Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, China.
Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, China.
J Biol Chem. 2022 Dec;298(12):102658. doi: 10.1016/j.jbc.2022.102658. Epub 2022 Nov 7.
Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel structure from S phase to late mitosis remains unclear. Here, we demonstrate mainly using cell synchronization, RNA interference, immunofluorescence and time-lapse-microscopy, biochemical analysis, and methods that the cartwheel persistently assembles and maintains centriole engagement and centrosome integrity during S phase to late G2 phase. Blockade of the continuous accumulation of centriolar Sas-6, a major cartwheel protein, after procentriole formation induces premature centriole disengagement and disrupts pericentriolar matrix integrity. Additionally, we determined that during mitosis, CDK1-cyclin B phosphorylates Sas-6 at T495 and S510, disrupting its binding to cartwheel component STIL and pericentriolar component Nedd1 and promoting cartwheel disassembly and centriole disengagement. Perturbation of this phosphorylation maintains the accumulation of centriolar Sas-6 and retains centriole engagement during mitotic exit, which results in the inhibition of centriole reduplication. Collectively, these data demonstrate that persistent cartwheel assembly after procentriole formation maintains centriole engagement and that this configuration is relieved through phosphorylation of Sas-6 by CDK1-cyclin B during mitosis in human cells.
车轮装配被认为是起始前体中心粒发生的第一步；然而，从 S 期到晚期有丝分裂，组装好的人类车轮结构持续存在的原因仍不清楚。在这里，我们主要通过细胞同步化、RNA 干扰、免疫荧光和延时显微镜、生化分析以及方法证明，车轮在 S 期到晚期 G2 期持续组装并保持中心粒结合和中心体完整性。在前体中心粒形成后，阻断中心粒 Sas-6 的连续积累，一种主要的车轮蛋白，会导致中心粒过早脱离，并破坏中心粒周围基质的完整性。此外，我们确定在有丝分裂期间，CDK1-周期蛋白 B 将 Sas-6 磷酸化在 T495 和 S510，破坏其与车轮成分 STIL 和中心粒周围成分 Nedd1 的结合，并促进车轮解体和中心粒脱离。这种磷酸化的干扰维持了中心粒 Sas-6 的积累，并在有丝分裂退出期间保持中心粒的结合，从而抑制了中心粒的复制。总之，这些数据表明，在前体中心粒形成后，车轮的持续组装维持了中心粒的结合，而这种构象通过 CDK1-周期蛋白 B 在人类细胞的有丝分裂过程中对 Sas-6 的磷酸化而得到缓解。
