Cilia pp 107-122 | Cite as

Kymographic Analysis of Transport in an Individual Neuronal Sensory Cilium in Caenorhabditis elegans

  • Robert O’HaganEmail author
  • Maureen M. Barr
Part of the Methods in Molecular Biology book series (MIMB, volume 1454)


Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules.

Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM-3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2.

Key words

Caenorhabditis elegans Kymograph Cell biology In vivo Sensory non-motile cilia PKD-2 Polycystins OSM-3 Kinesin-2 KLP-6 Kinesin-3 Male 



The authors were supported by NJCSCR Grant CSCR15IRG014 (R.O.) and NIH Grants DK059418 and DK074746 (M.B.).


  1. 1.
    Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL (1993) A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci U S A 90(12):5519–5523CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Pedersen LB, Geimer S, Rosenbaum JL (2006) Dissecting the molecular mechanisms of intraflagellar transport in Chlamydomonas. Curr Biol 16(5):450–459. doi: 10.1016/j.cub.2006.02.020 CrossRefGoogle Scholar
  3. 3.
    Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL (1998) Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol 141(4):993–1008CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Scholey JM (2008) Intraflagellar transport motors in cilia: moving along the cell's antenna. J Cell Biol 180(1):23–29CrossRefPubMedCentralGoogle Scholar
  5. 5.
    Inglis PN, Ou G, Leroux MR, Scholey JM (2006) The sensory cilia of Caenorhabditis elegans. WormBook:1–22Google Scholar
  6. 6.
    Ou G, Blacque OE, Snow JJ, Leroux MR, Scholey JM (2005) Functional coordination of intraflagellar transport motors. Nature 436(7050):583–587. doi: 10.1038/nature03818 CrossRefGoogle Scholar
  7. 7.
    Snow JJ, Ou G, Gunnarson AL, Walker MR, Zhou HM, Brust-Mascher I, Scholey JM (2004) Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons. Nat Cell Biol 6(11):1109–1113. doi: 10.1038/ncb1186 CrossRefGoogle Scholar
  8. 8.
    Jenkins PM, Hurd TW, Zhang L, McEwen DP, Brown RL, Margolis B, Verhey KJ, Martens JR (2006) Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17. Curr Biol 16(12):1211–1216. doi: 10.1016/j.cub.2006.04.034 CrossRefGoogle Scholar
  9. 9.
    Insinna C, Humby M, Sedmak T, Wolfrum U, Besharse JC (2009) Different roles for KIF17 and kinesin II in photoreceptor development and maintenance. Dev Dyn 238(9):2211–2222CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Jiang L, Tam BM, Ying G, Wu S, Hauswirth WW, Frederick JM, Moritz OL, Baehr W (2015) Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function. Faseb J. doi: 10.1096/fj.15-275677 Google Scholar
  11. 11.
    Miki H, Okada Y, Hirokawa N (2005) Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol 15(9):467–476. doi: 10.1016/j.tcb.2005.07.006 CrossRefGoogle Scholar
  12. 12.
    Morsci NS, Barr MM (2011) Kinesin-3 KLP-6 regulates intraflagellar transport in male-specific cilia of Caenorhabditis elegans. Curr Biol 21(14):1239–1244. doi: 10.1016/j.cub.2011.06.027 CrossRefPubMedCentralGoogle Scholar
  13. 13.
    Perkins LA, Hedgecock EM, Thomson JN, Culotti JG (1986) Mutant sensory cilia in the nematode Caenorhabditis elegans. Dev Biol 117(2):456–487CrossRefGoogle Scholar
  14. 14.
    Mukhopadhyay S, Lu Y, Qin H, Lanjuin A, Shaham S, Sengupta P (2007) Distinct IFT mechanisms contribute to the generation of ciliary structural diversity in C. elegans. Embo J 26(12):2966–2980CrossRefPubMedCentralGoogle Scholar
  15. 15.
    O'Hagan R, Piasecki BP, Silva M, Phirke P, Nguyen KC, Hall DH, Swoboda P, Barr MM (2011) The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans. Curr Biol 21(20):1685–1694. doi: 10.1016/j.cub.2011.08.049 CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Rand JB (2007) Acetylcholine. WormBook:1–21. doi:10.1895/wormbook.1.131.1Google Scholar
  17. 17.
    Qin H, Burnette DT, Bae YK, Forscher P, Barr MM, Rosenbaum JL (2005) Intraflagellar transport is required for the vectorial movement of TRPV channels in the ciliary membrane. Curr Biol 15(18):1695–1699CrossRefGoogle Scholar
  18. 18.
    Warburton-Pitt SR, Silva M, Nguyen KC, Hall DH, Barr MM (2014) The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans. PLoS Genet 10(12), e1004866. doi: 10.1371/journal.pgen.1004866 Google Scholar
  19. 19.
    Cevik S, Sanders AA, Van Wijk E, Boldt K, Clarke L, van Reeuwijk J, Hori Y, Horn N, Hetterschijt L, Wdowicz A, Mullins A, Kida K, Kaplan OI, van Beersum SE, Man Wu K, Letteboer SJ, Mans DA, Katada T, Kontani K, Ueffing M, Roepman R, Kremer H, Blacque OE (2013) Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain. PLoS Genet 9(12), e1003977. doi: 10.1371/journal.pgen.1003977 CrossRefPubMedCentralGoogle Scholar
  20. 20.
    Granato M, Schnabel H, Schnabel R (1994) pha-1, a selectable marker for gene transfer in C. elegans. Nucleic Acids Res 22(9):1762–1763CrossRefPubMedCentralGoogle Scholar
  21. 21.
    Barr MM, Sternberg PW (1999) A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401(6751):386–389Google Scholar
  22. 22.
    Barr MM, DeModena J, Braun D, Nguyen CQ, Hall DH, Sternberg PW (2001) The C. elegans autosomal dominant polycystic kidney disease gene homologs lov-1 and pkd-2 act in the same pathway. Curr Biol 11(17):1341–1346Google Scholar
  23. 23.
    Jauregui AR, Nguyen KC, Hall DH, Barr MM (2008) The C. elegans nephrocystins act as global modifiers of cilium structure. J Cell Biol 180(5):973–988CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Prelich G (2012) Gene overexpression: uses, mechanisms, and interpretation. Genetics 190(3):841–854. doi: 10.1534/genetics.111.136911 CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Frokjaer-Jensen C (2013) Exciting prospects for precise engineering of C. elegans genomes with CRISPR/Cas9. Genetics 195(3):635–642. doi: 10.1534/genetics.113.156521 CrossRefPubMedCentralGoogle Scholar
  26. 26.
    Bae YK, Qin H, Knobel KM, Hu J, Rosenbaum JL, Barr MM (2006) General and cell-type specific mechanisms target TRPP2/PKD-2 to cilia. Development 133(19):3859–3870CrossRefGoogle Scholar
  27. 27.
    O'Hagan R, Wang J, Barr MM (2014) Mating behavior, male sensory cilia, and polycystins in C. elegans. Seminars in Cell & Developmental Biology 33:25–33. doi: 10.1016/j.semcdb.2014.06.001 CrossRefGoogle Scholar
  28. 28.
    Wang J, Silva M, Haas LA, Morsci NS, Nguyen KC, Hall DH, Barr MM (2014) C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol 24(5):519–525. doi: 10.1016/j.cub.2014.01.002 CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Human Genetics Institute of New Jersey, RutgersThe State University of New JerseyPiscatawayUSA

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