DBT-DFG Indo-German Research Proposal: The recruitment of the γ-TuRC to the Sas-6 cartwheel for centriole microtubule assembly

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Centrioles are microtubule-based subcellular structures that constitute the core of centrosomes, the main microtubule-nucleating organelles in animal cells. In G1 phase of the cell cycle two centrioles surrounded by the pericentriolar material give rise to a functional centrosome. During S phase, each of the two mother centrioles initiates assembly of a new daughter centriole at its proximal end. The orthogonally arranged mother-daughter centriole pair stays connected during mitosis when each pair associates with one of the two poles of the mitotic spindle. With cytokinesis each cell inherits one centriole pair that then disengages. Centrioles consist of 9 microtubule triples that each contain radially arranged A, B and C microtubules. The initial assembly of centriolar microtubules requires a cartwheel-like nine-fold symmetric scaffold consisting of the protein Sas-6 that provides the base for assembly of the A-microtubules. A-microtubule assembly is a crucial step for the duplication of centrioles, the biogenesis of centrosomes and the correct formation of the mitotic spindle and its failure has been linked to chromosome missegregation and cancer. Therefore, it is of fundamental importance to understand how centrioles and in particular the A-tubules, as a key event in early centriole biogenesis, assemble mechanistically. Cryo-electron microscopy (cryo-EM) of human procentrioles previously indicated presence of cap-like structure at the base of the centriolar microtubule resembling the γ-tubulin ring complex (γ-TuRC), the major microtubule-nucleating entity. Recently, Manna has shown interaction of the γ-TuRC with SAS-6 and Schiebel reported the structure of the γ-TuRC based on cryo-EM analysis. Although, these studies indicate a molecular link of the cartwheel with the centriolar microtubules, how the γ-TuRCs are recruited, organized and activated for assembly of the A-microtubules are not understood and will be addressed in this collaborative study as part of DBT-DFG between India and Germany. Our working model is that the Sas-6 cartwheel recruits the γ-TuRC in S phase through its C-terminus with the subsequent activation of the γ-TuRC and the nucleation of the A-tubules followed by the assembly of the B- and C-tubules. To decipher A-tubule formation in greater detail, we will study the interaction of Sas-6 with the γ-TuRC using negative stain EM and cross-link mass spectrometry analysis (2.3.1), the role of PLK4-mediated phosphorylation in γ-TuRC-C-Sas-6 interaction (2.3.2), cryo-EM analysis of the γ-TuRC-Sas-6 complex (2.3.3), how events at the γ-TuRC-Sas-6 super-complex lead to A-tubule formation using super resolution microscopy (2.3.4) and biochemical approaches (2.3.5). Altogether, our proposal will unravel the mechanisms of centriolar microtubule assembly from a novel perspective, by combining ultra-structural analyses of the key protein complexes with regulation of their interactions that lead to the biogenesis of centrioles.