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A ciliopathy complex builds distal appendages to initiate ciliogenesis

Dhivya Kumar, Addison Rains, Vicente Herranz-Pérez, Quanlong Lu, Xiaoyu Shi, Danielle L. Swaney, Erica Stevenson, Nevan J. Krogan, Bo Huang, Christopher Westlake, Jose Manuel Garcia-Verdugo, Bradley Yoder, Jeremy F. Reiter

Preprint posted on March 01, 2021 https://www.biorxiv.org/content/10.1101/2021.03.01.433418v1.article-info

A new centriolar module; MNR and CEP90 are required to initiate distal appendage assembly at the mother centriole during ciliogenesis

Selected by Nicola Stevenson

Categories: biochemistry, cell biology

Background

The centrosome is a cellular organelle composed of two barrel-shaped centrioles embedded in a dense mesh of proteins called the pericentrosomal matrix (PCM) 1. Clustered around the centrosome are dense, non-membranous protein granules called centriolar satellites that contain hundreds of proteins required for numerous cellular processes2. The centrioles themselves are typically composed of nine radially arranged microtubule triplets that form a cylindrical structure. This cylinder contains a central protein rod with nine ‘spokes’ extending out to the microtubule walls like a cartwheel. Centrioles are always paired, with the most mature centriole termed the mother and the youngest the daughter. Due to its role in organising the mitotic spindle and building cilia, the centrosome is often referred to as the main microtubule organising centre of the cell1.

Centrosomes are essential for ciliogenesis. Cilia are ubiquitous microtubule-based organelles that extend out from the cell surface to sense developmental and environmental signals (primary cilia) or to beat and provide propulsive movement (motile cilia). Their construction begins when the mother centriole acquires distal appendages that recruit pre-ciliary vesicles to the centrosome. These vesicles fuse together to form a single large vesicle wrapped around the distal end of the mother centriole. The inner two microtubules of each centriolar triplet then extend to form the axoneme – the main structural component of the cilium. Once the axoneme reaches the cell surface, the ciliary vesicle surrounding it fuses with the plasma membrane to form the cilium. Throughout this process the distal appendages anchor the basal body to the ciliary membrane3.

Due to the pivotal role of the mother centriole in ciliogenesis, many mutations in centriolar proteins have been linked to diseases relating to cilium dysfunction. Despite this, many centriolar components are yet to be characterised with respect to this process. In this study Kumar et al identify CEP90 as a centriolar protein required for the recruitment of distal appendages to the mother centriole.

Key findings

Using a powerful combination of expansion microscopy and 3D SIM to look at centrosomes with ~30nm resolution, the authors begin this study by showing that CEP90 localises to both centriolar satellites and the distal end of each centriole where it forms a ring of nine puncta. Satellite-resident CEP90 has already been investigated4, so to specifically study the function of centriole-localised CEP90, satellites were removed by depletion of PCM1. Under these conditions, CEP90 was found to interact with proteins OFD1 and MNR suggesting that they form a functional module at this site.

Examination of the spatial organisation of these proteins showed that they arrange in concentric rings on the distal end of both centrioles. MNR forms the smallest ring, then CEP90, then OFD1. Intriguingly, the diameters of the CEP90 and OFD1 rings are smaller on the daughter centriole than on the mother centriole. Furthermore, the location of a fourth centriolar protein, TALPID3, completely changes between centrioles, sitting between MNR and CEP90 on the daughter but outside the OFD1 ring on the mother. This likely reflects different functional requirements between the centrioles.

To determine the role of centriolar CEP90 and MNR, RPE1 knockout (KO) cell lines were generated using CRISPR/Cas9. In both these cell lines and in mutant mice obtained from the IMPC, loss of either protein resulted in a loss of cilia. Consequently, mutant mice died at E9.5 with pericardial oedema and unlooped, midline hearts, likely as a result of deficient ciliary Hedgehog signalling. Attempts to rescue the ciliary defects in CEP90 KO RPE1 showed that both the N- and C-terminus of CEP90 are required for ciliogenesis, consistent with a role as a protein scaffold. Three CEP90 constructs containing known patient mutations were also tested for their rescue capabilities. CEP90E89Q, which causes microcephaly5, correctly localised to both centrioles and satellites but did not rescue ciliogenesis, indicating the importance of this residue. CEP90R405Q and CEP90D637A, which cause Joubert syndrome6, also failed to rescue ciliogenesis but in this case they also disrupted centriolar satellite morphology.

Whilst investigating the cause of the ciliogenesis defect in the KO cells, the authors discovered that the distal appendage proteins CEP83, FBF1, SCLT1, ANKRD26 and CEP164 were not recruited to the mother centriole in CEP90 and MNR KO cells, a key first step in initiating cilium formation. MNR KO cells also failed to recruit CEP90 to the centriole, whilst CEP90 was found to interact directly with CEP83 – the most proximal distal appendage protein. The authors therefore conclude that these proteins act in a hierarchical manner to orchestrate the very earliest steps of ciliogenesis; MNR recruits CEP90, which then recruits CEP83 to initiate distal appendage formation and facilitate the recruitment of pre-ciliary vesicles.

