Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in Ciliogenesis

Abstract

Centrioles are evolutionarily conserved macromolecular structures that are fundamental to form cilia, flagella, and centrosomes. Centrioles are 9-fold symmetrical microtubule-based cylindrical barrels comprising three regions that can be clearly distinguished in the Chlamydomonas reinhardtii organelle: an ∼100-nm-long proximal region harboring a cartwheel; an ∼250-nm-long central core region containing a Y-shaped linker; and an ∼150-nm-long distal region ending at the transitional plate. Despite the discovery of many centriolar components, no protein has been localized specifically to the central core region in Chlamydomonas thus far. Here, combining relative quantitative mass spectrometry and super-resolution microscopy on purified Chlamydomonas centrioles, we identified POB15 and POC16 as two proteins of the central core region, the distribution of which correlates with that of tubulin glutamylation. We demonstrated that POB15 is an inner barrel protein within this region. Moreover, we developed an assay to uncover temporal relationships between centriolar proteins during organelle assembly and thus established that POB15 is recruited after the cartwheel protein CrSAS-6 and before tubulin glutamylation takes place. Furthermore, we discovered that two poc16 mutants exhibit flagellar defects, indicating that POC16 is important for flagellum biogenesis. In addition, we discovered that WDR90, the human homolog of POC16, localizes to a region of human centrioles that we propose is analogous to the central core of Chlamydomonas centrioles. Moreover, we demonstrate that WDR90 is required for ciliogenesis, echoing the findings in Chlamydomonas. Overall, our work provides novel insights into the identity and function of centriolar central core components.

Expansion stimulated emission depletion microscopy (ExSTED)

“…To investigate if this is possible in principle, we investigated a number of structures, whose organization is not accessible in conventional light microscopy, but whose size is well established by super-resolution and electron microscopy methods. Specifically, we investigated the periodic actin-spectrin ring structure found along neuronal axons 2,20 , centrioles 9,21 , primary cilia of mammalian cells [10][11][12]22 and motile cilia 13,23 .…”

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

“…Motivated by the results, we set out to explore the molecular organization of centriole organelles by Ex-SMLM. We used isolated Chlamydomonas centrioles, which have a characteristic 9-fold microtubule triplet-based symmetry, forming a polarized cylinder ~ 500 nm long and ~ 220 nm wide 28 (Supplementary Fig. 8).…”

Microtubule Inner Proteins: A Meshwork of Luminal Proteins Stabilizing the Doublet Microtubule

“…Possible correlations of these proteins (tektin, Rib43a, Rib72, FAP20, and PACRG) with MIP structures are summarized in Table . All these proteins are also found in the proteome of the basal body in Chlamydomonas . In addition, the MIP structures of the A‐ and B‐tubules of triplet microtubules from the basal body are similar to those of doublet microtubules (Figure A).…”