Deciphering and engineering centriole assembly

ended

Deciphering and engineering the assembly of cellular organelles is a key pursuit in biology. The centriole is an evolutionarily conserved organelle well suited for this goal, and which is crucial for cell signaling, motility and division. The centriole exhibits a striking 9-fold radial symmetry of microtubules around a likewise symmetrical cartwheel containing stacked ring-bearing structures. Components essential for generating this remarkable architecture from alga to man have been identified. A next critical step is to engineer assays to probe the dynamics of centriole assembly with molecular precision to fully understand how these components together build a functional organelle. Our ambitious research proposal aims at taking groundbreaking steps in this direction through four specific aims: 1) Reconstituting cartwheel ring assembly dynamics. We will use high-speed AFM (HS-AFM) to dissect the biophysics of SAS-6 ring polymer dynamics at the root of cartwheel assembly. We will also use HS-AFM to analyze monobodies against SAS-6, as well as engineer surfaces and DNA origamis to further dissect ring assembly. 2) Deciphering ring stacking mechanisms. We will use cryo-ET to identify SAS-6 features that direct stacking of ring structures and set cartwheel height. Moreover, we will develop an HS-AFM stacking assay and a reconstituted stacking assay from human cells. 3) Understanding peripheral element contributions to centriole biogenesis. We will dissect the function of the peripheral centriole pinhead protein Cep135/Bld10p, as well as identify and likewise dissect peripheral A-C linker proteins. Furthermore, we will further engineer the HS-AFM assay to include such peripheral components. 4) Dissecting de novo centriole assembly mechanisms. We will dissect de novo centriole formation in human cells and water fern. We will also explore whether de novo formation involves a phase separation mechanism and repurpose the HS-AFM assay to probe de novo organelle biogenes