Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation
- Brandeis University, United States
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
- New England Biolabs, United States
Figures
Figure 1
Pathway of Arp2/3 complex mediated actin branch formation activated by WASP protein dimers on the inside surface of a cell membrane, as deduced from previous studies.
Within the white arrow Arp2/3 complex is activated by VCA, detaches from the membrane and initiates daughter filament elongation. The order of these steps and how they are coordinated remains unclear.
Figure 2 with 4 supplements
Rapid release of dimeric VCA from the nascent branch precedes nucleation.
(A) Design of an experiment to observe diVCA-activated branch nucleation by Arp2/3 complex on the sides of surface-immobilized actin filaments. Blue, green and red stars denote fluorescent dye …
Figure 2—figure supplement 1
N-WASP VCA dimers bind tightly to Arp2/3 complex and stimulate its actin nucleation activity.
(A) Schematic of Glutathione-S-transferase (GST) fusions with N-WASP VCA-containing domains. N-terminal GST-fusions, which are naturally tight dimers, were made to VVCA (N-WASP residues 393–505), …
Figure 2—figure supplement 2
VCA dimers crosslinked through Cy3 stimulate Arp2/3 to a similar extent as GST-VCA dimers.
(A) Schematics of N-WASP VVCA and VCA dimers made via fusion to GST (as in Figure 2—figure supplement 1) or by covalent crosslinking of single-cysteine mutant subunits with a bis-maleimide Cy3. The …
Figure 2—figure supplement 3
Arp2/3 complex and diVCA usually bind to and release from filaments as a unit when no daughter filament is formed.
Left: Merged images (Figure 2B) and fluorescence intensity records (Figure 2C) showing an example of simultaneous binding (t = 0) and release (dashed vertical line) of diVCA and Arp2/3 complex on an …
Figure 2—figure supplement 4
Association with Arp2/3 complex does not affect binding of VCA to actin.
(A) Binding, measured using fluorescence anisotropy (points), during titration of rabbit muscle actin into 20 nM VCA-AF488. Fit to a binding isotherm (line) yielded KD 220 ± 10 (68% C.I.) nM. (B) …
Figure 3
VCA dimers form long-lived complexes with Arp2/3 complex before filament binding but not after branch formation.
(A) Experimental design to observe diVCA binding and nucleation of actin filaments on immobilized Arp2/3 complexes. Arp2/3-SNAP was tethered to the slide surface via a bi-functional SNAP substrate …
Figure 4 with 2 supplements
diVCA mutations alter the stability of Arp2/3 complex-diVCA-actin monomer assemblies.
(A) Arrangement of V, C, and A domains in native N-WASP and in the diVCA constructs used in this study (w.t. is wild-type). Asterisks mark the domains bearing targeted mutations (substitution of one …
Figure 4—figure supplement 1
Design of the diVCA mutant constructs and characterization of Arp2/3 complex binding by the diVCA-C* mutant.
(A) Sequence alignments showing the substitution (colored residues) and deletion (dash) mutations in the diVCA mutant constructs. Lines mark the regions of VCA that interact with G-actin and with …
Figure 4—figure supplement 2
Single molecule analysis of wild-type and mutant diVCA binding to and dissociating from tethered Arp2/3 complex.
(A)–(C) Binding rate measurements. (A) Example curves from single experiments showing the cumulative distribution of the lifetimes of Arp2/3 complexes unoccupied by diVCA constructs. diVCA …
Figure 5 with 3 supplements
diVCA constructs differ in the rate but not the pathway of activity in stimulating branch formation.
(A) Rate (±S.E.) of initiation of daughter filament growth by Arp2/3 complex in the absence or presence of diVCA wild-type and mutant constructs. kB, the second order rate constant for the …
Figure 5—figure supplement 1
Analysis of diVCA mutant activities in bulk actin polymerization assays.
(A) Stimulation of actin filament assembly by 10 nM Arp2/3-SNAP complex activated by 25 nM wild-type or mutant diVCA constructs. Plots show records of pyrene fluorescence in assays containing 2 µM …
Figure 5—figure supplement 2
Saturation of stimulation of Arp2/3 complex actin nucleation activity by diVCA constructs.
(A) Actin nucleation activity measured as in Figure 5—figure supplement 1 with 10 nM yeast Arp2/3 complex and specified diVCA construct concentrations. Activity saturates above 50 nM for three …
Figure 5—figure supplement 3
Relationship relation between diVCA release from the nascent branch and daughter filament initiation for each of the three mutant diVCA constructs.
