Molecular Architecture of the Centriole Proteome: The Conserved WD40 Domain Protein POC1 Is Required for Centriole Duplication and Length Control

Abstract

Centrioles are intriguing cylindrical organelles composed of triplet microtubules. Proteomic data suggest that a large number of proteins besides tubulin are necessary for the formation and maintenance of a centriole's complex structure. Expansion of the preexisting centriole proteome from the green alga Chlamydomonas reinhardtii revealed additional human disease genes, emphasizing the significance of centrioles in normal human tissue homeostasis. We found that two classes of ciliary disease genes were highly represented among the basal body proteome: cystic kidney disease (especially nephronophthisis) syndromes, including Meckel/Joubert-like and oral-facial-digital syndrome, caused by mutations in CEP290, MKS1, OFD1, and AHI1/Jouberin proteins and cone-rod dystrophy syndrome genes, including UNC-119/HRG4, NPHP4, and RPGR1. We further characterized proteome of the centriole (POC) 1, a highly abundant WD40 domaincontaining centriole protein. We found that POC1 is recruited to nascent procentrioles and localizes in a highly asymmetrical pattern in mature centrioles corresponding to sites of basal-body fiber attachment. Knockdown of POC1 in human cells caused a reduction in centriole duplication, whereas overexpression caused the appearance of elongated centriole-like structures. Together, these data suggest that POC1 is involved in early steps of centriole duplication as well as in the later steps of centriole length control. INTRODUCTIONCentrioles are barrel-shaped structures composed of nine triplet microtubules. They are necessary for recruitment of pericentriolar material (PCM) to form a complete centrosome and they act as basal bodies during the formation of cilia and flagella. In most quiescent cells, centrioles move to and dock on the apical plasma membrane during ciliogenesis and provide a template for the extension of doublet microtubules, which make up the ciliary axoneme (Ringo, 1967;Sorokin, 1968;Snell et al., 1974;Vorobjev and Chentsov, 1982;Dawe et al., 2007). Centrioles that template cilia are known as basal bodies, and the proteins that compose them have received increased attention in recent years because of their role in ciliary diseases. Ciliary diseases, or ciliopathies, result in symptoms ranging from obesity and retinal degeneration to polydactyly and cystic kidneys (Pazour and Rosenbaum, 2002;Afzelius, 2004;Badano et al., 2006;Yoder, 2007;Marshall, 2008). Ciliopathies arise from mutations in not only ciliary genes but also in genes encoding proteins within the basal body (Ansley et al., 2003;Keller et al., 2005;Marshall, 2008). Proteomic analyses of centrioles from a number of diverse organisms reveal the presence of ciliary disease genes (Keller et al., 2005;Broadhead et al., 2006;Kilburn et al., 2007). Because having a properly anchored basal body is required for a functional cilium, mutations in any protein involved in the formation and maintenance of centriole or basal body integrity have the potential to lead to ciliary diseases.The structure of the centriole is complex and highly preci...

A Proximal Centriole-Like Structure Is Present in Drosophila Spermatids and Can Serve as a Model to Study Centriole Duplication

“…This is consistent with the localization study in Tetrahymena, which shows that POC1 associates with the site of daughter centriole assembly (Kilburn et al 2007). In addition, a very recent study shows that, in Chlamydomonas, POC1 localizes to the forming basal body (pro-basal body) and that, in human cells, RNAi against POC1 reduces centriole overduplication (Keller et al 2008). This demonstrates that studying PCL formation can serve to identify novel mutation in conserved centriolar genes.…”

“…We performed a genetic screen finding that the Drosophila ortholog of POC1 is essential for the formation of normal PCL. POC1 was identified previously in a proteomic screen as a centriolar protein and is localized to the early intermediate structure in centriole/basal body formation (Keller et al 2005(Keller et al , 2008Kilburn et al 2007). We propose to use PCL formation as a model to study the molecular pathway for centriole initiation.…”

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“…It has been reported that Ahi1 participates in the process of intracellular vesicle trafficking and is co-localized with microtubules and the microtubuleorganizing center (33,34). We postulated that Ahi1 could participate in 5-HT 2C R vesicles sorting to degradation after endocytosis.…”

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

“…However, there are growing examples of cell biologists developing different quantitative microscopy assays for fixed cells in recent years 27,[34][35][36] . Importantly, progress in understanding centrosome biology often requires understanding of the centrosome-specific function of proteins whose centrosomal pools may be regulated differently than other pools.…”