SAS-6 Association with γ-Tubulin Ring Complex Is Required for Centriole Duplication in Human Cells

Abstract

Centrioles are essential components of centrosome, the main microtubule-organizing center of animal cellsrequired for robust spindle bipolarity [1, 2]. They are duplicated once during the cell cycle [3], and the duplication involves assembly of a cartwheel on the pre-existing centriole followed by assembly of triplet microtubules around the cartwheel [4, 5]. Although the molecular details of cartwheel formation are understood [6-13], the mechanisms initiating the formation of centriolar microtubules are not known. Here, we show that the central component of cartwheel, HsSAS-6 plays a crucial role in the formation of centriolar microtubules by interacting with the microtubule nucleation machinery, γ-tubulin ring complex (γ-TuRC) in human cells. The globular N terminus and the central coiled-coil domain of SAS-6 are required for formation of the cartwheel [7, 14], whereas the function of its C-terminal outer cartwheel region in centriole duplication remains unclear. We find that deletion of HsSAS-6 C terminus disrupts microtubule formation in daughter centriole, and as a result, cells fail to form the new centriole. Consequently, this results in mitotic cells having only two centrioles localized at a single site. Detailed molecular analyses showed that HsSAS-6 interacts with the γ-TuRC proteins and associates with the γ-TuRC at the centrosome, and furthermore, the C terminus isessential for this association. High-resolution microscopy revealed localization of the γ-TuRC protein, γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub in the centriole. Together, theresults indicate that HsSAS-6 regulates centriolar microtubule assembly by anchoring γ-TuRCs to the pro-centriole at the onset of daughter centriole formation.