The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography

Overview

Journal bioRxiv

Date 2024 Sep 24

PMID 39314311

Authors

Sam Li

Jose-Jesus Fernandez

Marisa D Ruehle

Rachel A Howard-Till

Amy Fabritius

Chad G Pearson

David A Agard

Mark E Winey

Affiliations

Soon will be listed here.

Abstract

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryotomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner Junctions, underscoring its precise spatial regulation.

References

Shimogawa M, Wijono A, Wang H, Zhang J, Sha J, Szombathy N . FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction. Nat Commun. 2023; 14(1):5225. PMC: 10460401. DOI: 10.1038/s41467-023-40230-z. View

Hodges M, Scheumann N, Wickstead B, Langdale J, Gull K . Reconstructing the evolutionary history of the centriole from protein components. J Cell Sci. 2010; 123(Pt 9):1407-13. PMC: 2858018. DOI: 10.1242/jcs.064873. View

Fernandez J, Li S, Agard D . Consideration of sample motion in cryo-tomography based on alignment residual interpolation. J Struct Biol. 2019; 205(3):1-6. PMC: 6420838. DOI: 10.1016/j.jsb.2019.01.005. View

Zhang Z, Moye A, He F, Chen M, Agosto M, Wensel T . Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography. Life Sci Alliance. 2024; 7(3). PMC: 10770417. DOI: 10.26508/lsa.202302409. View

Carvalho-Santos Z, Azimzadeh J, Pereira-Leal J, Bettencourt-Dias M . Evolution: Tracing the origins of centrioles, cilia, and flagella. J Cell Biol. 2011; 194(2):165-75. PMC: 3144413. DOI: 10.1083/jcb.201011152. View

Sood P, Lin A, Yan C, McGillivary R, Diaz U, Makushok T . Modular, cascade-like transcriptional program of regeneration in . Elife. 2022; 11. PMC: 9371601. DOI: 10.7554/eLife.80778. View

Le Guennec M, Klena N, Gambarotto D, Laporte M, Tassin A, van den Hoek H . A helical inner scaffold provides a structural basis for centriole cohesion. Sci Adv. 2020; 6(7):eaaz4137. PMC: 7021493. DOI: 10.1126/sciadv.aaz4137. View

Gao J, Tong M, Lee C, Gaertig J, Legal T, Bui K . DomainFit: Identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models. Structure. 2024; 32(8):1248-1259.e5. PMC: 11316655. DOI: 10.1016/j.str.2024.04.017. View

Kubo S, Black C, Joachimiak E, Yang S, Legal T, Peri K . Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins. Nat Commun. 2023; 14(1):2168. PMC: 10105768. DOI: 10.1038/s41467-023-37868-0. View

10.

Laporte M, Gambarotto D, Bertiaux E, Bournonville L, Louvel V, Nunes J . Time-series reconstruction of the molecular architecture of human centriole assembly. Cell. 2024; 187(9):2158-2174.e19. PMC: 11060037. DOI: 10.1016/j.cell.2024.03.025. View

11.

Reiter J, Leroux M . Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol. 2017; 18(9):533-547. PMC: 5851292. DOI: 10.1038/nrm.2017.60. View

12.

Bruns P, Cassidy-Hanley D . Biolistic transformation of macro- and micronuclei. Methods Cell Biol. 1999; 62:501-12. DOI: 10.1016/s0091-679x(08)61553-8. View

13.

Geimer S, Melkonian M . The ultrastructure of the Chlamydomonas reinhardtii basal apparatus: identification of an early marker of radial asymmetry inherent in the basal body. J Cell Sci. 2004; 117(Pt 13):2663-74. DOI: 10.1242/jcs.01120. View

14.

Kitagawa D, Vakonakis I, Olieric N, Hilbert M, Keller D, Olieric V . Structural basis of the 9-fold symmetry of centrioles. Cell. 2011; 144(3):364-75. PMC: 3089914. DOI: 10.1016/j.cell.2011.01.008. View

15.

Scheres S, Chen S . Prevention of overfitting in cryo-EM structure determination. Nat Methods. 2012; 9(9):853-4. PMC: 4912033. DOI: 10.1038/nmeth.2115. View

16.

Owa M, Uchihashi T, Yanagisawa H, Yamano T, Iguchi H, Fukuzawa H . Inner lumen proteins stabilize doublet microtubules in cilia and flagella. Nat Commun. 2019; 10(1):1143. PMC: 6408466. DOI: 10.1038/s41467-019-09051-x. View

17.

Yanagisawa H, Mathis G, Oda T, Hirono M, Richey E, Ishikawa H . FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas. Mol Biol Cell. 2014; 25(9):1472-83. PMC: 4004596. DOI: 10.1091/mbc.E13-08-0464. View

18.

Carvalho-Santos Z, Machado P, Branco P, Tavares-Cadete F, Rodrigues-Martins A, Pereira-Leal J . Stepwise evolution of the centriole-assembly pathway. J Cell Sci. 2010; 123(Pt 9):1414-26. DOI: 10.1242/jcs.064931. View

19.

Allen R . The morphogenesis of basal bodies and accessory structures of the cortex of the ciliated protozoan Tetrahymena pyriformis. J Cell Biol. 1969; 40(3):716-33. PMC: 2107651. DOI: 10.1083/jcb.40.3.716. View

20.

Ichikawa M, Liu D, Kastritis P, Basu K, Hsu T, Yang S . Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins. Nat Commun. 2017; 8:15035. PMC: 5418579. DOI: 10.1038/ncomms15035. View