Véronique Pizon1, Noémie Gaudin1, Marion Poteau2, Carmen Cifuentes-Diaz3, Roland Demdou1, Vincent Heyer4,5,6,7, Bernardo Reina San Martin4,5,6,7, Juliette Azimzadeh1. 1. Université de Paris, Institut Jacques Monod, 75013, Paris, France. 2. Institut Gustave Roussy, CNRS UMR 8200/Université Paris-Sud, 94 805, Villejuif, France. 3. Institut du Fer à Moulin, Inserm UMR-S U839/UPMC, 75005, Paris, France. 4. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France. 5. Institut National de la Santé et de la Recherche Médicale (INSERM), U1258, Illkirch, France. 6. Centre National de la Recherche Scientifique (CNRS), UMR7104, Illkirch, France. 7. Université de Strasbourg, Illkirch, France.
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 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. 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.
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 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. 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.
Authors: Takashi Ochi; Valentina Quarantotti; Huawen Lin; Jerome Jullien; Ivan Rosa E Silva; Francesco Boselli; Deepak D Barnabas; Christopher M Johnson; Stephen H McLaughlin; Stefan M V Freund; Andrew N Blackford; Yuu Kimata; Raymond E Goldstein; Stephen P Jackson; Tom L Blundell; Susan K Dutcher; Fanni Gergely; Mark van Breugel Journal: Structure Date: 2020-05-05 Impact factor: 5.006