High-content screening and profiling of drug activity in an automated centrosome-duplication assay.
Maintenance of centrosome number is essential for cell-cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image-based centrosome-duplication assay that is accurate and robust enough for both careful cell-biology studies and high-throughput screening, and employ this assay in a series of chemical-genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform-specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320-member library of uncharacterized small molecules, we identified five potent centrosome-duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.
Perlman
Zachary E
ZE
Department of Systems Biology and Institute for Chemistry and Cell Biology, Harvard Medical School, Boston, MA 02115, USA. zperlman@fas.harvard.edu
Mitchison
Timothy J
TJ
Mayer
Thomas U
TU
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D018385:Centrosome / Q000187:drug effects* / Q000378:metabolism; D019295:Computational Biology; D004353:Drug Evaluation, Preclinical / Q000379:methods*