Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets Ulrike Eggert, Amy Kiger, Constance Richter, Zachary Perlman, Norbert Perrimon, Tomothy Mitchison, Christine Field Pat Ekpanyaskun Mar 3, 2005

To study protein function Small molecule inhibitors Small molecule inhibitors – directly inhibit protein fx. – search by phenotypic screening -> i.d. targets / pathway – binding of irrelevant prot.

RNA interference RNAi RNAi –dsRNA targets destruction of RNA –inhibit prot. fx in a genome-wide basis

Mitosis daughter cells are separated by constriction of a cleavage furrow

This paper Goal Complete list of prot. required for cytokinesis in Drosophila cells Complete list of prot. required for cytokinesis in Drosophila cellsApproach Genome-wide RNAi screening & small molecule inhibitors Genome-wide RNAi screening & small molecule inhibitors

Screening dsRNA screening dsRNA screening - >90% annotated genome dsRNAs - incubate ~4 d Small molecule screening Small molecule screening - 51,000 inhibitors 2 d 2 d Immunofluorescence -dsRNAs of 214 genes -1 new gene (Borr/CG4454) -50 inhibitors (commercial lib, bioactive, nat prod. extract)

Predicted Functional Annotations of 214 Genes Associated with RNAi Binucleate Phenotypes

32% 44% 24% 74% 20% 6% 40% 29% 51% 28% Penetrance of binucleate phenotype Inhibitor RNAi

24% 40% 29% 51% 28% 8% 12% Inhibitor RNAi Low cell count Phenotypic classes Binucleate binucleate w large, diffuse DNA Low cell countBinucleate with MT extension

Two phenotypes correlate in both methods dsRNA targeting Act5C OR Cytochalasin D dsRNA targeting Act5C OR Cytochalasin D RNAi inhibitors

Two phenotypes correlate in both methods Binucleate with diffuse DNA Chromosomal passenger protein that co-localize with Aurora B throughout mitosis & cytokinesis

Aurora B Chromosomal passenger prot Chromosomal passenger prot Localize on centromeres (prophase-metaphase) Localize on centromeres (prophase-metaphase) Relocalize to spindle midzone and cortex (anaphase) Relocalize to spindle midzone and cortex (anaphase) Disruption causes loss of phosphorylation on histone H3 and failure in cytokinesis Disruption causes loss of phosphorylation on histone H3 and failure in cytokinesis Andrews et al 2003

Two phenotypes correlate in both methods Chromosomal passenger protein that co-localize with Aurora B throughout mitosis & cytokinesis

Borr/CG4454 co-localizes with Aurora B

Cells treated to aurora B dsRNA, borr/CG4454 dsRNA or Binucleine 2 results in an absence of H3 Borr is required for INCENP localization Aurora B RNAi and Binucleine 2 shares same phenotype and affect localization of INCENPBorr is required for INCENP localization Aurora B RNAi and Binucleine 2 shares same phenotype and affect localization of INCENP

Summary i.d. new prot. involved in cytokinesis and new small molecule that inhibits it i.d. new prot. involved in cytokinesis and new small molecule that inhibits it 214 prot. important for cytokinesis in Drosophila 214 prot. important for cytokinesis in Drosophila Borr/CG4454 is a chromosomal passenger prot that co-localize with Aurora B Borr/CG4454 is a chromosomal passenger prot that co-localize with Aurora B Depletion had a profound effect on cytokinesis Depletion had a profound effect on cytokinesis Binucleine 2 as a cancer drug ? Binucleine 2 as a cancer drug ?

Message Integration of data from different tools to answer biological questions Integration of data from different tools to answer biological questions Subjective to visual inspection Subjective to visual inspection Comment