hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning

Abstract

Background. 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.Results. We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MTanchoring 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, hVFL3depletion 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.Conclusions and significance. 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.

CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions

“…CCDC61/VFL3 might interact with a centrosomal and basal body-specific protein that is yet to be identified, and scaffold the protein with microtubules to construct regularly aligned basal body-associated structures. Two groups recently proposed that CCDC61 interacts with CEP170 and might play a role in the subdistal appendage function of centrioles (B€ arenz et al, 2018;Pizon et al, 2020). While our manuscript was under review, Pizon and colleagues also reported CCDC61 association with microtubules (Pizon et al, 2020), in agreement with our data.…”

“…SAS6 assembly, for instance, is probably aided by its interaction with CEP135 and STIL (Dzhindzhev et al, 2014;Lin et al, 2013;Ohta et al, 2014), whereas for the XRCC4/XLF complex this function is exerted by its associations with DNA ligase IV, Ku70/80 and DNA (Ochi et al, 2014). Putative CCDC61 binding proteins, such as CEP170 (B€ arenz et al, 2018;Pizon et al, 2020) might play an equivalent role in CCDC61. Thus, the overarching principles of higher-order oligomerization and stabilization by other proteins appears to be conserved among the XRCC4 superfamily members.…”

“…These studies point toward a potential role of CCDC61 in the organization of basal bodies in cells with multiple cilia. A recent report suggests that CCDC61 might also be involved in chromatin alignment and mitotic spindle assembly, possibly by anchoring CEP170 (B€ arenz et al, 2018;Pizon et al, 2020). However, how CCDC61 functions mechanistically is currently unknown.…”

With Age Comes Maturity: Biochemical and Structural Transformation of a Human Centriole in the Making

“…Subdistal appendages, which are formed just below DAs, are usually robust electron densities that are highly variable in shape and in number ( Figure 1 C) [ 35 , 36 ]. On their periphery, SDAs are associated with MTs, which regulate MT centrosome positioning [ 37 , 38 , 39 , 40 , 41 ] and cohesion [ 42 ].…”

“…The number and shape of SDAs is highly variable [ 35 , 36 ] even between the cells of the same cell type. The ends of SDAs are associated with MTs [ 39 , 40 ], which mediate centrosome positioning, directional cell migration, and centrosome cohesion [ 37 , 204 , 205 ]. DAs are positioned distally to SDAs [ 19 , 20 , 21 , 36 , 79 ] and look like nine finger-like protrusions, slightly curved toward the centriole distal end [ 19 ] ( Figure 1 A).…”