Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers ofSAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellateTrichonymphaspp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) inT. spp. andTrichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report similar to 25 angstrom resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore,STAof centrioles from the distant flagellateTeranympha mirabilisuncovers similar cartwheel architecture and a distinct filamentousCID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generatedin vitroindicate thatSAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improvedSTAmaps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.