Final Report Summary - CENFOR (Dissecting the mechanisms governing centriole formation) Our research program funded by the ERC aimed at further understanding the mechanisms underlying assembly of the centriole, a fundamental organelle that is key for cell signaling, cell motility and cell division. The centriole harbors a signature nine-fold radial symmetry of microtubules and usually assembles around a likewise symmetric cartwheel element, which contains stacks of ring-containing structures constituted of 9 dimers of SAS-6 proteins. The mechanisms directing assembly of the cartwheel and, thus, of the entire centriole, remain incompletely understood. In this project, we made important progress in uncovering these mechanisms, often in collaboration with partner laboratories from allied fields. Some of the most salient findings that were achieved during the course of this project are summarized below. First, we altered SAS-6 proteins to change the symmetry of cartwheel ring-containing structures and assayed the consequences in cells. Our findings indicate that SAS-6 proteins play a major role in determining centriole symmetry, but that peripheral components such as centriolar microtubules are also important. Second, we developed a cell-free assay for cartwheel assembly, which enabled us to uncover that SAS-6 proteins possess an intrinsic ability not only to assemble ring-containing structures, but also to stack them into cartwheel-like elements. Third, we developed a complementary cell-free assay relying on a novel high-speed atomic force microscopy modality to uncover the detailed kinetics of SAS-6 self-assembly. Fourth, we used the unconventional Trichonympha agilis model system to further dissect the architecture of the centriole, as well as to conduct proteomic analysis to identify novel organelle constituents, an approach that we also pursued in the green alga Chlamydomonas reinhardtii. Overall, these and complementary lines of work enabled us to better understand the mechanisms leading to the assembly of the centriole organelle.
