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

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...

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Bibliographic Details
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
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Summary: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.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.201307049