Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Published byAugustus Stephens Modified over 9 years ago

Similar presentations

Presentation on theme: "Profilin is a molecular switch that allows formin to compete with Arp2/3 complex for actin monomers Cristian Suarez, postdoc in Kovar Lab."— 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? 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

4 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

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 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

11 Profilin facilitates formin elongation over Arp2/3 complex
FLOW Green-Actin Profilin 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 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

16

17 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

18 Profilin facilitates formin elongation over Arp2/3 complex

19 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

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 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

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

23 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

24

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

26

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LifeAct-GFP [CK666] DMSO + LatA CK666 X Tom Burke, unpublished

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Tom Burke, unpublished

29 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

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100 μM CK666 WT cdc12-112 for3Δ cdc for3Δ 25°C 36°C Double Formin Mutant LifeAct-GFP Tom Burke, unpublished

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 Fimbrin Fim1 Cofilin Adf1 Capping Protein Acp Alpha-actinin Ain1 Myosin Myo2 IQGAP Rng2 Transgelin Stg1 Tom Burke & Jenna Christensen, unpublished

32 No rescue at 36˚C

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

34

35

36

37

38

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

Similar presentations

Alpha-Actinin is Required for Tightly Regulated Remodeling of the Actin Cortical Network during Cytokinesis Svetlana Mukhina Speaker: Jerry Qiu, Kunkka.

Alpha-Actinin is Required for Tightly Regulated Remodeling of the Actin Cortical Network during Cytokinesis Svetlana Mukhina Speaker: Jerry Qiu, Kunkka.
Regulation of actin filament dynamics in vivo Fig. 16-52, Alberts G-actin F-actin Drugs that destabilize actin filaments e.g. cytochalasins –Cytochalasin.

Regulation of actin filament dynamics in vivo Fig , Alberts G-actin F-actin Drugs that destabilize actin filaments e.g. cytochalasins –Cytochalasin.
Chapter 16 The Cytoskeleton.

Chapter 16 The Cytoskeleton.
How does actin polymerization drive protrusion? Polymerization at tip? Expansion of actin meshwork? Increase in hydrostatic pressure? Hypothesis #1 Hypothesis.

How does actin polymerization drive protrusion? Polymerization at tip? Expansion of actin meshwork? Increase in hydrostatic pressure? Hypothesis #1 Hypothesis.
Cytoskeleton and Cell Motility

Cytoskeleton and Cell Motility
The role of tropmyosin in muscle contraction. 9.7 Nonmuscle motility (1) Actin-binding proteins affect the localized assembly or disassembly of the actin.

The role of tropmyosin in muscle contraction. 9.7 Nonmuscle motility (1) Actin-binding proteins affect the localized assembly or disassembly of the actin.
CYTOSKELETON (I) Actin filaments

CYTOSKELETON (I) Actin filaments
Regulation of Cytoskeletal Filaments

Regulation of Cytoskeletal Filaments
The Cytoskeleton. Intermediate Filaments 8-10 nm in diameter Four types: basis of aa sequence actin & tubulin -> globular protein, cell type.

The Cytoskeleton. Intermediate Filaments 8-10 nm in diameter Four types: basis of aa sequence actin & tubulin -> globular protein, cell type.
Www.soran.edu.iq Cell and Molecular Biology Behrouz Mahmoudi Cytoskeleton-1 1.

Cell and Molecular Biology Behrouz Mahmoudi Cytoskeleton-1 1.
Molecular Cell Biology Actin, including Principles of Assembly Cooper.

Molecular Cell Biology Actin, including Principles of Assembly Cooper.
Cytoskeleton II Chapter 16.

Cytoskeleton II Chapter 16.
Cytoskeletal dynamics in vitro Assembly of actin filaments in vitro –The Critical concentration –Treadmilling The regulation of actin filament dynamics.

Cytoskeletal dynamics in vitro Assembly of actin filaments in vitro –The Critical concentration –Treadmilling The regulation of actin filament dynamics.
APBs involved in regulating actin dynamics (Cont.) 2. How high rates of actin polymerization are maintained at the protruding edge? Thymosin  -4 Profilin.

APBs involved in regulating actin dynamics (Cont.) 2. How high rates of actin polymerization are maintained at the protruding edge? Thymosin  -4 Profilin.
Microfilaments In this chapter of our web text, we will examine the architecture of the Actin Microfilament Cytoskeleton. Microfilaments are polymers of.

Microfilaments In this chapter of our web text, we will examine the architecture of the Actin Microfilament Cytoskeleton. Microfilaments are polymers of.
APBs involved in regulating actin dynamics (Cont.) 2. How high rates of actin polymerization are maintained at the protruding edge? Thymosin  -4 Profilin.

APBs involved in regulating actin dynamics (Cont.) 2. How high rates of actin polymerization are maintained at the protruding edge? Thymosin  -4 Profilin.
The tail of Listeria monocytogenes : Lessons learned from a bacterial pathogen (cont.) 1. How do Listeria make tails Nucleation, growth 2. Role of ABPs.

The tail of Listeria monocytogenes : Lessons learned from a bacterial pathogen (cont.) 1. How do Listeria make tails Nucleation, growth 2. Role of ABPs.
Actin By Enrique M. De La Cruz & E. Michael Ostap

Actin By Enrique M. De La Cruz & E. Michael Ostap
Luke Peyralans Nolen Lab (IMB) August 2012 Courtesy UCSF.

Luke Peyralans Nolen Lab (IMB) August 2012 Courtesy UCSF.
CB201_12 Tim Mitchison Lecture 3 Force generation by polymerization dynamics Nucleation: controlling where and when polymers form.

CB201_12 Tim Mitchison Lecture 3 Force generation by polymerization dynamics Nucleation: controlling where and when polymers form.

Similar presentations

Ads by Google