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

被引:11
作者
Marshall, Wallace F. [1 ]
机构
[1] Univ Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94158 USA
基金
美国国家卫生研究院;
关键词
ciliogenesis; gravitaxis; intraflagellar transport; dynein; inhibitor; FLUORESCENCE CORRELATION SPECTROSCOPY; INTRAFLAGELLAR TRANSPORT; CILIA; REINHARDTII; CELLS; MUTATION; PROTEIN; KINASE; MUTANT; LACKS;
D O I
10.1177/1087057108328131
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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)
引用
收藏
页码:133 / 141
页数:9
相关论文
共 31 条
[1]   Cilia-related diseases [J].
Afzelius, BA .
JOURNAL OF PATHOLOGY, 2004, 204 (04) :470-477
[2]  
Blackman R. B., 1958, MEASUREMENT POWER SP
[3]   The bld1 mutation identifies the Chlamydomonas osm-6 homolog as a gene required for flagellar assembly [J].
Brazelton, WJ ;
Amundsen, CD ;
Silflow, CD ;
Lefebvre, PA .
CURRENT BIOLOGY, 2001, 11 (20) :1591-1594
[4]  
DUTCHER SK, 1988, GENETICS, V120, P965
[5]   Fluorescence correlation spectroscopy measures molecular transport in cells [J].
Elson, EL .
TRAFFIC, 2001, 2 (11) :789-796
[6]  
Harris E. H., 1989, CHLAMYDOMONAS SOURCE
[7]  
KAMIYA R, 1985, J CELL SCI, V74, P181
[8]   INCREASED LEVELS OF MESSENGER-RNAS FOR TUBULIN AND OTHER FLAGELLAR PROTEINS AFTER AMPUTATION OR SHORTENING OF CHLAMYDOMONAS FLAGELLA [J].
LEFEBVRE, PA ;
SILFLOW, CD ;
WIEBEN, ED ;
ROSENBAUM, JL .
CELL, 1980, 20 (02) :469-477
[9]   STATISTICAL PROPERTIES OF SEQUENCES OF STOCHASTIC PULSES [J].
LUKES, T .
PROCEEDINGS OF THE PHYSICAL SOCIETY OF LONDON, 1961, 78 (500) :153-&
[10]  
Mahjoub MR, 2004, MOL BIOL CELL, V15, P5172, DOI 10.1091/mbc.E04-07-0571