Quantitative high-throughput assays for flagella-based motility in chlamydomonas using plate-well image analysis and transmission correlation spectroscopy.
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.
Marshall
Wallace F
WF
Department of Biochemistry & Biophysics, University of California, San Francisco 94158, USA. wallace.marshall@ucsf.edu
D000818:Animals; D001681:Biological Assay / Q000191:economics / Q000379:methods; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002465:Cell Movement / Q000502:physiology*; D016825:Chlamydomonas reinhardtii / Q000166:cytology* / Q000502:physiology*; D003362:Cost-Benefit Analysis; D004526:Efficiency; D005407:Flagella / Q000502:physiology*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008954:Models, Biological; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors