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Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii

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Plant Chemical Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1795))

Abstract

Flagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of vertebrate cells and provide an excellent model to study ciliogenesis. Cilia and flagella are important organelles used for motility and sensing the extracellular environment. Abnormalities in cilia structure or ciliary dysfunction can have devastating consequences ranging from diabetes and obesity to polycystic kidney disease and mental retardation. Small-molecule inhibitor libraries can be used to screen for flagellum-associated phenotypes in assembly, length, motility, deflagellation, and cellular toxicity. These phenotypes can be assessed from direct microscopic visualization and custom-designed assays. These methods identify fundamental regulators of ciliary biology as well as potential therapeutic interventions for ciliopathies.

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References

  1. Ishikawa H, Marshall WF (2011) Ciliogenesis: building the cell’s antenna. Nat Rev Mol Cell Biol 12:222–234

    Article  CAS  Google Scholar 

  2. 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  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fuhrmann M, Stahlberg A, Govorunova E, Rank S, Hegemann P (2001) The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses. J Cell Sci 114:3857–3863

    PubMed  CAS  Google Scholar 

  4. Molnar A, Bassett A, Thuenemann E, Schwach F, Karkare S, Ossowski S et al (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58:165–174

    Article  CAS  PubMed  Google Scholar 

  5. Zhao T, Wang W, Bai X, Qi Y (2009) Gene silencing by artificial microRNAs in Chlamydomonas. Plant J 58:157–164

    Article  CAS  PubMed  Google Scholar 

  6. Avasthi P, Marley A, Lin H, Gregori-Puigjane E, Shoichet BK, von Zastrow M et al (2012) A chemical screen identifies class a g-protein coupled receptors as regulators of cilia. Am Soc Chem Biol 7:911–919

    CAS  Google Scholar 

  7. Hutner SH, Provasoli L, Scahtz A, Haskins P (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94:152–170

    CAS  Google Scholar 

  8. Rosenbaum JL, Moulder JE, Ringo DL (1969) Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins. J Cell Biol 41:600–619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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 12:222–234

    Google Scholar 

  10. Marshall WF, Qin H, Rodrigo Brenni M, Rosenbaum JL (2005) Flagellar length control system: testing a simple model based on intraflagellar transport and turnover. Mol Biol Cell 16:270–278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Marshall WF, Rosenbaum JL (2001) Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J Cell Biol 155:405–414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Parker JD, Quarmby LM (2003) Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport. BMC Cell Biol 4:11

    Article  PubMed  PubMed Central  Google Scholar 

  13. Bradley BA, Quarmby LM (2005) A NIMA-related kinase, Cnk2p, regulates both flagellar length and cell size in Chlamydomonas. J Cell Sci 118:3317–3326

    Article  CAS  PubMed  Google Scholar 

  14. Nakamura S, Takino H, Kojima MK (1987) Effect of lithium on flagellar length in Chlamydomonas-reinhardtii. Cell Struct Funct 12:369–374

    Article  CAS  Google Scholar 

  15. Marshall WF (2009) Quantitative high-throughput assays for flagella-based motility in chlamydomonas using plate-well image analysis and transmission correlation spectroscopy. J Biomol Screen 14:133–141

    Article  CAS  PubMed  Google Scholar 

  16. Dutta S, Avasthi P (2017) Flagellar synchronization is a simple alternative to cell cycle synchronization for ciliary and flagellar studies. mSphere 2(2). https://doi.org/10.1128/mSphere.00003-17

  17. Sato M, Murata Y, Mizusawa M, Iwashashi H, Shu-ichi O (2004) A simple and rapid dual-fluorescence viability assay for microalgae. Micrbiol Cult Coll 20:53–59

    Google Scholar 

  18. Lefebvre P, Nordstrom S, Moulder J, Rosenbaum JL (1978) Flagellar elongation and shortening in Chlamydomonas. J Cell Biol 78:8–27

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We would like to thank all the members of the Avasthi Lab for the critical reading of the manuscript. This work was funded by P20 GM104936-09 (PA) and NSF GRFP 1518767 (BJ). Thanks to Soumita Dutta for providing images of healthy and contaminated cells for Fig. 1.

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Correspondence to Prachee Avasthi .

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Jack, B., Avasthi, P. (2018). Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii. In: Fauser, F., Jonikas, M. (eds) Plant Chemical Genomics. Methods in Molecular Biology, vol 1795. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7874-8_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7874-8_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7873-1

  • Online ISBN: 978-1-4939-7874-8

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