Molecular architecture of the centriole proteome.

摘要

Centrioles were first described over a century ago as small, geometrically precise cellular entities composed of nine triplet microtubule blades arranged in a pinwheel-like array. The structure of the centriole is complex and highly precise, with centriole geometry and dimensions being tightly controlled, but the molecular mechanisms governing centriole assembly, length control, and maturation into basal bodies remain mysterious. The biological significance of centrioles is demonstrated by the fact that they are necessary for recruitment of pericentriolar material (PCM) to form a complete centrosome and that they act as basal bodies, during the formation of cilia and flagella. The remarkable centriole duplication cycle in which new daughter centrioles form at an intriguing right angle to the pre-existing mother centriole, along with unique ability of centrioles to act as basal bodies to initiate ciliogenesis have perplexed researchers for decades.;Much of the aura of mystery that surrounds centrioles stems from the fact that the protein composition of centrioles is largely unknown. To gain molecular insights into the function and assembly of centrioles, I set out to identify a parts list for the centriole through direct proteomic analysis of isolated centrioles. Prior to this study there were only eleven known core centriole proteins. I developed a purification protocol allowing for the isolation of virtually "naked" centrioles, with little to no obscuring PCM, from the green alga, Chlamydomonas. Proteomic analysis of these isolated centrioles provided the first opportunity to reveal the specific parts of the centriole and constitutes the centriole proteome. This investigation into centriole composition has helped elucidate the function and properties of this unique organelle, which has remained mysterious for more than a century.;Remarkably, this proteomic analysis of centrioles has provided not only a full and complete parts list for the centriole but has also demonstrated that human disease proteins are highly represented in the centriole proteome suggesting that multiple human disease gene products encode protein components of the centriole. In fact, known human disease genes encode over seventeen percent of the cross-validated Chlamydomonas centriole proteins. In particular, two classes of ciliary disease genes are highly represented among the centriole proteome: cystic kidney disease syndrome genes and cone-rod dystrophy syndrome genes. One possibility is that the human disease genes found in the centriole proteome encode for structurally conserved core centriole proteins that are necessary for establishing or maintaining the integrity of the complex triplet microtubules structure. Another possibility is that these proteins are involved in ciliogenesis-related functions. This research has laid a foundation for future studies of this enigmatic organelle.;Our published centriole proteome identified proteins predicted to compose the centriole, although we had no concrete evidence that these proteins were actually centriolar. To validate their in vivo localization to centrioles I created GFP-fusion proteins for candidate centriole proteins and have localized over sixty percent of all the proteins that we feel are core centriole components based our bioinformatics approach. To begin to learn how the centriole proteome is put together, I investigated POC1, one of the most abundant proteins from our centriole proteome, which is conserved in all organisms with triplet microtubules. I found that POC1 is a proximal and very early marker of centriole duplication and has a unique localization on intact mature centrioles, being found to colocalize with attachment points of multiple distinct fiber systems that contact the centriole. This is the first protein to date that localizes to both early duplicating centrioles and to places of centriole fiber attachment, indicating that POC1 may be involved in multiple distinct aspects of centriole biology. Furthermore, POC1 is involved in the early stages of centriole duplication and also plays a role in the enigmatic process of centriole length control.;Together this research signifies a cohesive body of work starting with the isolation and purification of Chlamydomonas centrioles leading to the first published centriole proteome and continuing with the detailed characterization of one particular protein involved in both centriole duplication and length control.