Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets

1. Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets Eggert et al. 2004

2. Cytokinesis

3. Cytokinesis (aurora B)

4. (Glotzer 2005)

5. RNAi and Chemical inhibitors • RNAi library • knockdown of specific genes • Chemical libraries • Cytokinesis inhibitors • Cytochalasin: inhibits actin • Blebbistatin: inhibits Myosin II

6. Design of parallel screens • Cultured Drosophila Kc167 cells • Added dsRNA or small molecules in 384-well plates • Grew for 4 days in dsRNA or 2 days with small molecules • Fixed, stained for cytoplasm and DNA or tubulin • Automated fluorescence microscopy

7. Potential small molecule inhibitors • Out of 50,000 possible molecules, 50 inhibited cytokinesis. • Tested cross-reactivity with HeLa cells, BSC-1 cells, and drug resistant S. cerevisiae • About 50% inhibited cytokinesis • 7 inhibit all three • Is this expected?

8. Genome wide RNAi screen • Out of 19470 dsRNAs, dsRNAs corresponding to 214 genes affected cytokinesis • 20% previously implicated • 1 new gene essential for cytokinesis

9. Figure 1 Small molecule inhibitors (25) RNAi (214)

10. Figure 1 Small molecule inhibitors (25) -Compare percentages of phenotype strength? RNAi (214)

11. Figure 2

12. Figure 2

13. Figure 2 7 of 9 cases tested showed weak actin staining

14. Compared with four other screens, results correlate • This screen missed ten genes • Since other screens were smaller, what does this indicate about these results?

15. Compared with four other screens, results correlate • This screen missed ten genes • Since other screens were smaller, what does this indicate about these results? • This screen was in Kc167 cells as opposed to S2 cells

16. Compared with four other screens, results correlate • This screen missed ten genes • Since other screens were smaller, what does this indicate about these results? • This screen was in Kc167 cells as opposed to S2 cells • 4 days may not be enough time for gene knockdown

17. Figure 3 Binucleate cells Diffuse DNA Low cell count Microtubule extension

18. Figure 4 Act5C dsRNA cells Cytochalasin D Similar phenotypes between Act5C dsRNA and cytochalasin D

19. Figure 5 Diffuse DNA, abnormal mitosis, mis-aligned chromosomes

20. Figure 5 Same phenotype=same pathway?

21. Figure 6 CG4454 colocalized with aurora B

22. Figure 7 -Depletion of CG4454 leads to INCENP localization -Depletion of CG4454 or aurora B leads to absence of phos. of Histone H3.

23. Chromosomal passenger complex • Since phenotype of CG4454 depletion matched phenotype of aurora B and INCENP, CG4454 could be member of chromosomal passenger complex (Glozter 2005)

24. Fig. S1 • RNAi depletion of aurora B and CG4454 had similar phenotypes in 14 out of 15 markers

25. Figure 8 Concentration and time dependent phosphorylation

26. Conclusions • 214 proteins important for cytokinesis • 25 previously uncharacterized predicted proteins • Identified 50 small molecule inhibitors of cytokinesis • Depleting CG4454/borr affects cytokinesis • Borr co-localizes with Aurora B