Building a cartwheel: Towards deciphering mechanisms of in cellulo SAS-6 assembly

Centrioles are evolutionarily conserved cylindrical structures characterized by 9-fold radial symmetry. SAS-6 is a conserved and key molecular player, which forms the basis of the cartwheel, critical for centriole assembly and establishing 9-fold symmetry. While SAS-6 self assembly, ring formation, and stacking are well characterized in vitro, these processes remain insufficently explored in the cellular context. This thesis addresses these questions using two approaches. First, a mechanistic approach where we probed human SAS-6 (HsSAS-6) function by modulation with binding monobodies and monitoring its integration into growing cartwheels in human cells. Second, a descriptive and structural approach where we leveraged the centrioles of Trichonympha agilis, a protist with thousands of centrioles featuring elongated cartwheel bearing proximal regions amenable to structural analysis. Addressing how SAS-6 integrates into the growing cartwheel during centriole duplication, pulse overexpression experiments using GFP tagged HsSAS-6 revealed complex integration patterns, suggesting both proximal and distal incorporation early in the assembly process and more distal incorporation later, though technical limitations prevented definitive conclusions. To further probe function of HsSAS-6 in the cellular context, we investigated modulating HsSAS-6 assembly using monobodies that bind HsSAS-6 and affect centriole duplication. Under-duplicating monobodies NMB5 and NMB571 reduced cartwheel length without altering overall procentriole length, suggesting these molecules modulate HsSAS-6 recruitment. In T. agilis, follow up studies on mass spectrometry candidates for proximal region components aimed to identify potential A-C linker, Pinhead, and cartwheel inner density (CID) components. Microtubule binding assays yielded inconclusive results, whereas co-IP assays for T. agilis SAS-6 (TaSAS-6) interactors suggested two potential binders. Ultra-expansion microscopy unexpectedly revealed asymmetric centrin distribution along T. agilis centriole length and width, with this asymmetry maintained across neighboring centrioles along the cell membrane. Furthermore, in situ cryo-electron tomography leveraging Serial Lift-Out and TEM tomography unveiled structural features never before observed in cryogenic conditions for centrioles, including curved spokes emanating from the cartwheel hub and previously unreported densities outside centrioles. A specific population of these densities was found to be radially asymmetrically distributed around the centriole. Together, this work provides an avenue to advance our understanding of in situ cartwheel architecture and paths to investigate how SAS-6 assembles and stacks into the highly organized structure that templates centriole formation.