Four principal lines of worked were pursued.
1) Reconstituting cartwheel ring assembly dynamics. In collaboration with Prof. Fantner, we developed photothermally-actuated off-resonance tapping high-speed atomic force microscopy (PORT-HS-AFM) to analyze the surface self-assembly reaction of SAS-6. By determining the effective dissociation constant of the system, we discovered that the surface catalyzes SAS-6 self-assembly, shifting the equilibrium ~104 fold compared to solution, providing a plausible mechanism for the signature orthogonal emergence of the nascent centriole from the surface of the resident centriole. Moreover, in collaboration with Prof. Hantschel, we developed monobodies to modulate centriole assembly. In particular, we characterized three monobodies that bind CrSAS-6 using structural, biophysical and cell biological assays. This enabled us to identify one monobody that severely impairs centriole formation, including in the cellular context. Moreover, we have generated DNA origamis intended to mimic the surface environment on which SAS-6 proteins self-assemble.
2) Deciphering ring stacking mechanisms. We set out to use HS-AFM to develop a cell-free stacking assay, but could achieve stacking of only two rings of CrSAS-6, even when DNA origami pillars were added. Therefore, we pursued two other lines of work to investigate SAS-6 ring stacking mechanisms, using Trichonympha agilis as a model, in which cartwheel stacks are exceptionally high. First, we developed a cryo-FIB pipeline to uncover high resolution information regarding stacking modalities in native specimens, without subjecting them to symmetrization or averaging, as had been done previously. Second, we set out to produce proteins of the four TaSAS-6 proteins to conduct biophysical analysis, which is anticipated to further the understanding of stacking mechanisms in general. These two lines of work are being pursued beyond the funding period.
3) Understanding peripheral element contributions to centriole biogenesis. We focused principally on the analysis of Cep135 in human cells. We used CRISPR/Cas9 to generate cell lines lacking the corresponding gene entirely, which enabled us to establish that Cep135 is important for organelle integrity during mentored centriole duplication, as well as essential for de novo centriole biogenesis. Moreover, localizing the N- and C-termini of Cep135 enabled us to establish that the protein is likely a Triplet base component, an element that connects the A-C linker with the pinhead of the centriole.
4) Dissecting de novo centriole assembly mechanisms. We have completed a targeted siRNA-based screen aimed at identifying genes essential for de novo centriole biogenesis in human cells, testing all proteins known to reside at centrioles and all proteins previously found to be essential for mentored centriole duplication. This enabled us to identify 14 genes that scored in duplicate above the significance threshold. Interestingly, three of these genes, Cep135, RTTN and ALMS1, are essential strictly for de novo centriole assembly, and not for mentored centriole duplication. In addition, we conducted live imaging experiments to determine where in the cell and when during the cell cycle centrioles emerge de novo. Moreover, we initiated work on physiological de novo centriole formation in the protist Naegeleria gruberi, which proved more tractable experimentally than Marsilea vestita, which was planned to be investigated initially.