Why I chose this preprint

I chose this preprint as I thought it was a visually stunning and scientifically compelling story about one of the most enigmatic stages of ciliogenesis. Unravelling the role of a specific pool of Cep90 amongst the chaos of the over-crowded centrosome is no mean feat and Kumar et al provide us with much needed insight into the hierarchy of protein interactions required for distal appendage assembly. Cilia are vital to development and health, as evidenced by the devastating consequences of genetic mutations affecting their formation and function. The existence of disease-causing mutations in CEP90 illustrates how important it is to understand the function of this protein.

This study also showcases the advantages of expansion microscopy. This technique has been used frequently in the context of cilium and centrosome organisation because of the highly dense protein networks present in these organelles. As with all techniques, expansion microscopy has its limits. However, unpicking the spatial organisation of protein machinery using corroborative combinations of super-resolution imaging can be a powerful way to make functional insights, as evidenced here.

Questions for authors

CEP90 and MNR are present on both the mother and daughter centriole yet distal appendage proteins are only recruited to the mother. Do you have any ideas on how this is restricted to the mother centriole? Perhaps the differential organisation you identify is important?

It is interesting that patient mutation CEP90E89Q does not affect CEP90 localisation yet prevents ciliogenesis – have you looked to see whether this mutation affects the ability of CEP90 to bind to CEP83? Or could you speculate on why this residue might be so key.

Similarly, with the Joubert syndrome mutations do you think the observed disruption to the centriolar satellites is impacting ciliogenesis or do you think it is having a separate role at the centrosome too?

When centriolar satellites are removed with nocodazole or PCM1 KO, the ring of CEP90 looks a lot more organised and prominent in your images then when satellites are present. Do you think some CEP90 could be relocating to and enriching on the centrioles under these conditions? This might suggest the satellites are acting as a store for centriolar proteins.

Did the loss of MNR or CEP90 affect cell cycle progression?

References

  1. Jaiswal, S., Kasera, H., Jain, S. et al. Centrosome: A Microtubule Nucleating Cellular Machinery. J Indian Inst Sci 101, 5–18 (2021). https://doi.org/10.1007/s41745-020-00213-1
  2. Prosser, S. L. Pelletier L.. Centriolar satellite biogenesis and function in vertebrate cells. Journal of Cell Science 133: jcs239566 (2020) doi: 10.1242/jcs.239566
  3. Ishikawa, H., Marshall, W. Ciliogenesis: building the cell’s antenna. Nat Rev Mol Cell Biol 12, 222–234 (2011). https://doi.org/10.1038/nrm3085
  4. Kim K, Rhee K. The pericentriolar satellite protein CEP90 is crucial for integrity of the mitotic spindle pole. J Cell Sci. 1;124(Pt 3):338-47 (2011). doi: 10.1242/jcs.078329.
  5. Kodani A, Yu TW, Johnson JR, Jayaraman D, Johnson TL, Al-Gazali L, Sztriha L, Partlow JN, Kim H, Krup AL, Dammermann A, Krogan NJ, Walsh CA, Reiter JF. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication. Elife. 22;4:e07519. (2015) doi: 10.7554/eLife.07519.
  6. Wheway G, Schmidts M, Mans DA, Szymanska K, Nguyen TT, Racher H, Phelps IG, Toedt G, Kennedy J, Wunderlich KA, Sorusch N, Abdelhamed ZA, Natarajan S, Herridge W, van Reeuwijk J, Horn N, Boldt K, Parry DA, Letteboer SJF, Roosing S, Adams M, Bell SM, Bond J, Higgins J, Morrison EE, Tomlinson DC, Slaats GG, van Dam TJP, Huang L, Kessler K, Giessl A, Logan CV, Boyle EA, Shendure J, Anazi S, Aldahmesh M, Al Hazzaa S, Hegele RA, Ober C, Frosk P, Mhanni AA, Chodirker BN, Chudley AE, Lamont R, Bernier FP, Beaulieu CL, Gordon P, Pon RT, Donahue C, Barkovich AJ, Wolf L, Toomes C, Thiel CT, Boycott KM, McKibbin M, Inglehearn CF; UK10K Consortium; University of Washington Center for Mendelian Genomics, Stewart F, Omran H, Huynen MA, Sergouniotis PI, Alkuraya FS, Parboosingh JS, Innes AM, Willoughby CE, Giles RH, Webster AR, Ueffing M, Blacque O, Gleeson JG, Wolfrum U, Beales PL, Gibson T, Doherty D, Mitchison HM, Roepman R, Johnson CA. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat Cell Biol. 17(8):1074-1087. (2015) doi: 10.1038/ncb3201.

Tags: appendage, centriole, centrosome, cilium, hh signalling

Posted on: 23rd March 2021 , updated on: 25th March 2021

doi: https://doi.org/10.1242/prelights.27843

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