1. Luke Peyralans Nolen Lab (IMB) August 2012 Courtesy UCSF

2. Actin G-actin F-actin Branched actin

3. The Actin Cytoskeleton Plays Critical Roles in Diverse Cellular Processes Crucial for movement, cell division, intracellular transport Immune system, wound healing C. elegans embryo undergoing mitosis Actin in red, coutesy of Bowerman lab (IMB)

4. Small Molecules can Inhibit Actin Branching Arp2/3 complex enables formation of branched filaments CK666 AI-015

5. Small Molecules can Inhibit Actin Branching Arp2/3 w/ bound inhibitor

6. Efficacy of inhibitors should be evaluated in vivo Cell permeability? Off-target effects?  Need in vivo assay for inhibitors

7. Summary 1. Branched actin is required for biologically important processes 2. Arp2/3 is the only known actin branch nucleator 3.Small molecules (AI015, CK666, etc) can inhibit Arp2/3 and serve as a tool What is the efficacy of these inhibitors in vivo?

8. Experiment Outline Speckle microscopy used to observe actin dynamics in live Drosophila m. S2 cells Compare dynamics before & after inhibitor addition

10. Velocity of speckles used as a measure of Arp2/3 inhibition Time Space Slope = velocity (dependent on activity of Arp2/3)

11. Effect of CK666 on Activity of Arp2/3 Complex, in vivo

12. Conclusions & Future Directions CK666 shows comparable activity in vivo and in vitro How does this compare to in vivo activity of AI015?

13. Acknowledgements National Institute of Child Health & Human Development NIH-1R25HD070817 Training Grant Dr. Peter O’Day Dr. Brad Nolen (IMB) Dr. Shih-Yuan Liu (Organic Chemistry) Su-Ling Liu, Byron Hetrick, Luke Hegelson, Julianna Han