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Profilin is a molecular switch that allows formin to compete with Arp2/3 complex for

Profilin is a molecular switch that allows formin to compete with Arp2/3 complex for actin monomers. Cristian Suarez, postdoc in Kovar Lab. Cells coordinate actin filament assembly for a wide range of processes. Formin.

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Profilin is a molecular switch that allows formin to compete with Arp2/3 complex for

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  1. Profilin is a molecular switch that allows formin to compete with Arp2/3 complex for actin monomers Cristian Suarez, postdoc in Kovar Lab

  2. Cells coordinate actin filament assembly for a wide range of processes Formin Arp2/3 complex and formin are in competition for the consumption of the same pool of actin monomers Arp2/3 complex Formin Arp2/3 complex

  3. 30-fold more Arp2/3 complex than formins in fission yeast Actin How can formin efficiently compete with Arp2/3 complex for actin monomers? ` Nucleatorsper cell ~50,000 Arp2/3 complex ~1,500 formins Actin per cell ~1,000,000 total Patches: 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

  4. Arp2/3 complex and formin generate actin filament networks from the same pool of profilin-actin Formin - profilin + profilin In vitro: TIRF Microscopy OG-actin Kovar and Pollard, PNAS, 2004

  5. Arp2/3 complex and formin generate actin filament networks from the same pool of profilin-actin Hypothesis: Profilin is a molecular switch that allows formin to compete with Arp2/3 complex

  6. 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

  7. Profilin allows formin to compete against Arp2/3 complex by: 1) increasing formin-mediated assembly rate 2) inhibiting Arp2/3 complex Formin

  8. Profilin facilitates formin elongation over Arp2/3 complex In vitro: TIRF Microscopy

  9. Profilin facilitates formin elongation over Arp2/3 complex Arp2/3 complex branches Formin-associated filaments In vitro: TIRF Microscopy

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

  11. Profilin facilitates formin elongation over Arp2/3 complex Green-Actin Profilin FLOW Red-Actin In vitro: TIRF Microscopy

  12. The ratio profilin/actin controls the competition between nucleators

  13. The ratio profilin/actin controls the competition between nucleators Actin over-express & WT Profilin over-express Actin over-express Profilin over-express

  14. 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.

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

  16. 30-fold more Arp2/3 complex than formins in fission yeast Nucleators per cell ~50,000 Arp2/3 complex ~1,500 formins Actin per cell ~1,000,000 total Patches (8,000 Arp2/3): 500,000 – 50% Ring (50 forminCdc12): 75,000 – 7.5% Cables (~100 forminFor3): 150,000? – 15% Tom Pollard, Jian-qiu Wu, VolodiaSirotkin, Fred Chang labs

  17. Profilin facilitates formin elongation over Arp2/3 complex

  18. Over expression of profilin rescue ring in cells overexpressingactin Tom Burke, unpublished

  19. 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

  20. 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

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

  22. Actin assembly from VCA-bead (Arp2/3 complex) Arp2/3 Complex Green-Actin +/- Profilin Arp2/3 Complex Red-Actin FLOW In vitro: TIRF Microscopy Flow Flow Photobleaching Photobleaching Photobleaching Photobleaching

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

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

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

  26. 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

  27. 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

  28. 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

  29. No rescue at 36˚C

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

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