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 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

30-fold more Arp2/3 complex than formins in fission yeast Actin How can formin efficiently compete with Arp2/3 complex for actin monomers? For Arp: 220 arp/patches for~35 patches per cell =~ 8,000 ` Nucleators per 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, Volodia Sirotkin, Fred Chang labs

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

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

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

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

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

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

Actin nucleators are spatially separated For Arp: 220 arp/patches for~35 patches per cell =~ 8,000 GFP-Rng2(CHD) Kovar et al., Trends Cell Biol., 2011

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

The ratio profilin/actin controls the competition between nucleators

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

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.

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

30-fold more Arp2/3 complex than formins in fission yeast For Arp: 220 arp/patches for~35 patches per cell =~ 8,000 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 formin Cdc12): 75,000 – 7.5% Cables (~100 formin For3): 150,000? – 15% Tom Pollard, Jian-qiu Wu, Volodia Sirotkin, Fred Chang labs

Profilin facilitates formin elongation over Arp2/3 complex

Over expression of profilin rescue ring in cells overexpressing actin Transition: Now if we decrease the concentration of profilin using a profilin ts… Tom Burke, unpublished

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

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

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

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

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

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

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

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 29

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

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

No rescue at 36˚C

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