1 / 38

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.

Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related