Download Parallel Chemical Genetic and Genome

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
– directly inhibit protein fx.
– search by phenotypic screening
-> i.d. targets / pathway
– binding of irrelevant prot.
RNA interference

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
Approach

Genome-wide RNAi screening & small
molecule inhibitors
Screening

dsRNA screening

Small molecule screening
- 51,000 inhibitors
- >90% annotated
genome dsRNAs
- incubate ~4 d
2d
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
Penetrance of binucleate phenotype
24%
40%
32%
44%
28%
Inhibitor
RNAi
6%
20%
29%
51%
74%
Phenotypic classes
24%
40%
28%
Binucleate
binucleate w
large, diffuse
DNA
8%
Inhibitor
RNAi
12%
Low cell
count
29%
51%
Low cell count
Binucleate with
MT extension
Two phenotypes correlate in both methods
RNAi

inhibitors
dsRNA targeting Act5C OR Cytochalasin D
Two phenotypes correlate in both methods
•Binucleate with diffuse DNA
•Chromosomal passenger protein that co-localize with Aurora B
throughout mitosis & cytokinesis
Aurora B
Andrews et al 2003

Chromosomal passenger prot

Localize on centromeres (prophase-metaphase)

Relocalize to spindle midzone and cortex (anaphase)

Disruption causes loss of phosphorylation on histone H3 and
failure in cytokinesis
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
•Borr is required for INCENP localization
Aurora B RNAi and Binucleine 2 shares same
phenotype and affect localization of INCENP
Cells treated to aurora B dsRNA, borr/CG4454
dsRNA or Binucleine 2 results in an absence of H3
Summary

i.d. new prot. involved in cytokinesis and new small
molecule that inhibits it

214 prot. important for cytokinesis in Drosophila

Borr/CG4454 is a chromosomal passenger prot that
co-localize with Aurora B

Depletion had a profound effect on cytokinesis

Binucleine 2 as a cancer drug ?
Message

Integration of data from different tools to
answer biological questions
Comment

Subjective to visual inspection