Advertisement

Cilia pp 83-96 | Cite as

Methods for Studying Movement of Molecules Within Cilia

  • Karl F. LechtreckEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1454)

Abstract

The assembly of cilia and eukaryotic flagella (interchangeable terms) requires the import of numerous proteins from the cell body into the growing organelle. Proteins move into and inside cilia by diffusion and by motor-based intraflagellar transport (IFT). Many aspects of ciliary protein transport such as the distribution of unloading sites and the frequency of transport can be analyzed using direct in vivo imaging of fluorescently tagged proteins. Here, we will describe how to use total internal reflection fluorescence microcopy (TIRFM) to analyze protein transport in the flagella of the unicellular alga Chlamydomonas reinhardtii, a widely used model for cilia and cilia-related disease.

Key words

Cilia Flagella Green fluorescent protein Intraflagellar transport (IFT) Total internal reflection fluorescence microscopy (TIRFM) 

Notes

Acknowledgement

This work was supported by a grant from the National Institutes of Health (GM110413).

References

  1. 1.
    Jiang YY, Lechtreck K, Gaertig J (2015) Total internal reflection fluorescence microscopy of intraflagellar transport in Tetrahymena thermophila. Methods Cell Biol 127:445–456. doi: 10.1016/bs.mcb.2015.01.001 CrossRefGoogle Scholar
  2. 2.
    Ishikawa H, Marshall WF (2015) Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia. Methods Cell Biol 127:189–201. doi: 10.1016/bs.mcb.2015.01.002 CrossRefGoogle Scholar
  3. 3.
    Lechtreck KF (2013) In vivo imaging of IFT in Chlamydomonas flagella. Methods Enzymol 524:265–284. doi: 10.1016/B978-0-12-397945-2.00015-9 CrossRefGoogle Scholar
  4. 4.
    Kozminski KG, Beech PL, Rosenbaum JL (1995) The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane. J Cell Biol 131:1517–1527CrossRefGoogle Scholar
  5. 5.
    Mueller J, Perrone CA, Bower R, Cole DG, Porter ME (2005) The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport. Mol Biol Cell 16:1341–1354. doi: 10.1091/mbc.E04-10-0931 CrossRefPubMedCentralGoogle Scholar
  6. 6.
    Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19:353–361. doi: 10.1046/j.1365-313X.1999.00526.x CrossRefGoogle Scholar
  7. 7.
    Craft JM, Harris JA, Hyman S, Kner P, Lechtreck KF (2015) Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism. J Cell Biol 208(2):223–237. doi: 10.1083/jcb.201409036 CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, Oyler GA, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74:545–556. doi: 10.1111/tpj.12165 CrossRefGoogle Scholar
  9. 9.
    Lechtreck KF, Johnson EC, Sakai T, Cochran D, Ballif BA, Rush J, Pazour GJ, Ikebe M, Witman GB (2009) The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella. J Cell Biol 187:1117–1132. doi: 10.1083/jcb.200909183 CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87:1228–1232CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Bower R, Tritschler D, Vanderwaal K, Perrone CA, Mueller J, Fox L, Sale WS, Porter ME (2013) The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes. Mol Biol Cell 24:1134–1152. doi: 10.1091/mbc.E12-11-0801 CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Collingridge P, Brownlee C, Wheeler GL (2013) Compartmentalized calcium signaling in cilia regulates intraflagellar transport. Curr Biol 23:2311–2318. doi: 10.1016/j.cub.2013.09.059 CrossRefGoogle Scholar
  13. 13.
    Huang K, Diener DR, Mitchell A, Pazour GJ, Witman GB, Rosenbaum JL (2007) Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella. J Cell Biol 179:501–514. doi: 10.1083/jcb.200704069 CrossRefPubMedCentralGoogle Scholar
  14. 14.
    Wren KN, Craft JM, Tritschler D, Schauer A, Patel DK, Smith EF, Porter ME, Kner P, Lechtreck KF (2013) A differential cargo-loading model of ciliary length regulation by IFT. Curr Biol 23:2463–2471. doi: 10.1016/j.cub.2013.10.044 CrossRefGoogle Scholar
  15. 15.
    Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang J (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10:407–409. doi: 10.1038/nmeth.2413 CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Pazour GJ, Agrin N, Leszyk J, Witman GB (2005) Proteomic analysis of a eukaryotic cilium. J Cell Biol 170:103–113. doi: 10.1083/jcb.200504008 CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Cellular BiologyUniversity of GeorgiaAthensUSA

Personalised recommendations