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

Abstract

HsSAS-6 homodimers are present in the cytoplasm and assemble into ninefold symmetrical arrays at centrosomes, thus initiating procentriole formation.

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

“…Electron microscopy and cryo‐electron tomography suggest that cartwheels in most species display a thickness in the order of 100 nm (Dippell, 1968; Cavalier‐Smith, 1974; Nakazawa et al , 2007; Guichard et al , 2012, 2013; O'Toole & Dutcher, 2014). A similar value (60–150 nm) was reported in a recent study using 3D‐STORM imaging of stable Sas‐6‐positive structures, likely reflecting cartwheels, in detergent‐extracted human U2OS cells (Keller et al , 2014). Assuming that stacked hubs display a vertical periodicity of 8.5 nm (Kitagawa et al , 2011; Guichard et al , 2012, 2013; Li et al , 2012), a cartwheel thickness of 60–150 nm would thus allow for ~7–17 stacks.…”

“…For U2OS cells, we thus estimate that Sas‐6 levels may reach roughly 50,000 copies per S‐ and G2‐phase cell. This number is about 10 times lower than a previous estimate (Keller et al , 2014). We cannot definitively explain this discrepancy, but would surmise that a combination of mass spectrometry‐based SRM with EGFP‐tagging of endogenous centrosomal proteins is more likely to produce accurate results than a combination of immunofluorescence staining with semi‐quantitative Western blotting.…”

“…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.…”

“…Assuming that 25% of cells are typically in late G2 (Keller et al , 2014), a weighted average of the above numbers results in 383 and 212 molecules of Sas‐6 and STIL, respectively, per S‐ and G2‐phase centrosome. Because total cellular fluorescence of Sas‐6 and STIL was too weak to be measured, these values could not be corroborated by comparing EGFP signal intensity with SRM data.…”

Correlative multicolor 3D SIM and STORM microscopy

“…Moreover, cryo-electron tomography revealed that the cartwheel in the flagellate Trichonympha could accommodate such ring-like structures [13]. Furthermore, STORM imaging using monoclonal antibodies recognizing the C-terminus of HsSAS-6 revealed that this part of the protein localizes towards the periphery of the cartwheel in human cells [14]. Despite these advances, a combined approach to accurately map in 3D the distribution of Bld12p, and in particular the N-terminus of the protein, is missing.…”