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Dueling nucleation factors: Competition between formin and Arp2/3 complex for actin monomers

Dueling nucleation factors: Competition between formin and Arp2/3 complex for actin monomers. Formin. Arp2/3 complex. Cells coordinate actin filament assembly for a wide range of processes. Correct -Time (signaling) -Place -Architecture -Dynamics.

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Dueling nucleation factors: Competition between formin and Arp2/3 complex for actin monomers

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  1. Dueling nucleation factors: Competition between formin and Arp2/3 complex for actin monomers Formin Arp2/3 complex

  2. Cells coordinate actin filament assembly for a wide range of processes Correct -Time (signaling) -Place -Architecture -Dynamics

  3. Cells coordinate actin filament assembly for a wide range of processes Coordinated action of many actin-binding Proteins -sequestering -nucleating -bundling -capping -severing -depolymerizing Profilin AdaptedfromPollard and Cooper, Science, 2009

  4. Profilin inhibits spontaneous nucleation of actin filament Pointed end Elongation Barbed end Nucleation Factor Nucleation

  5. Arp2/3 complex generates a branched actin filament network Collins et al., Current Biology, 2011 OG-actin TIRF,

  6. Formin nucleates long-straight actin filaments -profilin + profilin TIRF OG-actin Yang et al., PlosBiol, 2007 Kovar and Pollard, PNAS, 2004

  7. The fission yeast actin cytoskeleton GFP-Rng2(CHD) Kovaret al., Trends Cell Biol., 2011

  8. 30-fold more Arp2/3 complex than formins in fission yeast Do Arp2/3 complex and formin compete for actin monomers? Nucleators per cell ~50,000 Arp2/3 complex ~1,500 formins Actin per cell ~1,000,000 total Patches (Arp2/3): 500,000 – 50% Ring (formin Cdc12): 75,000 – 7.5% Cables (formin For3): 150,000? – 15% Tom Pollard, Jian-qiu Wu, VolodiaSirotkin, Fred Chang labs

  9. Depleting Arp2/3 complex (actin patches) leads to increased formin-mediated actin assembly. Arp2/3 complex outcompetes formin for actin monomers. CK666 X Thomas Burke, unpublished

  10. How do ~1,500 formins successfully compete with ~50,000 Arp2/3 complexes for actin monomers?

  11. How do ~1,500 formins successfully compete with ~50,000 Arp2/3 complexes for actin monomers? Profilin-ts(cdc3-124) WT LifeAct-mCherry Andrew Bestul, unpublished

  12. How do ~1,500 formins successfully compete with ~50,000 Arp2/3 complexes for actin monomers?

  13. Does profilin inhibit Arp2/3 complex-generated actin filament network?

  14. Profilin inhibits actin filament assembly induced by Arp2/3 complex Pyrene-actin assembly

  15. Profilin inhibits actin filament nucleation induced by Arp2/3 complex Actin, 1.5 µM + Arp2/3 complex + profilin, 1.5 µM + profilin, 5 µM TIRF, OG-actin

  16. Profilin mediates competition between Arp2/3 complex and formin.

  17. Inhibition of Arp2/3 complex suppresses fission yeast profilin-ts cells Fission yeast profilin temperature sensitive cells Genetic version of this experiment: Kathy Gould’s lab mid 90’s

  18. Addition of formin makes the inhibition of Arp2/3 complex more pronounced + red-SNAP-formin + profilin, 1.5 µM Actin, 1.5 µM + Arp2/3 complex + profilin, 1.5 µM TIRF, OG-actin

  19. Profilin allows formin to efficiently compete with an excess of Arp2/3 complex FLOW Arp2/3 complex, 100 nM Profilin, 5µM Arp2/3 complex, 100 nM Red-SNAP-Cdc12, 1 nM TIRF, OG-actin

  20. Actinnucleators are spatially separated GFP-Rng2(CHD) Kovaret al., Trends Cell Biol., 2011

  21. Actin assembly from VCA-bead and formin-beads Formin-coated bead Profilin, 4 µM VCA-coated bead Arp2/3 complex, 20 nM TIRF, OG-actin

  22. Profilin allows formin to compete with Arp2/3 complex for actin consumption Formin-bead VCA-bead Arp2/3 complex, 10 nM Arp2/3 complex, 10 nM Profilin, 4 µM FLOW TIRF OG-actin

  23. Conclusion • Without profilin: • Slow formin-mediated actin assembly • No Arp2/3 complex inhibition Arp2/3 complex-mediated assembly dominates. • With profilin: • - Fast formin-mediated actin assembly • Arp2/3 complex inhibition Formin can efficiently compete with Arp2/3 complex for actin consumption.

  24. SUNY Upstate: Vladimir Sirotkin Michael L. James Kovar Lab: David Kovar Andrew Bestul Tom Burke Jenna Christensen Yujie Li Jen Sees Jon Winkelman Dennis Zimmermann

  25. Profilin inhibit actin filament assembly induced by Arp2/3 complex Wsp1 domain organization:

  26. Fission yeast profilin-ts cells have severe formin-mediated cytokinesis defects Profilin-ts(cdc3-124) WT WT cdc3-124 Rlc1-GFP Andy Bestul, unpublished

  27. The Arp2/3 complex inhibitor CK666 depletes actin patches while forming ectopic F-actin LifeAct-GFP [CK666] DMSO + LatA CK666 X Tom Burke, unpublished

  28. The quantity of patches decreases as ectopic F-actin increases Tom Burke, unpublished

  29. The fission yeast actin cytoskeleton X Actin Binding Proteins Tropomyosin Cdc8 Transgelin Stg1 Myosin Myo52 Profilin Cdc3 Actin Binding Proteins Tropomyosin Cdc8 Fimbrin Fim1 Cofilin Adf1 Capping Protein Acp Alpha-actinin Ain1 Myosin Myo2 IQGAP Rng2 Transgelin Stg1 Profilin Cdc3 +CK666

  30. Ectopic F-actin is formin-mediated 100 μM CK666 for3Δ cdc12-112 for3Δ WT cdc12-112 25°C 36°C Double Formin Mutant LifeAct-GFP Tom Burke, unpublished

  31. Formin associated actin binding proteins localize to ectopic F-actin X Actin Binding Proteins Tropomyosin Cdc8 Transgelin Stg1 Myosin Myo52 Profilin Cdc3 Actin Binding Proteins Tropomyosin Cdc8 Fimbrin Fim1 Cofilin Adf1 Capping Protein Acp Alpha-actinin Ain1 Myosin Myo2 IQGAP Rng2 Transgelin Stg1 Profilin Cdc3 Formin Cdc12 Formin For3 Profilin Cdc3 Tropomyosin Cdc8 Myosin Myo52 +CK666 Tom Burke & Jenna Christensen, unpublished

  32. No rescue at 36˚C

  33. Fission yeast profilin-ts cells have fairly normal Arp2/3 complex-mediated actin patch dynamics WT Profilin-ts(cdc3-124) LifeAct-mCherry Does profilin inhibit Arp2/3 complex-generated actin filament network? Andrew Bestul, unpublished

  34. Profilin inhibits actin filament assembly induced by Arp2/3 complex Pyrene-actin assembly

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