Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

• Published in: The EMBO journal 2016-10, Vol.35 (19), p.2152-2166
• Main Authors: Bauer, Manuel, Cubizolles, Fabien, Schmidt, Alexander, Nigg, Erich A
• Format: Article
• Language: English
• Subjects: Cell cycle; Cell Cycle Proteins - analysis; Cell Line; centriole; centrosome; Centrosome - chemistry; EMBO05; EMBO06; EMBO31; Epithelial Cells - chemistry; Fluorescence; fluorescence imaging; Humans; Optical Imaging; Proteins; Proteomics; Resource; selected reaction monitoring
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201694462

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function. Synopsis Constituents and assembly of centrosomes have been well studied, but quantitative information on the abundance of their components is needed to fully understand the pathophysiological consequences of structural and numerical centrosome aberrations. Here, a combination of approaches estimates the abundance of key centrosomal proteins in human cells. Quantitative estimates are based on a combination of targeted proteomics and fluorescence imaging. Quantitative information is provided for whole‐cell lysates and for isolated centrosomes. We predict 1 STIL molecule for every Sas‐6 dimer at centrioles. We estimate that cartwheel structures of human centrioles harbor up to 16 stacked hubs. Graphical Abstract A combination of quantitative approaches allows to estimate the absolute and relative abundance of key centrosomal and centriolar proteins in human cells.