Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP

  1. Luke A Helgeson
  2. Brad J Nolen  Is a corresponding author
  1. University of Oregon, United States
9 figures, 6 videos and 2 tables

Figures

Schematic overview of branching nucleation and the proteins involved.

(A) Overview of branching nucleation depicting the required reaction components (Arp2/3 complex, NPF, actin monomers and actin filaments) and the resultant Y-shaped branches. The barbed and pointed …

https://doi.org/10.7554/eLife.00884.003
Cortactin synergizes with GST-N-WASP-VCA.

(A) Time course of pyrene-actin polymerization showing synergistic activation of Arp2/3 complex by cortactin and GST-VCA. Reactions contain 2 µM 15% pyrene-actin, 20 nM Arp2/3 complex and cortactin …

https://doi.org/10.7554/eLife.00884.004
Figure 3 with 1 supplement
Actin filament recruitment cannot explain cortactin-mediated synergy.

(A) Cartoon pathway of steps optimized in the kinetic model of branching nucleation. (B) Representative pyrene-actin polymerization time courses of Arp2/3 complex activated by GST-VCA (dashed lines) …

https://doi.org/10.7554/eLife.00884.006
Figure 3—figure supplement 1
Mathematical modeling of actin polymerization in the presence or absence of GST-VCA, Arp2/3 complex and cortactin.

(A) Cartoon pathway of mathematical models used to fit four independent sets of pyrene-actin polymerization assays. The conditions of each reaction set and its associated model are described in Table…

https://doi.org/10.7554/eLife.00884.007
Figure 4 with 1 supplement
The oligomerization state of VCA is an important determinant of synergy.

(A) Plot of the fold activation vs cortactin concentration for reactions containing 2 µM 15% pyrene-actin, 20 nM Arp2/3 complex and 250 nM GST-VCA (black), 750 nM VCA (blue), 750 nM VVCA (green), …

https://doi.org/10.7554/eLife.00884.010
Figure 4—figure supplement 1
The level of synergy is the same at saturating and subsaturating concentrations of the type I NPF.

Plot of fold activation vs cortactin concentration for reactions containing 2 µM 15% pyrene-actin, 20 nM Arp2/3 complex and subsaturating (open symbols and dotted fit line) or saturating (closed …

https://doi.org/10.7554/eLife.00884.011
Figure 5 with 1 supplement
VCA affinity for the nascent branch junction is an important determinant of synergy.

(A) Maximum polymerization rates verses (monomer) concentration of N-WASP constructs for reactions containing 20 nM Arp2/3 complex and 2 µM 15% pyrene-actin. (B) Cartoon depicting obligatory …

https://doi.org/10.7554/eLife.00884.012
Figure 5—figure supplement 1
The E455R mutation in N-WASP is predicted to provide additional favorable electrostatic interactions with actin monomers.

(A) Alignment of WH2/V sequences (sequence accession numbers): Tβ4 (NP_001106702.1), Ciboulot isoform D (NP_001245516.1), MIM (O43312.2), WIP (O43516.3), N-WASP (Q95107.1) and WASP (P42768.4). …

https://doi.org/10.7554/eLife.00884.013
Figure 6 with 1 supplement
Cortactin binds statically to actin filaments.

(A) smTIRF microscopy images of single cortactin molecules (red) bound to polymerizing actin filaments (green). TIRF reactions contained 1 µM 33% Oregon-Green actin and 2 nM Alexa568-cortactin …

https://doi.org/10.7554/eLife.00884.014
Figure 6—figure supplement 1
Validation of single molecule data.

(A) Plot of fold activation vs cortactin concentration for reactions containing 20 nM Arp2/3 complex and 250 nM GST-VCA and either full length cortactin (circle) or Alexa568-cortactin residues 1–336 …

https://doi.org/10.7554/eLife.00884.015
Cortactin directly targets branch junctions with a fast on rate.

