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
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