Sage Journals: Discover world-class research

Abstract

Cilia are motile and sensory organelles with important roles in human development, physiology, and disease. Genetic defects in cilia produce a host of disease symptoms, including polycystic kidney disease, hydrocephalus, retinal degeneration, chronic bronchiectasis, infertility, and polydactyly. Currently, there are no known drugs for pharmacological remediation of ciliary defects. Small-molecule modulators of ciliary assembly or function would provide potential lead compounds for drug discovery efforts and would immediately be invaluable tools for a chemical biology approach to studying cilia. Here the author describes 2 assays for ciliary motility that are quantitative, automatable, cost-effective, and simple to implement. Both assays exploit cell-based strategies using the model organism Chlamydomonas reinhardtii. The first assay scores cilia-dependent gravitaxis by analyzing the cell distribution in wells of U-bottom microplates, using a simple and robust image analysis algorithm. The second assay measures motility directly by estimating the time required for cells to swim across a small illuminated aperture using a method equivalent to fluorescence correlation spectroscopy adapted to transmitted-light microscopy. The 2 assays have different advantages in terms of speed and sensitivity to small reductions in motility and may be most efficiently used in combination. (Journal of Biomolecular Screening 2009:133-141)

Keywords

ciliogenesis gravitaxis intraflagellar transport dynein inhibitor

References

Sloboda RD , Rosenbaum JL : Making sense of cilia and flagella. J Cell Biol 2007;179:575-582.

Satir P. , Christensen ST : Overview of the structure and function of mammalian cilia. Ann Rev Physiol 2007;69:377-400.

Marshall WF , Nonaka S. : Cilia: tuning in to the cell's antenna. Curr Biol 2006;16:R604-R614.

Mitchell DR : Cell and molecular biology of flagellar dyneins. Int Rev Cytol 1994;155:141-180.

Afzelius BA : Cilia-related diseases. J Pathol 2004;204:470-477.

Zariwala MA , Knowles MR , Omran H. : Genetic defects in ciliary structure and function. Ann Rev Physiol 2007;69:423-450.

Marshall WF : The cell biological basis of ciliary disease. J Cell Biol 2008;180:17-21.

Mahjoub MR , Qasim Rasi M. , Quarmby LM : A NIMA-related kinase, Fa2p, localizes to a novel site in the proximal cilia of Chlamydomonas and mouse kidney cells. Mol Biol Cell 2004;15:5172-5186.

Wilson NF , Lefebvre PA : Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii. Eukaryot Cell 2004;3: 1307-1319.

10.

Pan J. , Snell WJ : Chlamydomonas shortens its flagella by activating axonemal disassembly, stimulating IFT particle trafficking, and blocking anterograde cargo loading. Dev Cell 2005;9:431-438.

11.

Smith EF , Yang P. : The radial spokes and central apparatus: mechanochemical transducers that regulate flagellar motility. Cell Motil Cytoskel 2004;57:8-17.

12.

Pazour GJ , Agrin N. , Leszyk J. , Witman GB : Proteomic analysis of a eukaryotic cilium. J Cell Biol 2005;170:103-113.

13.

Qin H. , Wang Z. , Diener D. , Rosenbaum J. : Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control. Curr Biol 2007;17: 193-202.

14.

Qin H. , Burnette DT , Bae YK , Forscher P. , Barr MM , Rosenbaum JL : Intraflagellar transport is required for the vectorial movement of TRPV channels in the ciliary membrane. Curr Biol 2005;15:1695-1699.

15.

Silflow CD , Lefebvre PA : Assembly and motility of eukaryotic cilia and flagella: lessons from Chlamydomonas reinhardtii. Plant Physiol 2001; 127:1500-1507.

16.

Snell WJ , Pan J. , Wang O. : Cilia and flagella revealed: from flagellar assembly in Chlamydomonas to human obesity disorders. Cell 2004;117: 693-697.

17.

Merchant S. , Prochnik SE , Vallon O. , Harris EH , Karpowicz SJ , Witman GB , et al: The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 2007;318:245-250.

18.

Racey TJ , Hallett R. , Nickel B. : A quasi-elastic light scattering and cinematographic investigation of motile Chlamydomonas reinhardtii. Biophys J 1981;35:557-571.

19.

Yoshimura K. , Matsuo Y. , Kamiya R. : Gravitaxis in Chlamydomonas reinhardtii studied with novel mutants. Plant Cell Physiol 2003 ;44:1112-1118.

20.

Harris EH : The Chlamydomonas Sourcebook. San Diego: Academic Press, 1989.

21.

Elson EL : Fluorescence correlation spectroscopy measures molecular transport in cells . Traffic 2001;2:789-796.

22.

Moore KJ , Turconi S. , Ashman S. , Ruediger M. , Haupts U. , Emerick VV , et al: Single molecule detection technologies in miniaturized high throughput screening: fluorescence correlation spectroscopy. J Biomol Screen 1999;4:335-354.

23.

Blackman RB , Tukey JW : The Measurement of Power Spectra from the Point of View of Communications Engineering. New York: Dover, 1958.

24.

Lukes T. : The statistical properties of sequences of stochastic pulses. Proc Royal Soc London 1961;78:153-168.

25.

Brazelton WJ , Amundsen CD , Silflow CD , Lefebvre PA : The bld1 mutation identifies the Chlamydomonas osm-6 homolog as a gene required for flagellar assembly. Curr Biol 2001;11:1591-1594.

26.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.

27.

Lefebvre PA , Silflow CD , Wieben ED , Rosenbaum JL : Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas flagella. Cell 1980;20:469-477.

28.

Kamiya R. , Okamoto M. : A mutant of Chlamydomonas reinhardtii that lacks the flagellar outer dynein arm but can swim. J Cell Sci 1985;74: 181-191.

29.

Mitchell DR , Rosenbaum JL : A motile Chlamydomonas flagellar mutant that lacks outer dynein arms. J Cell Biol 1985;100:1228-1234.

30.

Zhang H. , Mitchell DR : Cpc1, a Chlamydomonas central pair protein with an adenylate kinase domain . J Cell Sci 2004;117:4179-4188.

31.

Dutcher SK , Gibbons W. , Inwood WB : A genetic analysis of suppressors of the PF10 mutation in Chlamydomonas reinhardtii . Genetics 1988;120: 965-976.

Quantitative High-Throughput Assays for Flagella-Based Motility in Chlamydomonas Using Plate-Well Image Analysis and Transmission Correlation Spectroscopy

Abstract

Keywords

References