH2突变导致的小头畸形的分子和细胞病理学仍不清楚。为阐明WDR62的生理作用，我们使用CRISPR/Cas9系统和单链寡核苷酸作为靶向点突变的供体，生成了敲入WDR62/MCPH2 c.731 C > T（p.Ser 244 Leu）错义突变的人类细胞系。在正常中期，有丝分裂纺锤体与基质平行形成，以确保细胞对称分裂，而WDR62/MCPH2突变的细胞则表现出由星状微管组装受损导致的纺锤体方向随机化。研究表明，有丝分裂激酶Polo样激酶1（PLK1）是维持纺锤体方向通过星状微管发育所必需的。在本研究中，我们证明WDR62是一种在Ser 897位点被磷酸化的PLK1底物，并且这种在纺锤体极的磷酸化促进星状微管组装以稳定纺锤体方向。我们的研究结果为PLK1-WDR62通路在维持适当纺锤体方向中的作用提供了见解。
