Université de Paris, Institut Jacques Monod, 75013, Paris, France.
Institut Gustave Roussy, CNRS UMR 8200/Université Paris-Sud, 94 805, Villejuif, France.
Biol Cell. 2020 Jan;112(1):22-37. doi: 10.1111/boc.201900038. Epub 2019 Dec 11.
The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes.
We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein.
Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.
中心体通过控制微管（MT）细胞骨架的结构来调节细胞的空间组织。相反，中心体在细胞内的位置取决于它帮助组织的细胞骨架网络。在哺乳动物细胞中，中心体的定位涉及一群稳定锚定在中心粒上的 MT，中心粒是中心体的核心组成部分。一种含有蛋白 ninein 和 Cep170 的 MT 锚定复合物在亚末端附属物（SAP）中富集，这些附属物装饰着较老的中心粒（称为母中心粒）和中心粒近端。在这里，我们研究了 hVFL3/CCDC61 在中心体中的作用，hVFL3/CCDC61 是真核生物中一组将不同的细胞骨架纤维锚定到中心粒的蛋白质的人类同源物。
我们表明 hVFL3 与 MT 锚定复合物的成分在 SAP 和中心粒近端共定位，并与 Cep170 发生物理相互作用。hVFL3 的耗竭增加了母中心粒和子中心粒之间的距离，而不影响连接中心粒并包含蛋白根蛋白和 C-Nap1 的丝状连接体的组装。当通过使 C-Nap1 失活破坏连接体时，hVFL3 耗竭加剧了中心粒分裂，这一表型也在 SAP 其他成分耗竭后观察到。这支持 hVFL3 是 SAP 功能所必需的，我们通过显示 hVFL3 耗竭的有丝分裂细胞中的中心体定位受到干扰进一步证实了这一点。最后，我们发现 hVFL3 是一种 MT 结合蛋白。
总的来说，我们的结果支持 hVFL3 在有丝分裂期间在 SAP 处锚定 MT，并确保适当的中心体凝聚和定位。因此，尽管不同真核生物物种的中心粒附属物的形状有很大差异，但 VFL3 蛋白家族的作用在进化中似乎得到了保守。
