(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 …
(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 …
(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) …
(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…
(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), …
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 …
(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 …
(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). …
(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 …
(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 …
(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 …
(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 …
(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 …
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 …
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 …
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 …
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, …
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.
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, …
Mathematical modeling parameters
Reaction # | Description | kon (M−1s−1) | koff (s−1) | KD (µM) | Reference |
---|---|---|---|---|---|
1 | Actin dimerization | 1.98 × 107 | 5.26 × 107 | 2.6 × 106 | Mullins 1998, This study |
2 | Actin trimerization | 1.16 × 107 | 4.07 × 105 | 3.5 × 104 | Mullins 1998, This study |
3 | Spontaneous nucleation | 1110 − 1220*,† | This study | ||
4 | Barbed end elongation | 1.16 × 107 | 1.4 | Pollard 1986 | |
5 | Barbed end elongation, actin monomer bound to GST-VCA | 1.16 × 107 | 1.4 | Pollard 1986, Higgs 1999 | |
6 | Barbed end elongation, two actin monomers bound to GST-VCA | 1.16 × 107 | 1.4 | Pollard 1986, Higgs 1999 | |
7 | Actin monomer binds GST-VCA | 5 × 106 | 3 | 0.6 | Marchand 2001, Beltzner 2008 |
8 | Actin monomer binds GST-VCA with bound actin | 5 × 106 | 3 | 0.6 | Marchand 2001, Beltzner 2008 |
9 | Actin monomer binds GST-VCA:actin2 | 4.2 × 104 | 74.4 | 1.8 × 103 | This study |
10 | Actin monomer binds GST-VCA:actin3 | 1.5 × 107 | 1.04 | 0.069 | This study |
11 | Actin monomer binds GST-VCA:actin4 | 2 × 107 | 0.062 | 0.003 | This study |
12 | GST-VCA nucleation | 6.1 × 10−8* | This study | ||
13 | Arp2/3 binds actin filament | 1.37 × 106 | 1.23 | 0.9 | Hetrick 2013 |
14 | GST-VCA binds Arp2/3 | 0.8 × 106 | 0.072 | 0.009 | Padrick 2008 |
15 | GST-VCA:actin binds Arp2/3 | 0.8 × 106 | 0.014 | 0.018 | Padrick 2008, Beltzner 2008, Kelly 2006 |
16 | GST-VCA:actin2 binds Arp2/3 | 0.8 × 106 | 0.029 | 0.028 | Padrick 2008, Beltzner 2008, Kelly 2006 |
17 | Actin monomer binds GST-VCA:Arp2/3 | 2.5 × 106 | 3 | 1.2 | Marchand 2001, Beltzner 2008 |
18 | Actin monomer binds GST-VCA:actin:Arp2/3 | 2.5 × 106 | 3 | 1.2 | Marchand 2001, Beltzner 2008 |
19 | GST-VCA:actin2:Arp2/3 binds actin filament (kfil_on) | 1.37 × 106 | 1.23 | 0.9 | Hetrick 2013 |
20 | GST-VCA binds Arp2/3:F-actin | 0.8 × 106 | 0.072 | 0.009 | Padrick 2008 |
21 | GST-VCA:Arp2/3 binds F-actin | 1.37 × 106 | 1.23 | 0.9 | Hetrick 2013 |
22 | GST-VCA:Arp2/3:actin binds F-actin | 1.37 × 106 | 1.23 | 0.9 | Hetrick 2013 |
23 | GST-VCA:Arp2/3:F-actin binds actin monomer | 2.5 × 106 | 3 | 1.2 | Marchand 2001, Beltzner 2008 |
24 | GST-VCA:Arp2/3:actin:F-actin binds actin monomer | 2.5 × 106 | 3 | 1.2 | Marchand 2001, Beltzner 2008 |
25 | Arp2/3 complex nucleation (knuc) | 0.004 − 0.006* | This study | ||
26 | Cortactin binds actin filament | 1.21 × 104 | 0.063 | 5.21 | This study |
27 | Cortactin binds nascent branch junction | 2.0 × 106 | 0.034 | 0.017 | This study |
28 | Synergy displacement activation of Arp2/3 complex (kdis) | 0.036* | This study | ||
29 | Synergy recycling, cortactin dissociates sequestered GST-VCA | 2.0 × 106 | 0.034 | 0.017 | This study |
Units are s−1.
This value was adjusted for each full set of reactions.
Underlined values were allowed to float during some optimizations, see Table 2.
Mathematical modeling reaction sets
Reaction set | Reactions | Initial concentrations | Variable concentrations (µM) | Floated parameters | Quality of fit |
---|---|---|---|---|---|
1 | 1–4 | – | 2.0, 3.0, 4.0, 5.0, 6.0 actin monomers | k1, k−1, k2, k−2, k3 | 1.75 × 10−11 |
2 | 1–12 | 3 µM actin monomers | 0, 0.02, 0.04, 0.08, 0.1, 0.2, 0.6, 0.8, 1.0 GST-VCA | k9, k−9, k10, k−10, k11, k−11, k12 | 2.32 × 10−11 |
3 | 1–25 | 3 µM actin monomers, 50 nM Arp2/3 complex | 0, 0.01, 0.025, 0.050, 0.1, 0.15, 0.25, 0.5, 1.0 GST-VCA | k25 (knuc) | 1.28 × 10−11 |
4a | 1–28 | 3 µM actin monomers, 20 nM Arp2/3 complex, 100 nM GST-VCA | 0, 0.005, 0.025, 0.075, 0.1, 0.25, 1.0 cortactin | kfil_on(k13= k19=k21=k22)*†, k25†, k28 (kdis) | 2.87 × 10−11 |
4b | 1–25, 29 | 3 µM actin monomers, 20 nM Arp2/3 complex, 100 nM GST-VCA | 0, 0.005, 0.025, 0.075, 0.1, 0.25, 1.0 cortactin | k29, k−29 | 4.26 × 10−10 |
kfil_on is a single global variable used for the indicated reaction rates.
Only optimized for the 0 µM cortactin reaction.