Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells

• Published in: The Journal of cell biology 2014-03, Vol.204 (5), p.697-712
• Main Authors: Keller, Debora, Orpinell, Meritxell, Olivier, Nicolas, Wachsmuth, Malte, Mahen, Robert, Wyss, Romain, Hachet, Virginie, Ellenberg, Jan, Manley, Suliana, Gönczy, Pierre
• Format: Article
• Language: English
• Subjects: Cell Cycle; Cell Cycle Proteins - analysis; Cell Cycle Proteins - metabolism; Cell Cycle Proteins - physiology; Cell Line, Tumor; Centrioles - metabolism; Centrioles - ultrastructure; Correlation analysis; Cytoplasm; Dimerization; Fluorescence; Fluorescence Recovery After Photobleaching; Humans; Models, Biological; Protein Transport; Proteins
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.201307049

SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.