Molecular mechanisms of compound microtubule structure and function - PROJECT SUMMARY The mammalian centrosome-cilium complex is involved in cell division, signaling, and motility. Defects in the structure or function of the complex are linked to a variety of human disease states, including cancer, microcephaly, and a group of inherited disorders known as ciliopathies that affect many tissues and organs. The centrosome is composed of two centrioles surrounded by pericentriolar material and is the major microtubule organizing center of animal cells. One of the centrioles within the centrosome templates the growth of a cilium, an antenna-like organelle involved in cell signaling and motility. While the field has catalogued many of the hundreds of proteins in the complex, how they work together to create functional centrosomes and cilia is unclear. We propose that specialized, compound (triplet and doublet) microtubules within the complex form dedicated scaffolds for the protein-protein interactions that define the organelle. Triplet microtubules in centrioles and doublet microtubules in cilia are conserved in almost all species with these organelles and are only found in centrioles and cilia. Little is known about how these microtubules are formed and regulated to create functional organelles. Delta-tubulin and epsilon-tubulin, two little-studied members of the tubulin superfamily, are key proteins involved in forming compound microtubules at the centrosome-cilium complex. Null mutations in either delta- or epsilon-tubulin in mammalian cells results in the formation of aberrant, unstable centrioles with singlet microtubules that fail to recruit many centrosome proteins. The molecular mechanisms by which these tubulins act on centrioles are unknown. Here, using a combination of high-resolution expansion microscopy, CRISPR/Cas9 gene editing, biochemistry, and proteomics, we will address outstanding questions about the structure, formation, and function of the compound microtubules. We will: 1) determine the molecular mechanisms by which delta-tubulin and epsilon- tubulin form or stabilize the compound microtubules, 2) define how the compound microtubules elongate, and 3) determine how the compound microtubules scaffold the centrosome-cilium complex. These studies will provide mechanistic insight into the assembly of these microtubule structures, improve our understanding of the centrosome-cilium complex, and provide insight into its dysregulation in disease.
