Regulated HsSAS-6 Levels Ensure Formation of a Single Procentriole per Centriole during the Centrosome Duplication Cycle

Abstract

Centrosome duplication involves the formation of a single procentriole next to each centriole, once per cell cycle. The mechanisms governing procentriole formation and those restricting its occurrence to one event per centriole are poorly understood. Here, we show that HsSAS-6 is necessary for procentriole formation and that it localizes asymmetrically next to the centriole at the onset of procentriole formation. HsSAS-6 levels oscillate during the cell cycle, with the protein being degraded in mitosis and starting to accumulate again at the end of the following G1. Our findings indicate that APC(Cdh1) targets HsSAS-6 for degradation by the 26S proteasome. Importantly, we demonstrate that increased HsSAS-6 levels promote formation of more than one procentriole per centriole. Therefore, regulated HsSAS-6 levels normally ensure that each centriole seeds the formation of a single procentriole per cell cycle, thus playing a fundamental role in driving the centrosome duplication cycle and ensuring genome integrity.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…Similarly, the number of Sas‐6 molecules at centrosomes rose from ~346 in G1/S arrested cells to 440 in G2/M cells (Fig 5B) and those of STIL from 167 to 318 (Fig 5C). Neither Sas‐6‐EGFP nor STIL‐EGFP could be detected at centrosomes in mitotic cells (Fig 5B and C), consistent with ubiquitin‐dependent proteolytic degradation of both proteins (Strnad et al , 2007; Tang et al , 2011a; Arquint et al , 2012; Vulprecht et al , 2012; Arquint & Nigg, 2014). …”

“…However, these numbers represent an underestimate, because ~50% of centrosomes purified from asynchronously growing KE37 cells are derived from G1‐phase cells. In contrast to most other proteins analyzed here, including the protein used for calibration (γ‐tubulin), both Sas‐6 and STIL show marked cycle regulation and very similar cell cycle profiles; as a consequence, they are barely detectable at most G1‐phase centrosomes (Strnad et al , 2007; Arquint & Nigg, 2014; Keller et al , 2014). Thus, an approximate doubling of the above numbers likely provides a better estimate of the absolute abundance of Sas‐6 and STIL on those centrosomes (S and G2 phases) that are positive for these proteins, resulting in 170 and 72 molecules for Sas‐6 and STIL, respectively.…”

“…Centriole biogenesis and maintenance of correct centrosome numbers in proliferating cells critically depend on the exact levels of the key duplication factors Plk4 (Bettencourt‐Dias et al , 2005; Habedanck et al , 2005), Sas‐6 (Leidel et al , 2005; Strnad et al , 2007), and STIL (Tang et al , 2011b; Arquint et al , 2012; Vulprecht et al , 2012). To fully understand the regulation of centriole duplication, it will thus be important to obtain quantitative data on the abundance of these proteins under physiological conditions.…”

The Centrosome and Its Duplication Cycle

“…Similar reductions occur for CPAP, STIL, and human Sas-6 and each of these molecules is targeted by APC/C-Cdh1 or Cdc20 (Strnad et al 2007;Tang et al 2009Tang et al , 2011Puklowski et al 2011;Arquint et al 2012;Arquint and Nigg 2014). Interplay between APC/C and SCF pathways has also been reported to regulate levels of Sas-6 (Puklowski et al 2011).…”

“…If the Plk4 sites in Ana2 are mutated to nonphosphorylatable residues, it can still bind to the daughter centriole but it cannot recruit Sas-6, and centriole duplication fails (Dzhindzhev et al 2014). In contrast to Drosophila, where some Sas-6 remains at the core of the centriole once it is incorporated, in human cells, Sas-6 is destroyed during G 1 (Strnad et al 2007) and is transiently recruited to the lumen of the mother centriole in S phase (Fong et al 2014). The human Sas-6 is then repositioned to the site of procentriole formation in a process that is dependent on STIL/Ana2) and Plk4 (Fong et al 2014).…”