An intermittent live cell imaging screen for siRNA enhancers and suppressors of a kinesin-5 inhibitor.
Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug's actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology.
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
Melody
M
Tsui
Tiao
T
Xie
James D
JD
Orth
Anne E
AE
Carpenter
Stewart
S
Rudnicki
Suejong
S
Kim
Caroline E
CE
Shamu
Timothy J
TJ
Mitchison
D015894:Genome, Human; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008954:Models, Biological; D015233:Models, Statistical; D009369:Neoplasms / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism*; D008941:Spindle Apparatus / Q000187:drug effects; D013997:Time Factors; D050257:Tubulin Modulators / Q000494:pharmacology
C470243:KIF11 protein, human; D034741:RNA, Small Interfering; D050257:Tubulin Modulators; D049452:Green Fluorescent Proteins; D016547:Kinesins; D017239:Paclitaxel; D015739:Nocodazole