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Purification and Localization of Intraflagellar Transport Particles and Polypeptides

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Cytoskeleton Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1365))

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Abstract

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.

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Notes

  1. 1.

    The term “flagella” is used throughout this chapter, but readers should realize that the process of IFT occurs—and is just as important—in cilia as well. Indeed, many developmental problems and diseases of humans are related to defects in primary cilia, some of which derive from defects in IFT (see ref. [1] for a recent overview of this field).

References

  1. Brown JM, Witman GB (2014) Cilia and diseases. Bioscience 64:1126–1137

    Article  PubMed Central  PubMed  Google Scholar 

  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 90:5519–5523

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 131:1517–1527

    Article  CAS  PubMed  Google Scholar 

  4. Kozminski KG (1995) High-resolution imaging of flagella. Methods Cell Biol 47:263–271

    Article  CAS  PubMed  Google Scholar 

  5. Orozco JT, Wedaman KP, Signor D, Brown H, Rose L, Scholey JM (1999) Movement of motor and cargo along cilia. Nature 398:674

    Article  CAS  PubMed  Google Scholar 

  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 16:1341–1354

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 187:81–89

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Pazour GJ, Dickert BL, Witman GB (1999) The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly. J Cell Biol 144:473–481

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 10:693–712

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 147:519–530

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Walther Z, Vashishtha M, Hall JL (1994) The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. J Cell Biol 126:175–188

    Article  CAS  PubMed  Google Scholar 

  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 141:993–1008

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Cole DG (2003) The intraflagellar transport machinery of Chlamydomonas reinhardtii. Traffic 4:435–442

    Article  CAS  PubMed  Google Scholar 

  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 187:135–148

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Johnson KA, Rosenbaum JL (1992) Polarity of flagellar assembly in Chlamydomonas. J Cell Biol 119:1605–1611

    Article  CAS  PubMed  Google Scholar 

  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 74:747–759

    Article  CAS  PubMed  Google Scholar 

  17. Dentler WL (1980) Structures linking the tips of ciliary and flagellar microtubules to the membrane. J Cell Sci 42:207–220

    CAS  PubMed  Google Scholar 

  18. Sale WS, Satir P (1977) The termination of the central microtubules from the cilia of Tetrahymena pyriformis. Cell Biol Int Rep 1:56–63

    Article  Google Scholar 

  19. Schneider MJ, Ulland M, Sloboda RD (2008) A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption. Mol Biol Cell 19:4319–4327

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 126:5018–5029

    Article  PubMed Central  PubMed  Google Scholar 

  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 54:1665–1669

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 94:152–170

    CAS  Google Scholar 

  23. Surzycki S (1971) Synchronously grown cultures of Chlamydomonas reinhardi. Meth Enzymol 23:67–84

    Article  Google Scholar 

  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 64:446–460

    Article  CAS  PubMed  Google Scholar 

  25. LaemmLi UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  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 114:281–284

    Article  CAS  PubMed  Google Scholar 

  27. Stephens RE (1998) Electrophoretic resolution of tubulin and tektin subunits by differential interaction with long-chain alkyl sulfates. Anal Biochem 265:356–360

    Article  CAS  PubMed  Google Scholar 

  28. Fairbanks G, Steck TL, Wallach DF (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617

    Article  CAS  PubMed  Google Scholar 

  29. Hunt GE (1947) A technique for aeration of sterile liquid culture medium. Science 105:184

    Article  CAS  PubMed  Google Scholar 

  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 54:507–539

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 13:1969–1974

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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

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Authors and Affiliations

  1. Department of Biological Sciences, Dartmouth College, Hanover, NH, 03755, USA

    Roger D. Sloboda

  2. The Marine Biological Laboratory, Woods Hole, MA, 02543, USA

    Roger D. Sloboda

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  1. Roger D. Sloboda
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Correspondence to Roger D. Sloboda .

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Editors and Affiliations

  1. Brooklyn College, Brooklyn, New York, USA

    Ray H. Gavin

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Sloboda, R.D. (2016). Purification and Localization of Intraflagellar Transport Particles and Polypeptides. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 1365. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3124-8_6

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  • DOI: https://doi.org/10.1007/978-1-4939-3124-8_6

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3123-1

  • Online ISBN: 978-1-4939-3124-8

  • eBook Packages: Springer Protocols

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