Parallel Chemical Genetic and Parallel Chemical Genetic and Genome-Wide RNAi Screens Genome-Wide RNAi Screens

Identify Cytokinesis Inhibitors Identify Cytokinesis Inhibitors and Targetsand Targets

Ulrike Eggert, Amy Kiger, Constance Richter, Zachary Perlman, Norbert Perrimon, Tomothy Mitchison, Christine FieldUlrike Eggert, Amy Kiger, Constance Richter, Zachary Perlman, Norbert Perrimon, Tomothy Mitchison, Christine Field

Pat EkpanyaskunPat EkpanyaskunMar 3, 2005Mar 3, 2005

To study protein functionTo study protein function Small molecule inhibitorsSmall molecule inhibitors

– directly inhibit protein fx. directly inhibit protein fx. – search by phenotypic screening search by phenotypic screening

-> i.d. targets / pathway-> i.d. targets / pathway– binding of irrelevant prot.binding of irrelevant prot.

RNA interferenceRNA interference RNAiRNAi

– dsRNA targets dsRNA targets destruction of RNAdestruction of RNA

– inhibit prot. fx in a inhibit prot. fx in a genome-wide basisgenome-wide basis

MitosisMitosis

• daughter cells are separated by constriction of a cleavage furrow

This paperThis paperGoalGoal

Complete list of prot. required for Complete list of prot. required for cytokinesis in cytokinesis in DrosophilaDrosophila cells cells

ApproachApproach Genome-wide RNAi screening & small Genome-wide RNAi screening & small

molecule inhibitorsmolecule inhibitors

ScreeningScreening dsRNA screeningdsRNA screening

- >90% annotated - >90% annotated genome dsRNAsgenome dsRNAs

- incubate ~4 d- incubate ~4 d

Small molecule Small molecule screeningscreening- 51,000 inhibitors- 51,000 inhibitors

2 d2 dImmunofluorescence

-dsRNAs of 214 genes-1 new gene (Borr/CG4454)

-50 inhibitors (commercial lib, bioactive, natprod. extract)

21Predicted Functional Annotations of 21Predicted Functional Annotations of G GGGG GGGGGGGGGG G GGG GG GG GGGGGGGGG4 G GGGG GGGGGGGGGG G GGG GG GG GGGGGGGGG4

G GGGGGGGGGGG GGGGGGGGGG

32%

44%

24%

74%

20%6%

40%

29%51%

28%

Penetrance of binucleate phenotype

Inhibitor

RNAi

24%40%

29%51%

28% 8%

12%

InhibitorRNAi

Low cell count

Phenotypic classesBinucleate

binucleate w large, diffuse

DNA

Low cell count Binucleate with MT extension

Two phenotypes correlate in both Two phenotypes correlate in both methodsmethods

dsRNA targeting dsRNA targeting Act5CAct5C OR Cytochalasin DOR Cytochalasin D

RNAi inhibitors

Two phenotypes correlate in both Two phenotypes correlate in both methodsmethods

•Binucleate with diffuse DNA•Chromosomal passenger protein that co-localize with Aurora B throughout mitosis & cytokinesis

Aurora BAurora B

Chromosomal passenger protChromosomal 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 cytokinesisDisruption causes loss of phosphorylation on histone H3 and failure in cytokinesis

Andrews et al 2003

Two phenotypes correlate in both Two phenotypes correlate in both methodsmethods

•Chromosomal passenger protein that co-localize with Aurora B throughout mitosis & cytokinesis

Borr/CG4454 co-localizes with Borr/CG4454 co-localizes with Aurora BAurora B

Cells treated to Cells treated to aurora Baurora B dsRNA, dsRNA, borr/CG4454borr/CG4454 dsRNA or Binucleine 2 results in an absence dsRNA or Binucleine 2 results in an absence

of H3of H3

•Borr is required for INCENP localizationBorr is required for INCENP localizationAurora B RNAi and Binucleine 2 shares Aurora B RNAi and Binucleine 2 shares same phenotype and affect localization same phenotype and affect localization of INCENPof INCENP

SummarySummary i.d. new prot. involved in cytokinesis and new i.d. new prot. involved in cytokinesis and new

small molecule that inhibits itsmall molecule that inhibits it

214 prot. important for cytokinesis in 214 prot. important for cytokinesis in DrosophilaDrosophila

Borr/CG4454 is a chromosomal passenger prot Borr/CG4454 is a chromosomal passenger prot that co-localize with Aurora Bthat co-localize with Aurora B

Depletion had a profound effect on cytokinesisDepletion had a profound effect on cytokinesis

Binucleine 2 as a cancer drug ? Binucleine 2 as a cancer drug ?

MessageMessage Integration of data from different tools Integration of data from different tools

to answer biological questions to answer biological questions

Subjective to visual inspectionSubjective to visual inspection

CommentComment