"Centrioles are critical for the formation of cilia, flagella and centrosomes, as well as for human health. The mechanisms governing centriole formation constitute a long-standing question in cell biology. We will pursue an innovative multidisciplinary research program to gain further insight into these mechanisms, using human cells, C. elegans and Trichonympha as model systems. This program is expected to also have a major impact by contributing a novel cell free assay to the field, thus paving the way towards making synthetic centrioles. Six specific aims will be pursued:1) Deciphering HsSAS-6/STIL distribution and dynamics. We will use super-resolution microscopy, molecular counting, photoconversion and FCS to further characterize these two key components required for centriole formation in human cells.2) The SAS-6 ring model as a tool to redirect centriole organization. Utilizing predictions from the SAS-6 ring model, we will assay the consequences for centrioles and cilia of altering the diameter and symmetry of the structure.3) Determining the architecture of C. elegans centrioles. We will conduct molecular counting and cryo-ET of purified C. elegans centrioles to determine if they contain a spiral or a cartwheel, as well as identify SAS-6-interacting components.4) Comprehensive 3D map and proteomics of Trichonympha centriole. We will obtain a ~35 Å 3D map of the complete T. agilis centriole, perform proteomic analysis to identify its constituents and test their function using RNAi.5) Regulation of cartwheel height and centriole length. We will explore whether cartwheel height is set by SAS-6 proteins and perform screens in human cells to identify novel components regulating cartwheel height and centriole length.6) Novel cell free assay for cartwheel assembly and centriole formation. Using SAS-6 proteins on a lipid monolayer as starting point, we will develop and utilize a cell-free assay to reconstitute cartwheel assembly and centriole format"