Native Architecture of the Centriole Proximal Region Reveals Features Underlying Its 9-Fold Radial Symmetry

Abstract

Our work provides unprecedented insight into the architecture of the centriole proximal region, which is key for a thorough understanding of the mechanisms governing centriole assembly.

The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor

“…This correlates with cellular studies in which Sas-6 and Ana2 dual overexpression was required for cartwheel formation, suggesting that Ana2 potentiates Sas-6 cartwheel formation, potentially through oligomerization (17). Recent cryotomographic studies of nascent centriole architecture reveal auxiliary protein density connecting the Sas-6-based cartwheel to Sas-4 and the distal microtubule triplets (3). Given the integral role of Ana2 in Sas-6 cartwheel formation as well as evidence that it binds both Sas-6 and Sas-4, Ana2 is a likely candidate for this density.…”

“…Here, we use x-ray crystallography, isothermal microtitration calorimetry (ITC), 3 and size exclusion chromatography with multiangle static light scattering (SEC-MALS) to characterize the interactions between LC8 and Ana2. Our results demonstrate that LC8 dimers bind Ana2 at two distinct sites, the first of which contains a high-affinity, canonical LC8-binding TQT motif (residues 159 -168), whereas the second contains a non-canonical TQC motif (residues 237-246).…”

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

“…Nevertheless, it is interesting to compare our prediction with estimates of cartwheel thickness, albeit collected from centrioles of different organisms. 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).…”

“…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. These numbers, albeit tentative, are encouragingly close to our estimate of 15–16 stacked hubs per cartwheel.…”

Correlative multicolor 3D SIM and STORM microscopy

“…Isolated basal bodies were first purified [7] and centrifuged at 10,000 g onto a 12 mm diameter round coverslip (Thomas Scientific, coverslips #1.5, ref 1217N79) in a 15ml Corex tube containing special adaptors as described previously (Fisher, ref 4550015) and filled with 5ml 10mM K-PIPES, pH 7. Coverslips were then fixed for 7 min in -20°C methanol, washed in PBS, incubated 1 hr at room temperature with primary antibodies in 1% bovine serum albumin and 0.05% Triton X-100, washed 5 min in PBS, and incubated 1 hr at room temperature with secondary antibodies.…”