This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 340227
Centrioles are evolutionarily conserved organelles that are essential for the formation of cilia, flagella, as well as centrosomes, and which are characterized by a striking 9-fold radial symmetry of microtubules (Fig. 1). Just like the genetic material, centrioles duplicate once and only once per cell cycle. Formation of a procentriole next to each existing centriole, around a structure called the cartwheel, marks the onset of centriole duplication. Despite their importance, the mechanisms governing centriole biogenesis have remained elusive until recently.
We and others identified five proteins required for centriole formation in C. elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6 and SPD-2 (Gönczy, 2012). We established that centriole formation is an orderly assembly process in which these proteins are recruited in a step-wise fashion (Delattre et al.; 2006). Relatives of these proteins are crucial for centriole formation also in other organisms. Thus, we found that HsSAS-6 is necessary for procentriole formation in human cells and that excess HsSAS-6 results in supernumerary procentrioles. Therefore, regulated levels of HsSAS-6 ensure formation of a single procentriole per existing centriole during the duplication cycle (Strnad et al.; 2007).
In collaboration with the Steinmetz laboratory, we revealed the crystal structure and the oligomerisation properties of Chlamydomonas SAS-6 (Bld12p) by combining structural, biochemical and cell biological methods. We discovered that Bld12p can form rings in vitro that ressemble the central part of the cartwheel. This lead us to propose that oligomerization of SAS-6 proteins dictates the near-universal 9-fold symmetry of centrioles (Fig. 3) (Kitagawa et al.; 2011).
The above findings left open the question whether SAS-6 rings exist in vivo. Thus, we sought to obtain a three-dimensional map of the cartwheel in the native state by using cryoelectron tomography (cryo-ET). We found that the cartwheel is a stack of central rings that exhibit a vertical periodicity of 8.5 nanometers and that is able to accommodate nine SAS-6 homodimers. Furthermore, we discovered that the spokes emanating from two such rings associate into a layer, with a vertical periodicity of 17 nanometers on the cartwheel margin (Fig. 4) (Guichard et al.; 2012).