Slide 1

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

Slide 2

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

Slide 3

Tom Pollard, Jian-qiu Wu, Volodia Sirotkin, Fred Chang labs 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: 500,000 50% Ring (Formin-Cdc12): 75,000 7.5% Cables (Formin For3): 150,000? 15% How can formin efficiently compete with Arp2/3 complex for actin monomers? ` Actin

Slide 4

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

Slide 5

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

Slide 6

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

Slide 7

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

Slide 8

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

Slide 9

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

Slide 10

Actin nucleators are spatially separated Kovar et al., Trends Cell Biol., 2011 GFP-Rng2(CHD)

Slide 11

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

Slide 12

The ratio profilin/actin controls the competition between nucleators

Slide 13

WTActin over-expressProfilin over-express Actin over-express &

Slide 14

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

Slide 15

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

Slide 16

Slide 17

Tom Pollard, Jian-qiu Wu, Volodia Sirotkin, Fred Chang labs 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 formin Cdc12): 75,000 7.5% Cables (~100 formin For3): 150,000? 15%

Slide 18

Profilin facilitates formin elongation over Arp2/3 complex

Slide 19

Over expression of profilin rescue ring in cells overexpressing actin Tom Burke, unpublished

Slide 20

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

Slide 21

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

Slide 22

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

Slide 23

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

Slide 24

Slide 25

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

Slide 26

Slide 27

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

Slide 28

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

Slide 29

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

Slide 30

Ectopic F-actin is formin-mediated 25C 36C 100 M CK666 WT cdc12-112 for3 cdc12-112 Double Formin Mutant for3 LifeAct-GFP Tom Burke, unpublished

Slide 31

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 Tropomyosin Cdc8 Fimbrin Fim1 Cofilin Adf1 Capping Protein Acp Alpha-actinin Ain1 Myosin Myo2 IQGAP Rng2 Transgelin Stg1 Profilin Cdc3 Actin Binding Proteins Tropomyosin Cdc8 Transgelin Stg1 Myosin Myo52 Profilin Cdc3 Tom Burke & Jenna Christensen, unpublished

Slide 32

No rescue at 36C

Slide 33

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

Slide 34

Slide 35

Slide 36

Slide 37

Slide 38