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First published online 27 November 2002
doi: 10.1242/jcs.00233

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Differential localization of WAVE isoforms in filopodia and lamellipodia of the neuronal growth cone

Motohiro Nozumi1, Hiroyuki Nakagawa1, Hiroaki Miki2, Tadaomi Takenawa2 and Shigeaki Miyamoto1,*

1 Department of Biochemical Science, Kyushu Institute of Technology, 680-4 Kawatsu, lizuka, Fukuoka 820-8502, Japan
2 Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan



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  		Fig. 1. Immunoblotting with anti-WAVEs. Anti-WAVE antibody (anti-WAVEs) reacting with all WAVE isoforms was recognized as a doublet at around 85 kDa of NG108 cell SDS-whole extract, corresponding to less- and hyper-phosphorylated forms of antigen as previously reported (Miki et al., 1999Go). Lane (A) shows the pattern of PAGE for SDS-whole extract and (B) shows immunoblotting pattern with anti-WAVEs.

 


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  		Fig. 2. Localization of WAVEs and reorganization of actin bundles. NG108 growth-cone lamellipodia of cells cultured for 24 hours in the presence of 1 mM Bt2cAMP were double stained with rhodamine-phalloidin (red) and anti-WAVEs (green). WAVEs show a continuous localization along the leading edge of lamellipodium. Many actin bundles orthogonally encounter the leading edge (arrowhead) and some diagonally running ones fuse with others at the leading edge (arrows). Bar, 1 µm.

 


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  		Fig. 3. Localization of WAVEs and reorganization of actin bundles. Growth cone was double stained with anti-WAVEs (green) and rhodamine-phalloidin (red) after 24 hours culture in the presence of 1 mM Bt2cAMP. WAVEs clearly localize at the tips of filopodia (arrowheads). The proximal portions of actin bundles extend into the cytoplasm and sometimes associate with others (thin arrow). Some actin bundles separate around the filopodial base and radially spread into the veil (thick arrows). Premature filopodia show a nub-like shape (square bracket). Bar, 1 µm.

 


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  		Fig. 4. Localization of WAVE1-EGFP, WAVE2-EGFP and WAVE3-EGFP along the leading edge of lamellipodia and at the tips of filopodia. The growth cones were double stained with anti-GFP antibody (green) and rhodamine-phalloidin (red). The upper panel shows the growth-cone lamellipodium (A,C,E) and the lower panel shows the growth-cone microspikes and filopodia (B,D,F). The fluorescence signal emitted from each WAVE-EGFP coincides with that from anti-GFP antibody labeled with fluorescence dye. (A,B) WAVE1-GFP. (C,D) WAVE2-EGFP. (E,F) WAVE3-EGFP. Bar, 1 µm.

 


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  		Fig. 5. Time-lapse images of localization of WAVE isoforms tagged with EGFP in the growth cone. (A,B) WAVE1-EGFP localized along the leading edge and concentrated in the extending region (asterisk) but disappeared in the retracting region (indicated by square bracket). Neither was found at the filopodial tips (arrows). The upper panel (A) shows fluorescence images of WAVE1-EGFP, whereas the lower panel (B) shows differential interference contrast (DIC) images corresponding to the upper images. The four columns show the image every 44 seconds. (C,D) WAVE2-EGFP condensed as dot-like spots at the sites where microspikes encounter the leading edge (arrowheads). They correspond to the tips of filopodia and project extracellularly with elongation (open arrows). WAVE2-EGFP remained at the tips of extending filopodia (open arrows) and sometimes moved with time laterally along the leading edge with the lateral motion of actin bundles through the lamellipodium (arrowheads). By contrast, WAVE2-EGFP spots disappeared at the tips of retracting filopodia in the region indicated by square brackets. The upper panel (C) shows fluorescence images from WAVE2-EGFP at the growth cone and the lower panel (D) indicates the corresponding DIC images. (E,F) WAVE3-EGFP localized at the tips of extending filopodia and remained there during elongation (arrowheads), and also distributed along the leading edge of extending lamellipodia (asterisks). The upper panel (E) shows fluorescence images of the WAVE3-EGFP distribution at the growth cone and the lower panel (F) shows the corresponding DIC images. Bar, 1 µm.

 


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  		Fig. 6. Localization of the EGFP-tagged domain and region fragments of WAVE1 and WAVE2. (A) EGFP-tagged constructs of WAVE1 and WAVE2. SHD, B, NT54, NT83 and Pro-rich indicate SCAR homology domain, basic region, N-terminal 54-amino-acid fragment of WAVE2, N-terminal 83-amino-acid fragment of WAVE2 and proline-rich region, respectively. Numbers show the position of amino acids relative to the N-terminus. (B-F) Localization of the truncated fragments of WAVE1 and WAVE2 at neuronal growth cone. The growth cone was double stained with anti-GFP (green) and rhodamine-phalloidin (red). WAVE1 SHD (B) and SHD-BR (C) localized at the filopodial tips (arrowheads). NT83 fragments of WAVE2 SHD (E) localized at the filopodial tips (arrowheads), whereas NT54 fragments (D) did not. WAVE2 Pro-rich domain (F) distributed diffusely at growth cone. Bar, 1 µm.

 


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  		Fig. 7. Filopodial formation and elongation. (A) WAVE localizes at the distal ends of actin bundles. WAVE localized at the initiation site of filopodia and moved along the leading edge (small arrows) with the lateral motion of actin bundles through lamellipodium cytoplasm as indicated by large arrows. (B) Filopodia formation and elongation. Several actin bundles are fused at the WAVE-concentrated points and form the filopodium core. Actin polymerization via WAVE at the barbed ends elongates the microspikes and filopodia in the direction indicated by the arrows. Some proximal portions of actin bundle extend radially into the actin network and others reach the microtubule in the cytoplasm. (C) Filopodia and lamellipodia retraction. When WAVEs diffuse into the cytoplasm from the leading edge and filopodial tips, actin bundles are detached from the membrane, and then lamellipodia and filopodia retract as indicated by the arrows.

 




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