Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia

Autoři: Hiroshi Yoke aff001;  Hironori Ueno aff002;  Akihiro Narita aff003;  Takafumi Sakai aff001;  Kahoru Horiuchi aff001;  Chikako Shingyoji aff001;  Hiroshi Hamada aff004;  Kyosuke Shinohara aff001
Působiště autorů: Department of Biotechnology & Life Science, Tokyo University of Agriculture & Technology, Koganei, Tokyo, Japan aff001;  Molecular Function & Life Sciences, Aichi University of Education, Kariya, Aichi, Japan aff002;  Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan aff003;  Center for Biosystems Dynamics Research, RIKEN, Kobe, Japan aff004
Vyšlo v časopise: Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia. PLoS Genet 16(3): e32767. doi:10.1371/journal.pgen.1008664
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008664


Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.

Klíčová slova:

Cilia – Dyneins – Immunofluorescence – Microtubules – Sperm – Subcellular localization – Trachea – Tubulins


1. Horani A, Ferkol TW, Dutcher SK, Brody SL. Genetics and biology of primary ciliary dyskinesia. Paediatric respiratory reviews. 2016; 18:18–24. Epub 2015/10/20. doi: 10.1016/j.prrv.2015.09.001 26476603

2. Zhao L, Yuan S, Cao Y, Kallakuri S, Li Y, Kishimoto N, DiBella L, Sun Z. Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility. Proc. Natl. Acad. Sci. USA 2013; 110(31):12697–702. Epub 2013/7/15. doi: 10.1073/pnas.1300968110 23858445

3. Li Y, Zhao L, Yuan S, Zhang J, Sun Z. Axonemal dynein assembly requires the R2TP complex component Pontin. Development 2017 Dec 15;144(24):4684–4693. Epub 2017 Nov 7. doi: 10.1242/dev.152314 29113992

4. Hartill et al 2018 DNAAF1 links heart laterality with the AAA1 ATPase RUVBL1 and ciliary intraflagellar transport. Hum Mol. Genet. 2018; 27(3):529–545. doi: 10.1093/hmg/ddx422 29228333

5. Omran etal Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins. Nature 2008; 456(7222):611–6. doi: 10.1038/nature07471 19052621

6. Loges N. T. etal 2009. Deletions and Point Mutations of LRRC50 Cause Primary Ciliary Dyskinesia Due to Dynein Arm Defects. Am. J. Hum. Genet. 2009; 85(6):883–9. doi: 10.1016/j.ajhg.2009.10.018 19944400

7. Mitchison etal 2012 Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia. Nat. Genet. 2012; 44(4):381–9, S1-2. doi: 10.1038/ng.1106 22387996

8. Tarkar etal 2013 DYX1C1 is required for axonemal dynein assembly and ciliary motility. Nat. Genet. 2013; 45(9):995–1003. Epub 2013/7/21. doi: 10.1038/ng.2707 23872636

9. Olblich et al 2002 Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left–right asymmetry Nat. Genet. 2002; 30(2):143–4. Epub 2002/1/14. doi: 10.1038/ng817 11788826

10. Fassad et al 2018 Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus. Am J Hum Genet. 2018; 103(6):984–994. Epub 2018/11/21. doi: 10.1016/j.ajhg.2018.10.016 30471717

11. Smith EF, Yang P. The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil. Cytoskeleton. 2004;57(1):8–17. doi: 10.1002/cm.10155 14648553

12. Lindemann C. B.,. Lesich K. A. 2010. Flagellar and ciliary beating: the proven and the possible. J. Cell Sci. 2010;123(Pt 4):519–28. doi: 10.1242/jcs.051326 20145000

13. Oda T., Yanagisawa H. Yagi T., Kikkawa M. Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity. J. Cell Biol. 2014 Mar 3;204(5):807–19. doi: 10.1083/jcb.201312014 24590175

14. Castleman VH, Romio L, Chodhari R, Hirst RA, de Castro SC, Parker KA, etal Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause Primary Ciliary Dyskinesia with central-microtubular-pair abnormalities. Am. J. Hum. Genet. 2009; 84(2):197–209. Epub 2009/2/5. doi: 10.1016/j.ajhg.2009.01.011 19200523

15. Burgoyne T, Lewis A, Dewar A, Luther P, Hogg C, Shoemark A, Dixon M. 2014. Characterizing the ultrastructure of primary ciliary dyskinesia transposition defect using electron tomography. Cytoskeleton 2014; 71(5):294–301. Epub 2014/3/25. doi: 10.1002/cm.21171 24616277

16. Knowles M.R. et al Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype. Am. J. Resp. Crit. Med. 2014; 189(6):707–17. doi: 10.1164/rccm.201311-2047OC 24568568

17. Lin J, Yin W, Smith MC, Song K, Leigh MW, Zariwala MA, Knowles MR, Ostrowski LE, Nicastro D. 2014. Cryo-electron tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia. Nat. Commun. 2014; 5:5727. doi: 10.1038/ncomms6727 25473808

18. Daniels ML, Leigh MW, Davis SD, Armstrong MC, Carson JL, Hazucha M, Dell SD, Eriksson M, Collins FS, Knowles MR, Zariwala MA. 2013. Founder mutation in RSPH4A identified in patients of hispanic descent with primary ciliary dyskinesia. Human Mutation 2013; 34(10):1352–6. Epub 2013 Aug 6. doi: 10.1002/humu.22371 23798057

19. Yiallouros PK, Kouis P, Pirpa P, Michailidou K, Hadjisavvas A, Loizidou M, Kyriacou K 2017. Clinical disease spectrum in RSPH9 Primary Ciliary Dyskinesia patients: a case series. European Respiratory J. 2017; 50: PA1853; https://doi: 10.1183/1393003.congress-2017.PA1853.

