Cell cycle dependent regulation of centrosome maturation by Drosophila Pericentrin

Authors

• R Varadarajan
• K Plevock
• CJ Fagerstrom
• NM Rusan

Abstract

Centrosomes are the major microtubule organizing centers in most eukaryotic cells. Centrosomes consist of a pair of centrioles encircled by pericentriolar material (PCM). Centrosome maturation is a key cell cycle-dependent process that facilitates recruitment of PCM proteins to the centrioles thereby enabling centrosome-driven functions, including microtubule nucleation. Mutations in PCM proteins (Pericentrin and Centrosomin) are associated with many human genetic disorders, including cancer and ciliopathies. Drosophila is an excellent model system to study the centrosome and diseases that are related to their dysfunction. Here, we focus on understanding the molecular mechanisms underlying PCM recruitment and mitotic microtubules nucleation. Pericentrin like protein (Plp) is a radially organized centrosome linker protein that bridges centrioles and PCM. Plp is shown to be required for PCM recruitment, however, its cell cycle dependent molecular regulation and the crosstalk between other PCM components including Asl, Sas4, CNN and Polo kinase remain unclear. To investigate the precise role of Plp during centrosome maturation, we performed a structure-function analysis by generating Plp protein truncation and characterized its function in vivo. We found that the C terminal region of Plp contains a promoting signal for PCM recruitment, while the N terminal region, which is proximal to the PCM, contains an inhibitory signal. We found that the newly identified Plp regulatory domains interact with essential PCM components such as CNN and Sas4. By performing in vivo mass spectrometric analysis on these regulatory regions, we identified the precise phosphorylation sites that are targeted by Polo, which is a key kinase of centrosome maturation. Combinatorial analysis of PCM interactions with the phospho modification on Plp regulatory domains suggests a sophisticated autoinhibition mechanism at play. Furthermore, we noticed that the N terminus of Plp extends further towards PCM during centrosome maturation (mitotic) and this conformational modification appears to be Polo driven. Collectively, our current work suggests that Plp functions as a molecular switch to regulate PCM recruitment. That is, in interphase Plp resides in a inhibited conformation that prevent PCM recruitment, and in mitosis, Polo relieves Plp from autoinhibition that in turn allows interaction with PCM components thereby activating centrosomes to nucleate microtubules.

Scientific Focus Area: Cell Biology

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