(A) smTIRF microscopy images showing interaction of cortactin with preformed branched networks. Reactions were initiated using 1 µM 33% Oregon-Green actin, 5 nM Arp2/3 complex and 30 nM VCA and …

https://doi.org/10.7554/eLife.00884.017
Cortactin remains at the branch junction during daughter filament elongation.

(A) smTIRF microscopy images of polymerizing branch networks containing 1 µM 33% Oregon-Green actin, 5 nM Arp2/3 complex, 50 nM VCA and 2 nM Alexa568-cortactin (red). Images show filament side (gray …

https://doi.org/10.7554/eLife.00884.019
Mathematical model of the obligatory displacement mechanism of cortactin-mediated synergy.

(A) Cartoon pathway of key reactions describing cortactin-mediated displacement of GST-VCA (see ‘Materials and methods’ for full model). (B) Pyrene-actin polymerization time courses of Arp2/3 …

https://doi.org/10.7554/eLife.00884.023

Videos

Video 1
Synergistic activation of Arp2/3 complex by GST-VCA and high concentrations of cortactin.

Video corresponds to images in Figure 2C. Reaction contains 1 µM 33% Oregon-Green actin, 10 nM Arp2/3 complex, 50 nM GST-VCA and 1 µM cortactin. Single-wavelength (488 nm) images were acquired at a …

https://doi.org/10.7554/eLife.00884.005
Video 2
Single molecules of cortactin interacting with polymerizing actin filaments.

Reaction contains 1 µM 33% Oregon-Green actin (green) and 2 nM Alexa568-cortactin (red). Images from both channels were acquired using a 50 ms exposure at a ratio of 5:1 and a frame rate of 3.7 …

https://doi.org/10.7554/eLife.00884.016
Video 3
Single molecules of cortactin binding to preformed branch junctions and filament sides.

Preformed branched filament networks were created by polymerizing 1 µM 33% Oregon-Green actin (green), 5 nM Arp2/3 complex and 30 nM VCA for 6.4 min, then flowing into the reaction chamber a …

https://doi.org/10.7554/eLife.00884.018
Video 4
Single molecules of cortactin binding to branching networks.

Reaction contains 1 µM 33% Oregon-Green actin (green), 5 nM Arp2/3 complex, 50 nM VCA and 2 nM Alexa568-cortactin (red). 561- and 488-channel images were exposed for 50 ms and 30 ms, respectively, …

https://doi.org/10.7554/eLife.00884.020
Video 5
Single molecule of cortactin binding to a nascent branch junction.

Visible in the video are nascent branch (left-center at 3 s), branch junction and filament side binding cortactin molecules. Reaction conditions are the same as video 4.

https://doi.org/10.7554/eLife.00884.021
Video 6
Single molecule of NtA binding to a nascent branch junction.

Reaction contains 1 µM 33% Oregon-Green actin (green), 10 nM Arp2/3 complex, 350 nM VCA and 10 nM Alexa568-NtA(1-48) (red). 561- and 488-channel images were exposed for 50 ms and 30 ms, …

