Self-assembling SAS-6 multimer is a core centriole building block.
Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a "cartwheel" with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with approximately 6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles.
Gopalakrishnan
Jayachandran
J
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Guichard
Paul
P
Smith
Andrew H
AH
Schwarz
Heinz
H
Agard
David A
DA
Marco
Sergio
S
Avidor-Reiss
Tomer
T
D000818:Animals; D030801:Animals, Genetically Modified; D029742:Caenorhabditis elegans Proteins / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000502:physiology*; D002502:Centrioles / Q000378:metabolism*; D018385:Centrosome / Q000648:ultrastructure*; D004330:Drosophila; D004926:Escherichia coli / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods; D010843:Pichia / Q000201:enzymology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D008941:Spindle Apparatus; D019076:Transgenes
D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; C497201:SAS-6 protein, C elegans; D049452:Green Fluorescent Proteins