WH2 and proline‐rich domains of WASP‐family proteins collaborate to accelerate actin filament elongation

Abstract WASP‐family proteins are known to promote assembly of branched actin networks by stimulating the filament‐nucleating activity of the Arp2/3 complex. Here, we show that WASP‐family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP‐family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline‐rich sequence that binds profilin–actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline‐rich sequences are required to support polymerase activity by (i) bringing polymerization‐competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin–actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP‐family proteins that create it. Collaboration between WH2 and proline‐rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP‐family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.

A Barbed end elongation rates determined from time-lapse TIRF microscopy of single-filament growth from 2 lM actin (30% Cy5 labeled) in the presence of indicated profilin concentrations. At least 40 filaments were analyzed per condition. B Barbed end elongation rates determined from time-lapse TIRF microscopy of single-filament growth at indicated actin (black) or profilin-actin (red) concentrations.
At least 40 filaments were analyzed per condition. C Snapshot images (top panels) or kymographs (bottom panels) from time-lapse TIRF microscopy of single-filament growth from Alexa568 phalloidin-stabilized seeds in the presence of 1 lM actin (30% Cy5 labeled) in the presence of 5 nM Alexa488-UTRN N . D Barbed end polymerization rates for either a control sample containing only 1 lM of actin alone (left bar) or with the addition of 5 nM Alexa488-UTRN N either visualized using labeled actin (middle bar) or labeled UTRN N (right bar). Note that the presence of the filament-binding probe at this very low concentration does not affect actin polymerization. Also, the growth velocities determined from either actin or UTRN N channel were indistinguishable, further demonstrating the validity of the probe. At least 40 filaments were analyzed per condition.
Data information: All error bars are SD. A Barbed end growth velocities in 1 lM profilin-actin as a function of measured mCherry-WAVE1 surface density either outside (green) or inside (magenta) of an NPF patch or a non-WAVE1-treated coverslip that was used as an independent control (black) as in Fig 2A-E. Note that the passivated region outside of the high-density patch still contains about 10% residual surface-bound WAVE1. Error bars are SD. At least 40 filaments were analyzed per condition. B Soluble Cy3-PWCA (0.5 lM) does not associate with the sides or barbed ends of phalloidin-stabilized actin filaments (30% Alexa488-labeled actin plus dark phalloidin). Actin filament-binding was characterized in the presence of both 50 mM and 100 mM KCl. C Soluble Cy3-PWCA (0.5 lM) does not associate with the sides or barbed ends of dynamically elongating actin filaments. Filaments were polymerized in the presence of either 2 lM actin or 2 lM actin-profilin (50 and 100 mM KCl). Actin filaments were visualized using soluble Cy5-UTRN actin-binding domain (5 nM). D TIRF microscopic images of streptavidin-functionalized polystyrene microspheres to which biotinylated version of the indicated proteins (magenta) were immobilized about 5 min after immersion in 1 lM actin (containing 10% Alexa488-actin, green). Note that VASP-coupled beads spawn filaments which are persistently attached through their elongating barbed ends, while control and WAVE1 (PWCA) beads to not bind to filaments. E Barbed end elongation rates (dots) determined from time-lapse TIRF microscopy of single-filament growth at indicated actin (black) or WAVE1(WCA)-actin (red) concentrations. Actin data are the same as in Fig EV1B). Off-rates of actin or WAVE1(WCA)-actin from filament ends were determined from the y-intercept of linear fit to the data (lines). Error bars are SD. At least 40 filaments were analyzed per condition.
The  Figure EV4. The WAVE1 PRD domain contains six proline-rich sites which interact with profilin with distinct affinities as determined by analytical ultracentrifugation.
A Primary sequence of the wt WAVE1 PRD (top) compared to single poly-proline site mutants (bottom) as indicated. B Molecular weight of indicated WAVE1 PRD variants measured by sedimentation equilibrium ultracentrifugation. Equilibrium radial distributions at 7 K, 10 K, and 14 K rpm are shown. C Interaction between WAVE1 PRD[B] and profilin measured by sedimentation equilibrium ultracentrifugation. Equilibrium radial distributions of WAVE1 PRD [B] in the presence of 91.2, 44.8, and 21.7 lM profilin at 7 K, 10 K, and 14 K rpm are shown. Equilibrium traces are fit using a monomer-dimer equilibrium model (see Materials and Methods). D WAVE1 PRD contains six proline-rich sites that each interact with profilin 1 and profilin 2 with distinct affinities as determined by analytical ultracentrifugation.
Affinities for profilin 1 or profilin 2 binding for either the profilin-binding deficient PRD mutant (PRD null) or indicated single site constructs with individual binding sites re-introduced. The residual interaction between profilin and PRD null mutant was modeled as a 1:1 interaction with a K D of 138 lM. We consider this lowaffinity interaction as non-specific. All other data with WAVE1 PRD single binding site variants were analyzed using a two-site, independent binding model with one site fixed to the measured non-specific affinities.
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