Luke Peyralans Nolen Lab (IMB) August 2012 Courtesy UCSF.
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Transcript of Luke Peyralans Nolen Lab (IMB) August 2012 Courtesy UCSF.
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Developing Small Molecule Inhibitors to Study the Actin Cytoskeleton
Luke PeyralansNolen Lab (IMB)
August 2012
Courtesy UCSF
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Actin
G-actinF-actin
Branched actin
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The Actin Cytoskeleton Plays Critical Roles in Diverse Cellular
ProcessesCrucial for movement, cell division, intracellular
transport
Immune system, wound healing
C. elegans embryo undergoing mitosisActin in red, coutesy of Bowerman lab (IMB)
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Small Molecules can Inhibit Actin Branching
Arp2/3 complex enables formation of branched
filaments
CK666 AI-015
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Small Molecules can Inhibit Actin Branching
Arp2/3 w/ bound inhibitor
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Efficacy of inhibitors should be evaluated in vivo
• Cell permeability?
• Off-target effects?
Need in vivo assay for inhibitors
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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?
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Experiment OutlineSpeckle microscopy used
to observe actin dynamics in live Drosophila m. S2 cells
Compare dynamics before & after inhibitor addition
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Velocity of speckles used as a measure of Arp2/3 inhibition
Time
Space
Slope = velocity (dependent on activity of Arp2/3)
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Effect of CK666 on Activity of Arp2/3 Complex, in vivo
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Conclusions & Future Directions
CK666 shows comparable activity in vivo and in vitro
How does this compare to in vivo activity of AI015?
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AcknowledgementsNational 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