Role of Sas-4 in centrosome maturation

• Applicant: Professor Jay Gopalakrishnan, Ph.D.
• Subject Area: Cell Biology
• Term: from 2013 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 242472216

Centrosomes consist of a pair of centrioles surrounded by Peri-Centriolar Material (PCM) acting as the major microtubule-organizing centers (MTOC) in animal cells. In order for a centrosome to nucleate microtubules at the onset of mitosis, it has to recruit PCM proteins, a process termed as centrosome maturation. Although centrosome maturation is essential for the functionality of centrosomes, the mechanisms of centrosome maturation remain unclear. Others and we have shown that Drosophila Sas-4 and its human counterpart CPAP plays a role in PCM recruitment to duplicated centrosomes by associating with PCM proteins such as Cnn, Asterless (Asl), Pericentrin (PLP) and components of g-tubulin ring complexes (g TuRCs). However, whether and how Sas-4 plays a role in triggering initial PCM recruitment at the onset of mitosis is not completely understood. In this grant, we aim to dissect the possible function of Sas-4 in centrosome maturation. Since, Sas-4 is a dynamically regulated protein in cells, first, we will apply biochemical methods to analyze the cell cycle dependent posttranslational modifications in Sas-4 and identify its phosphorylation status at the onset of mitosis. To dissect the functional significance of modifications during mitotic PCM recruitment, we will introduce respective point mutations and generate transgenic Drosophila. Upon identifying the significance of Sas-4 in mitotic PCM assembly in vivo, we will purify Sas-4-mediated PCM from fly embryonic extracts to functionally reconstitute MTOC functions of centrosomes in vitro. The proposed experiments will identify how Sas-4 is regulated during centrosome maturation and the essential PCM complexes required to functionally reconstituting microtubule-nucleating activity of a centrosome in vitro. Thus, the current study will provide new insights into how mitotic centrosomes are built to maintain spindle poles for accurate cell division.Results obtained from the previous funding period (2014 to 2016) We were granted a partial funding (GO2301/2-1), which supported a doctoral student to study the mechanisms of centrosome biogenesis. Experiments from this funding period resulted in two original publications (Zheng and Gooi et al., PNAS 2014 and Zheng and Ramani et al., Nature Commun 2016)1, 2. In the first work, we demonstrated that Sas-4 plays a role in tethering Sas-4-PCM complexes to a centriole. In the second work, we showed the significance of Sas-4-tubulin interaction in centriole and primary cilium length control. In addition, this grant partially helped us in performing cilia and centrosome-related experiments, which resulted in two more original works (Gabriel et al., EMBO J 20163 and Mariappan et al., Mol Cell, Under review). Overall, this grant greatly helped in setting up our laboratory and pursuing experiments related to centrosome biogenesis.

• DFG Programme: Research Grants