Purification and Localization of Intraflagellar Transport Particles and Polypeptides

  • Roger D. SlobodaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1365)


The growth and maintenance of almost all cilia and flagella are dependent on the proper functioning of the process of intraflagellar transport (IFT). This includes the primary cilia of most human cells that are in the Go phase of the cell cycle. The model system for the study of IFT is the flagella of the biflagellate green alga Chlamydomonas. It is in this organism that IFT was first discovered, and genetic data from a Chlamydomonas mutant first linked the process of IFT to polycystic kidney disease in humans. The information provided in this chapter addresses procedures to purify IFT particles from flagella and localize these particles, and their associated motor proteins, in flagella using light and electron microscopic approaches.

Key words

Flagella Cilia Intraflagellar transport IFT Motility Immunofluorescence Immunogold EM 



Work in the author’s lab is supported by the NSF (MCB 0950402). This support is greatly appreciated.


  1. 1.
    Brown JM, Witman GB (2014) Cilia and diseases. Bioscience 64:1126–1137PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    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 90:5519–5523PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Kozminski KG, Beech PL, Rosenbaum JL (1995) The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with flagellar membrane. J Cell Biol 131:1517–1527CrossRefPubMedGoogle Scholar
  4. 4.
    Kozminski KG (1995) High-resolution imaging of flagella. Methods Cell Biol 47:263–271CrossRefPubMedGoogle Scholar
  5. 5.
    Orozco JT, Wedaman KP, Signor D, Brown H, Rose L, Scholey JM (1999) Movement of motor and cargo along cilia. Nature 398:674CrossRefPubMedGoogle Scholar
  6. 6.
    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 16:1341–1354PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Engel BD, Ludington WB, Marshall WF (2009) Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model. J Cell Biol 187:81–89PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Pazour GJ, Dickert BL, Witman GB (1999) The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly. J Cell Biol 144:473–481PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Porter ME, Bower R, Knott JA, Byrd P, Dentler W (1999) Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas. Mol Biol Cell 10:693–712PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Signor D, Wedaman KP, Orozco JT, Dwyer ND, Bargmann CI, Rose LS, Scholey JM (1999) Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans. J Cell Biol 147:519–530PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Walther Z, Vashishtha M, Hall JL (1994) The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. J Cell Biol 126:175–188CrossRefPubMedGoogle Scholar
  12. 12.
    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 141:993–1008PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Cole DG (2003) The intraflagellar transport machinery of Chlamydomonas reinhardtii. Traffic 4:435–442CrossRefPubMedGoogle Scholar
  14. 14.
    Pigino G, Geimer S, Lanzavecchia S, Paccagnini E, Cantele F, Diener DR, Rosenbaum JL, Lupetti P (2009) Electron-tomographic analysis of intraflagellar transport particle trains in situ. J Cell Biol 187:135–148PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Johnson KA, Rosenbaum JL (1992) Polarity of flagellar assembly in Chlamydomonas. J Cell Biol 119:1605–1611CrossRefPubMedGoogle Scholar
  16. 16.
    Dentler WL, Rosenbaum JL (1977) Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly. J Cell Biol 74:747–759CrossRefPubMedGoogle Scholar
  17. 17.
    Dentler WL (1980) Structures linking the tips of ciliary and flagellar microtubules to the membrane. J Cell Sci 42:207–220PubMedGoogle Scholar
  18. 18.
    Sale WS, Satir P (1977) The termination of the central microtubules from the cilia of Tetrahymena pyriformis. Cell Biol Int Rep 1:56–63CrossRefGoogle Scholar
  19. 19.
    Schneider MJ, Ulland M, Sloboda RD (2008) A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption. Mol Biol Cell 19:4319–4327PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Satish Tammana TV, Tammana D, Diener DR, Rosenbaum J (2013) Centrosomal protein CEP104 (Chlamydomonas FAP256) moves to the ciliary tip during ciliary assembly. J Cell Sci 126:5018–5029PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 54:1665–1669PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Hutner SH, Provasoli L, Schatz A, Haskins CP (1950) Some approaches to the study of the role of metals in the metabolism of microoganisms. Proc Am Phil Soc 94:152–170Google Scholar
  23. 23.
    Surzycki S (1971) Synchronously grown cultures of Chlamydomonas reinhardi. Meth Enzymol 23:67–84CrossRefGoogle Scholar
  24. 24.
    Sloboda RD, Howard L (2007) Localization of EB1, IFT polypeptides, and kinesin-2 in Chlamydomonas flagellar axonemes via immunogold scanning electron microscopy. Cell Motil Cytoskeleton 64:446–460CrossRefPubMedGoogle Scholar
  25. 25.
    LaemmLi UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  26. 26.
    Best D, Warr PJ, Gull K (1981) Influence of the composition of commercial sodium dodecyl sulfate preparations on the separation of alpha- and beta-tubulin during polyacrylamide gel electrophoresis. Anal Biochem 114:281–284CrossRefPubMedGoogle Scholar
  27. 27.
    Stephens RE (1998) Electrophoretic resolution of tubulin and tektin subunits by differential interaction with long-chain alkyl sulfates. Anal Biochem 265:356–360CrossRefPubMedGoogle Scholar
  28. 28.
    Fairbanks G, Steck TL, Wallach DF (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617CrossRefPubMedGoogle Scholar
  29. 29.
    Hunt GE (1947) A technique for aeration of sterile liquid culture medium. Science 105:184CrossRefPubMedGoogle Scholar
  30. 30.
    Witman GB, Carlson K, Berliner J, Rosenbaum JL (1972) Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol 54:507–539PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Pedersen LB, Geimer S, Sloboda RD, Rosenbaum JL (2003) The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii. Curr Biol 13:1969–1974CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biological SciencesDartmouth CollegeHanoverUSA
  2. 2.The Marine Biological LaboratoryWoods HoleUSA

Personalised recommendations