https://doi.org/10.7554/eLife.00884.022

Tables

Table 1

Mathematical modeling parameters

https://doi.org/10.7554/eLife.00884.008
Reaction #Descriptionkon (M−1s−1)koff (s−1)KD (µM)Reference
1Actin dimerization1.98 × 1075.26 × 1072.6 × 106Mullins 1998, This study
2Actin trimerization1.16 × 1074.07 × 1053.5 × 104Mullins 1998, This study
3Spontaneous nucleation1110 − 1220*,This study
4Barbed end elongation1.16 × 1071.4Pollard 1986
5Barbed end elongation, actin monomer bound to GST-VCA1.16 × 1071.4Pollard 1986, Higgs 1999
6Barbed end elongation, two actin monomers bound to GST-VCA1.16 × 1071.4Pollard 1986, Higgs 1999
7Actin monomer binds GST-VCA5 × 10630.6Marchand 2001, Beltzner 2008
8Actin monomer binds GST-VCA with bound actin5 × 10630.6Marchand 2001, Beltzner 2008
9Actin monomer binds GST-VCA:actin24.2 × 10474.41.8 × 103This study
10Actin monomer binds GST-VCA:actin31.5 × 1071.040.069This study
11Actin monomer binds GST-VCA:actin42 × 1070.0620.003This study
12GST-VCA nucleation6.1 × 10−8*This study
13Arp2/3 binds actin filament1.37 × 1061.230.9Hetrick 2013
14GST-VCA binds Arp2/30.8 × 1060.0720.009Padrick 2008
15GST-VCA:actin binds Arp2/30.8 × 1060.0140.018Padrick 2008, Beltzner 2008, Kelly 2006
16GST-VCA:actin2 binds Arp2/30.8 × 1060.0290.028Padrick 2008, Beltzner 2008, Kelly 2006
17Actin monomer binds GST-VCA:Arp2/32.5 × 10631.2Marchand 2001, Beltzner 2008
18Actin monomer binds GST-VCA:actin:Arp2/32.5 × 10631.2Marchand 2001, Beltzner 2008
19GST-VCA:actin2:Arp2/3 binds actin filament (kfil_on)1.37 × 1061.230.9Hetrick 2013
20GST-VCA binds Arp2/3:F-actin0.8 × 1060.0720.009Padrick 2008
21GST-VCA:Arp2/3 binds F-actin1.37 × 1061.230.9Hetrick 2013
22GST-VCA:Arp2/3:actin binds F-actin1.37 × 1061.230.9Hetrick 2013
23GST-VCA:Arp2/3:F-actin binds actin monomer2.5 × 10631.2Marchand 2001, Beltzner 2008
24GST-VCA:Arp2/3:actin:F-actin binds actin monomer2.5 × 10631.2Marchand 2001, Beltzner 2008
25Arp2/3 complex nucleation (knuc)0.004 − 0.006*This study
26Cortactin binds actin filament1.21 × 1040.0635.21This study
27Cortactin binds nascent branch junction2.0 × 1060.0340.017This study
28Synergy displacement activation of Arp2/3 complex (kdis)0.036*This study
29Synergy recycling, cortactin dissociates sequestered GST-VCA2.0 × 1060.0340.017This study
  1. *

    Units are s−1.

  2. This value was adjusted for each full set of reactions.

  3. Underlined values were allowed to float during some optimizations, see Table 2.

Table 2

Mathematical modeling reaction sets

https://doi.org/10.7554/eLife.00884.009
Reaction setReactionsInitial concentrationsVariable concentrations (µM)Floated parametersQuality of fit
11–42.0, 3.0, 4.0, 5.0, 6.0 actin monomersk1, k−1, k2, k−2, k31.75 × 10−11
21–123 µM actin monomers0, 0.02, 0.04, 0.08, 0.1, 0.2, 0.6, 0.8, 1.0 GST-VCAk9, k−9, k10, k−10, k11, k−11, k122.32 × 10−11
31–253 µM actin monomers, 50 nM Arp2/3 complex0, 0.01, 0.025, 0.050, 0.1, 0.15, 0.25, 0.5, 1.0 GST-VCAk25 (knuc)1.28 × 10−11
4a1–283 µM actin monomers, 20 nM Arp2/3 complex, 100 nM GST-VCA0, 0.005, 0.025, 0.075, 0.1, 0.25, 1.0 cortactinkfil_on(k13= k19=k21=k22)*, k25, k28 (kdis)2.87 × 10−11
4b1–25, 293 µM actin monomers, 20 nM Arp2/3 complex, 100 nM GST-VCA0, 0.005, 0.025, 0.075, 0.1, 0.25, 1.0 cortactink29, k−294.26 × 10−10
  1. *

    kfil_on is a single global variable used for the indicated reaction rates.

  2. Only optimized for the 0 µM cortactin reaction.

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