These panels show the data presented in Figure 5B replotted separately to make the error bars more visible. V*, C*, and A* data sets contain 41, 49, and 27 observations, respectively, where the …
Figure 6 with 1 supplement
Release of diVCA from nascent branches is rare and limits the rate of daughter nucleation.
(A) Schematic mechanism of diVCA stimulated branch formation (see text). The key activation step, release of diVCA from the nascent branch, is highlighted. (B) Classification of nascent branch fates …
Figure 6—figure supplement 1
Correlation between the actin nucleation activity of Arp2/3 activated by wild-type and mutant diVCA constructs (from Figure 5—figure supplement 1B) and the rate constant of diVCA release from the nascent branch (from Figure 4F).
Dotted line is a linear fit constrained to pass through the origin. The correlation coefficient r = 0.9598 is unlikely to be coincidental (p=0.027; ‘Materials and methods’). Error bars indicate S.E.s.
Figure 7
Model of WASP-Arp2/3 complex stimulated actin branch formation at cell membranes (see text).
https://doi.org/10.7554/eLife.01008.021Videos
Video 1
The diVCA-stimulated actin branch formation event shown in Figure 2B–C.
Red: Arp2/3-SNAP649 (5 nM in solution); green: Cy3-diVCA (5 nM); blue: actin-AF488 (1 µM, 10% labeled). Arp2/3 complex and diVCA images were recorded every 0.05 s; actin images were recorded every …
Tables
Table 1
Colocalization kinetics and activities of diVCA and Arp2/3 complex
| no VCA | diVCA w.t. | diVCA V* | diVCA C* | diVCA A* | |
|---|---|---|---|---|---|
| Arp2/3 complex off filament (Figure 4D, Figure 4—figure supplement 2) | |||||
| N (groups) | 5 | 3 | 3 | 1 | |
| kV+ (107 M−1 s−1) | 16±5 (SEM) | 7±3 (SEM) | 6±1 (SEM) | 16±1 (SE) | |
| NV | 1528 | 957 | 1339 | 1262 | |
| τV1 (s) | 8±1 | 3.6±0.4 | 2.4±0.1 | 40±10 | |
| AV1 | 24±3% | 42±2% | 98±2% | 28±9% | |
| τV2 (s) | 61±3 | 56±3 | 14±6 | 250±30 | |
| Arp2/3 complex on filament (Figure 6B) | |||||
| NA | 715 | 877 | 407 | 1089 | 597 |
| kA+ (104 M−1 s−1) | 2.0±0.3 | 2.1±0.3 | 1.3±0.2 | 2±1 | 0.8±0.2 |
| fAV | 0.83±0.09 | 0.7±0.1 | 0.59±0.08 | 0.7±0.1 | |
| fV− | 0.026±0.004 | 0.041±0.009 | 0.018±0.003 | 0.015±0.004 | |
| fB | 0.006±0.002 | 0.008±0.002 | 0.029±0.008 | 0.015±0.003 | 0.013±0.003 |
| Arp2/3 complex at branch sites (Figure 5A, Figure 6C–E) | |||||
| NB | 57 | 69 | 69 | 40 | |
| τV* (s) | 0.7±0.1 | 0.54±0.08 | 0.37±0.04 | 0.7±0.2 | |
| kV* (s−1) | 0.04±0.01 | 0.08±0.02 | 0.05±0.01 | 0.022±0.008 | |
| kB (M−1 s−1) | 120±40 | 160±50 | 320±90 | 200±100 | 100±30 |
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Parameter descriptions:
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N = number of groups of observations used to calculate binding rate of diVCA to isolated Arp2/3 complexes.
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kV+ = second order rate constant for diVCA binding to Arp2/3 complexes.
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NV = number of observations of diVCA on isolated Arp2/3 complexes.
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τV1 = first characteristic lifetime of diVCA on isolated Arp2/3 complexes.
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AV1 = percent of diVCA that dissociate from Arp2/3 complexes with time constant τV1.
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τV2 = second characteristic lifetime of diVCA on isolated Arp2/3 complexes.
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NA = number of observations of Arp2/3 complexes on the sides of select filaments.
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kA+ = second order rate constant for Arp2/3 complex binding filament sides (per filament subunit).
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fAV = fraction of Arp2/3 complexes that bind filament sides coincident with diVCA.
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fV- = fraction of diVCA-Arp2/3-filament complexes that release diVCA.
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fB = fraction of diVCA-Arp2/3-filament complexes that nucleate a daughter filament.
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NB = number of observations of branch formation from diVCA-Arp2/3-filament complexes.
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τV* = mean lifetime of diVCA on nascent branches.
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kV* = rate constant for diVCA release from the nascent branch.
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kB = second order rate constant for branch formation (per mother filament subunit).