20. Frommer et al 2015. Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects. Am. J. Respir. Cell Mol. Biol. 2015; 53(4):563–73. doi: 10.1165/rcmb.2014-0483OC 25789548

21. Jeanson etal 2015. RSPH3 Mutations Cause Primary Ciliary Dyskinesia with Central-Complex Defects and a Near Absence of Radial Spokes. Am J. Hum. Genet. 2015;97(1):153–62. Epub 2015 Jun 11. doi: 10.1016/j.ajhg.2015.05.004 26073779.

22. Abbasi F, Miyata H, Shimada K, Morohoshi A, Nozawa K, Matsumura T, Xu Z, Pratiwi P, Ikawa M. 2018. RSPH6A is required for sperm flagellum formation and male fertility in mice J. Cell Sci. 2018; 131(19). pii: jcs221648. doi: 10.1242/jcs.221648 30185526

23. Pigino G., Ishikawa T. 2012 Axonemal radial spokes. 3D structure, function and assembly. BioArchitecture 2012; 2(2):50–58. doi: 10.4161/bioa.20394 22754630

24. Warner F. D. 1970. New observations on flagellar fine structure. The relationship between matrix structure and the microtubule component of the axoneme. J. Cell Biol. 1970; 47(1):159–82. doi: 10.1083/jcb.47.1.159 4935335

25. Pigino G, Bui KH, Maheshwari A, Lupetti P, Diener D, Ishikawa T. 2011 Cryoelectron tomography of radial spokes in cilia and flagella J. Cell Biol. 2011; 195(4):673–87. Epub 2011/11/7. doi: 10.1083/jcb.201106125 22065640

26. Lin J, Heuser T, Carbajal-González BI, Song K, Nicastro D 2012. The structural heterogeneity of radial spokes in Cilia and Flagella is Conserved. Cytoskeleton 2012; 69(2):88–100. Epub 2012/1/12. doi: 10.1002/cm.21000 22170736

27. Yamaguchi H., Oda T, Kikkawa M, Takeda H. 2018. Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly eLife 2018; 7. pii: e36979. doi: 10.7554/eLife.36979 29741156

28. Zhu X., Liu Y, Yang PF. 2017. Radial Spokes-A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella. Cold Spring Harb. Perspect. Biol. 2017; 9(5). pii: a028126. doi: 10.1101/cshperspect.a028126 27940518

29. Shinohara K., Chen D, Nishida T, Misaki K, Yonemura S, Hamada H. 2015. Absence of radial spokes in mouse node cilia is required for rotational movement but confers ultrastructural instability as a trade-off. Dev. Cell 2015; 35(2):236–46. doi: 10.1016/j.devcel.2015.10.001 26506310

30. Ueno H., Ishikawa T, Bui KH, Gonda K, Ishikawa T, Yamaguchi T. Mouse respiratory cilia with the asymmetric axonemal structure on sparsely distributed ciliary cells can generate overall directional flow. Nanomedicine 2012; 8(7):1081–7. Epub 2012/1/31 doi: 10.1016/j.nano.2012.01.004 22306160

31. Patel-King R. S., Gorbatyuk O, Takebe S, King SM, SM 2004 Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase. Mol. Biol. Cell 2004; 15(8):3891–902. Epub 2004/6/11. doi: 10.1091/mbc.E04-04-0352 15194815

32. Kohno T, Wakabayashi K, Diener DR, Rosenbaum JL, Kamiya R. Subunit Interactions Within the Chlamydomonas Flagellar Spokehead. Cytoskeleton 2011; 68(4):237–46. Epub 2011 Mar 9. doi: 10.1002/cm.20507 21391306

33. Sivadas P.J. Dienes M, St. Maurice M, Meek WD, Yang P. A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex. J. Cell Biol. 2012; 199(4):639–51. doi: 10.1083/jcb.201111042 23148234

34. Anderegg L, Im Hof Gut M, Hetzel U, Howerth EW, Leuthard F, Kyöstilä K etal. NME5 frameshift variant in Alaskan Malamutes with primary ciliary dyskinesia. PLoS Genetics 15(9):e1008378. doi: 10.1371/journal.pgen.1008378 31479451

35. Wooley DM. Studies on the eel sperm flagellum I. The structure of the inner dynein arm complex. J. Cell Sci. 1997; 110 (Pt 1):85–94. 9010787

36. Wooley DM. Studies on the eel sperm flagellum III. Vibratile motility and rotatory bending. Cell Motil.Cytoskeleton 1998; 39(3):246–55. doi: 10.1002/(SICI)1097-0169(1998)39:3<246::AID-CM7>3.0.CO;2-2 9519905

37. Kremer JR., Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 1996; 116(1):71–6. doi: 10.1006/jsbi.1996.0013 8742726

38. Yasunaga T, Wakabayashi T. Extensible and object-oriented system Eos supplies a new environment for image analysis of electron micrographs of macromolecules. J. Struct. Biol. 1996; 116(1):155–60. doi: 10.1006/jsbi.1996.0025 8742738

39. Heymann JB. Bsoft: Image and molecular processing in electron microscopy. J. Struct. Biol. 2001; 133(2–3):156–69. doi: 10.1006/jsbi.2001.4339 11472087

40. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 2004; 25(13):1605–12. doi: 10.1002/jcc.20084 15264254

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