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Inhibiting Arp2 /3 complex stimulates formin mediated actin assembly

Inhibiting Arp2 /3 complex stimulates formin mediated actin assembly. Thomas Burke Molecular Biosciences Retreat 11/3/12. Cells coordinate actin filament assembly for a wide range of processes. Correct -Time (signaling) -Place -Architecture -Dynamics. Coordinated action

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Inhibiting Arp2 /3 complex stimulates formin mediated actin assembly

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  1. Inhibiting Arp2/3 complex stimulates formin mediated actin assembly Thomas Burke Molecular Biosciences Retreat 11/3/12

  2. Cells coordinate actin filament assembly for a wide range of processes Correct -Time (signaling) -Place -Architecture -Dynamics Coordinated action of many actin- binding proteins -sequestering -nucleating -bundling -capping -severing -depolymerizing

  3. F-actin cytoskeleton infission yeast Schizosaccharomycespombe GFP-CHD Arp2/3 complex Formin Cdc12 Formin For3

  4. How does the F-actin cytoskeleton self organize into different structures with a specific amount of actin? • Nucleator concentration/cell • ~50,000 Arp2/3 complexes • ~1,500 formins • Actin monomer available: ~1,000,000 total • Patches (Arp2/3 complex): 300,000 – 33% • Ring (formin Cdc12): 75,000 – 7.5% • Cables (formin For3): 150,000 – 15% ??? A Arp2/3 complex For3 Cdc12

  5. How does the F-actin cytoskeleton self organize into different structures with a specific amount of actin? • Nucleator concentration/cell • ~50,000 Arp2/3 complexes • ~1,500 formins • Actin monomer available: ~1,000,000 total • Patches (Arp2/3 complex): 300,000 – 33% • Ring (formin Cdc12): 75,000 – 7.5% • Cables (formin For3): 150,000 – 15% ??? • Hypothesis: actin monomers are rate limiting • Balance of nucleation factors controls amount of actin incorporated into specific process

  6. The Arp2/3 complex inhibitor CK666 depletes actin patches while forming ectopic F-actin Lifeact-GFP [CK666] DMSO X CK666

  7. The quantity of patches decreases as ectopic F-actin increases

  8. Ectopic F-actin formation and reversibility

  9. F-actin cytoskeleton in S. pombe X Actin Binding Proteins Tropomyosin Cdc8 TransgelinStg1 Myosin Myo52 Profilin Cdc3 Actin Binding Proteins Tropomyosin Cdc8 Fimbrin Fim1 Cofilin Adf1 Capping Protein Acp Alpha-actinin Ain1 Myosin Myo2 IQGAP Rng2 TransgelinStg1 Profilin Cdc3 +CK666

  10. Ectopic F-actin is formin mediated 100 μM CK666 cdc12-112 for3Δ Lifeact Lifeact WT cdc12-112 Lifeact 25°C 36°C Double Formin Mutant

  11. Formin associated actin binding proteins localize to ectopic F-actin X ForminCdc12 Formin For3 Profilin Cdc3 Tropomyosin Cdc8 Myosin Myo52 Actin Binding Proteins Tropomyosin Cdc8 TransgelinStg1 Myosin Myo52 Profilin Cdc3 Actin Binding Proteins Tropomyosin Cdc8 Fimbrin Fim1 Cofilin Adf1 Capping Protein Acp Alpha-actinin Ain1 Myosin Myo2 IQGAP Rng2 TransgelinStg1 Profilin Cdc3 +CK666

  12. Conclusions • Discovered an important regulatory mechanism for the self assembly of the F-actin cytoskeleton • Arp2/3 complex limits access of actin monomer to formin • Depleting Arp2/3 complex (actin patches) leads to a gain of formin mediated ectopic F-actin • For3 and Cdc12 • Profilin dependent • Formin filament associated ABPs localize to ectopic F-actin • Tropomyosin • Myo52

  13. Acknowledgments Kovar Lab Cristian Suarez Andy Bestul Jenna Christensen Jon Winkelman Dennis Zimmermann Jen Sees Yujie Li Thesis Committee Dr. Benjamin Glick Dr. Mohan Gupta Dr. Aaron Turkewitz Funding: Molecular and Cellular Biology Training Program Grant #: T32 GM0007183

  14. Inhibition of Arp2/3 complex in vivo • Gmf1 • Inhibits Arp2/3 mediated actin branching in vitro • Gmf1 over expression in vivo leads to loss of actin patches, gain of ectopic F-actin Nakano et al, 2010

  15. Hypothesis: actin monomers are rate limiting • Balance of nucleation factors controls amount of actin incorporated into specific process A

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