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Enhancement of branching efficiency by the actin filament-binding activity of N-WASP/WAVE2

Shiro Suetsugu1,2, Hiroaki Miki1, Hideki Yamaguchi1,2, Takeshi Obinata3 and Tadaomi Takenawa1,2,*

1 Department of Biochemistry, Institute of Medical Science, University of Tokyo and
2 CREST, JST, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3 Department of Biology, Faculty of Science, Chiba University Faculty of Science, Chiba University, Yayoicho, Inageku, Chiba, 263-8522, Japan



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  		Fig. 1. F-actin binding ability of N-WASP/WAVE2. (a) Schematic structure of N-WASP and WAVE2. The number of amino acids from rat N-WASP or human WAVE2 is indicated. Abbreviations: A, acidic region; B, basic region; C, cofilin homology domain; CRIB, Cdc42/Rac interactive binding region; IQ, IQ motif; Pro, proline-rich region; SHD, SCAR/WAVE homology region; V, verprolin homology domain; WH1, WASP homology 1 domain. (b,c) Association of F-actin with the basic region of N-WASP (b) and WAVE2 (c). Wild-type protein and {Delta}basic N-WASP (1 µM) or WAVE2 (0.3 µM) were mixed with F-actin (2 µM). The association with F-actin was assayed by co-sedimentation with F-actin after ultracentrifugation. The precipitates were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. The upper arrows indicate N-WASP or WAVE2, and the lower arrows indicate actin.

 


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  		Fig. 2. F-actin binding region of N-WASP/WAVE2. (a) The association of isolated basic region with filaments was analyzed by co-sedimentation assay. The amino acid numbers of these fragments from N-WASP or WAVE2 are shown. Association was analyzed by western blotting to either glutathione-S-transferase tag or His tag. (b) Dose dependence of actin filaments (which are composed mostly of ADP filaments and small amounts of filaments with ATP and ADP-Pi caps) against association with the isolated basic region by co-sedimentation assay. The association was monitored by western blotting followed by densitometry. (c) Comparison of the affinity of isolated basic region against ADP-Pi or ADP filaments by co-sedimentation assay. Association was analyzed by western blotting. ADP-Pi filaments were made by polymerizing actin in the presence of phalloidin. ADP filaments were made by hexokinase treatment. To estimate the effect of phalloidin staining, some of the ADP filaments were mixed with phalloidin and also used in the assays.

 


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  		Fig. 3. Basic region of N-WASP competed for actin filament binding. The association of full-length N-WASP with actin filaments in the presence or absence of protein fragments of the N-WASP binding region was analyzed by co-sedimentation assay. Association of full-length N-WASP with actin filaments was monitored with anti-N-WASP antibody. The concentrations of isolated N-WASP fragment are indicated.

 


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  		Fig. 4. Increase in association of Arp2/3 with actin filaments by wild-type WASP family proteins. Association of Arp2/3 (0.25 µM) with actin filaments (2 µM) in the presence or absence of N-WASP, its mutants or WAVE2 (1 µM) was analyzed by co-sedimentation. Filaments were made by polymerizing G-actin. Phalloidin filaments were made by adding phalloidin after 1 hour of polymerization. Filaments and phalloidin filaments are composed of ATP actin caps of <100 actin molecules (<0.6 µm per 5 µm (average) filament) and ADP filaments. ADP-Pi/phalloidin filaments were made by polymerizing G-actin with phalloidin. Co-sedimented Arp2/3 complex was monitored by western blotting with anti-Arp3 antibody (a). The extent of association at 2 µM of F-actin was quantified by densitometry and expressed as the fold increase in comparison with Arp2/3 only (b). Bars show the standard deviations.

 


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  		Fig. 5. Essential role of the basic region in branch formation on pre-existing actin filaments. (a) Visualization of actin filaments after actin polymerization for 15 minutes. Total actin filaments were visualized by green phalloidin staining. Preformed pre-incubated actin filaments are shown in red. Filaments were made by polymerizing rhodamine-labeled G-actin, which were composed of ATP actin caps of ~100 actin molecules (<0.6 µm per 5 µm (average) filament) and ADP filaments. ADP filaments were made by treating ordinary filaments with hexokinase overnight to hydrolyze ATP completely. Arrows indicate the points of elongation from pre-existing filaments. Concentration were as follows: VCA, WT and {Delta}basic N-WASP or WAVE2, 100 nM; GTP{gamma}S-loaded Cdc42, 500 nM; PIP2-containing vesicles, 1 µM; Arp2/3, 60 nM; F-actin, 300 nM; ADP-F-actin, 300 nM; G-actin, 2 µM. Scale bar, 5 µm. (b,c) Plots of length of newly-formed (green) (b) or pre-existing (red) (c) ‘mother’ filaments between branch points and presumed barbed ends against the length of ‘daughter’ filaments induced by wild-type N-WASP, Cdc42 and PIP2. The lengths of red mother filaments do not include end-elongating green filaments. (d) Values for branching per µm filaments are the mean of at least three independent experiments. Values that are significantly different, which are indicated on the graph, were determined by Student’s t test. Error bars indicate standard deviation.

 


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  		Fig. 6. Actin polymerization in the absence or presence of preformed actin filaments. Actin polymerization is shown induced by Arp2/3 and VCA fragment (a) and wild-type (b) or {Delta}basic mutant (c) N-WASP. All assays were performed with N-WASP or N-WASP mutants at 100 nM and Arp2/3 at 60 nM. F-actin was added to a final concentration of 300 nM. GTP{gamma}S-loaded Cdc42 and PIP2-containing vesicles were added at final concentrations of 500 nM and 1 µM, respectively. After pre-incubation for 5 minutes, actin was added (0.5 µM, 9% pyrene-labeled) and fluorescence was monitored. Pyrene fluorescence was converted to the amount of actin filaments. The rates of increase of actin filaments (the value obtained by differentiating the plot curve shown in a-c) are also shown: without Arp2/3 and N-WASP (d), VCA fragment (e), wild-type (f) and {Delta}basic (g). (h) The barbed end concentration of actin filaments at points where polymerization is 80% complete (see Materials and Methods). Pre-existence of side of actin filament (gelsolin-capped filaments) increased the number of the barbed ends only in the case of wild-type N-WASP. (i) Depolymerization of gelsolin-capped actin filaments. Gelsolin-capped filaments were diluted to 0.1 µM in the presence of N-WASP (200 nM) or N-WASP (100 nM) with Cdc42 (500 nM), PIP2 (1 µM vesicles) and Arp2/3 (60 nM) and the fluorescence was monitored.

 

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