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doi: 10.1242/10.1242/jcs.00362

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Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure

Mariko Sawa1,3, Shiro Suetsugu1,3, Asako Sugimoto5, Hiroaki Miki2,4, Masayuki Yamamoto6 and Tadaomi Takenawa1,3,*

1 Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 Cancer Genomics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3 CREST, Japan Science and Technology Corporation (JST), Minato-ku, Tokyo 108-8639, Japan
4 PRESTO, Japan Science and Technology Corporation (JST), Minato-ku, Tokyo 108-8639, Japan
5 Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
6 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan



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  		Fig. 1. arx-2(RNAi), arx-5(RNAi) and wsp-1(RNAi) show defects in morphogenesis. (A-D) Time-lapse differential interference contrast (DIC) images of wild-type (A), arx-2(RNAi) (B), arx-5(RNAi) (C) and wsp-1(RNAi) (D) embryos. Images were obtained at 280, 360, 420 and 500 minutes after the first cleavage. Representative hypodermal cells are indicated with arrowheads. In wild-type embryos, hypodermal cells migrated toward the ventral midline between 280 and 360 minutes. By 420 minutes, the embryo had rotated 90° and started elongation (A). These processes were not observed in RNAi-treated embryos (B-D). (E-H) Localization of adherens junction marker AJM-1 fused with GFP is shown for wild-type embryos at approximately 420 minutes (E). The terminal phenotype of arx-2(RNAi) (F), arx-5(RNAi) (G) and wsp-1(RNAi) (H) embryos. An arrowhead indicates a hypodermal cell, and an arrow indicates the pharynx. All embryos are shown with the anterior positioned to the left. The wild-type embryos in A at approximately 420 and 500 minutes and in E are lateral views; all other images are ventral views. Bar in H, 10 µm.

 


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  		Fig. 2. arx-2(RNAi) and wsp-1(RNAi) embryos show defects in hypodermal cell migration. (A-C) Ventral view of time-lapse recording of hypodermal cell marker AJM-1 fused with GFP in wild-type (A), arx-2(RNAi) (B) and wsp-1(RNAi) (C) embryos. The 3D images of AJM-1 localization were obtained at approximately 340, 360, 380, 400 and 430 minutes after the first cleavage. Some of the contralateral pairs of hypodermal cells are numbered. The pharynx is indicated with arrows. (D,E) Dorsal view of AJM-1 expression in wild-type (D) and arx-2(RNAi) (E) embryos. Treatment by arx-2(RNAi), which is lethal, showed normal AJM-1 expression on the dorsal side, although the width of the dorsal cells was narrowed due to the inability of the ventral cells to close (marked by asterisk). Bar, 10 µm.

 


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  		Fig. 3. Reduction of F-actin formation by arx-2(RNAi) and wsp-1(RNAi). Phalloidin staining of embryos carrying the jcIs1[ajm-1::GFP]. (A-D) Wild-type, (E-H) arx-2(RNAi) embryo during ventral enclosure and (I-L) wsp-1(RNAi) embryo at the terminal phenotype. Phalloidin staining (A,E,I), expression of AJM-1::GFP (B,F,J) and merged images (C,G,K) are shown. In merged images, phalloidin staining is red, and expression of AJM-1::GFP is green. Corresponding magnified images of phalloidin (left), AJM-1GFP (center), merged image (right) are also provided (D,H,L). The leading edge is indicated with arrows (A-L) and with dotted lines (D,H,L). In the wildtype, F-actin accumulation is observed at the leading edge (A,C,D). In arx-2(RNAi) and wsp-1(RNAi) embryos, F-actin accumulation is reduced at the leading edge (E,G,H,I,K,L).

 


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  		Fig. 4. Expression of the differentiation marker of hypodermal cells. (A) Immunostaining by anti-LIN-26 antibodies (a,d) and MH27 antibodies (b,e) in wild-type (a-c) and arx-1(RNAi) embryos (d-f). Merged images are shown in c and f. Expression of LIN-26 is green, and staining by MH27 antibody is red. (B) Phalloidin staining of embryos expressing the seam cell marker; SCM::GFP. (a-c) Wild-type. (d-f) arx-2(RNAi). SCM::GFP (a,d), Phalloidin staining (b,e) and merged images (c,f) are shown. In merged images, expression of SCM::GFP is green and phalloidin staining is red.

 


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  		Fig. 5. Amino-acid sequence of WSP-1. (A) Amino-acid sequences of WSP-1 (C. elegans N-WASP) isoforms 1 and 2 are given with domains indicated by underlining. The sequence of WSP-1 isoform 2 continues to amino acid 171 of WSP-1 isoform 1. The asterisk in isoform 2 indicates the putative first methionine found in the cDNA sequence. It is possible that isoform 2 contains additional amino acids upstream of the sequence presented. (B) Domain structures of WSP-1 isoform 1 and 2. The splicing variant point is indicated with an arrowhead, and the bar indicates 100 amino acids. WH1, WASP homology 1 domain; CRIB, Cdc42/Rac interactive binding region; V, verprolin homology (VPH) domain; C, cofilin homology domain; A, acidic region. (C) Western blotting of mixed stage C. elegans lysates with an anti-WSP-1 antibody. A single band at 64 kDa is indicated with an arrowhead, indicating specific recognition of WSP-1 by the anti-WSP-1 antibody. (D) Activation of Arp2/3-complex-mediated actin polymerization by WSP-1. The actin polymerization assay using 60 nM bovine Arp2/3 complex and either 100 nM WSP-1 VCA or bovine N-WASP VCA. G-actin was added to a final concentration of 2 µM (9% pyrene-labeled).

 


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  		Fig. 6. Cell-specific RNAi of arx-1 results in morphological defects. Expression of dsRNA of arx-1 under the expression of the lin-26 promoter caused morphological defects in embryogenesis. (A) DIC image. (B) Confocal image detecting AJM-1. The pharynx is indicated with an arrowhead; hypodermal cells are indicated with arrows.

 


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  		Fig. 7A. Localization of the Arp2/3 complex. (A) Western blotting of total protein lysates from mixed-stage wild-type embryos with an anti-ARX-1 antibody (left), an anti-ARX-7 antibody (center) and a monoclonal antibody against actin (right). The band corresponding to each protein is indicated with an arrowhead. ARX-1 is at 48 kDa, ARX-7 is at 16 kDa and actin is at 45 kDa. (B) ARX-1 localization during early embryogenesis. Immunostaining with an anti-ARX-1 antibody (a,d,g) and an anti-actin antibody (b,e,h) with merged images shown (c,f,i) for wild-type (a-c), arx-1(RNAi) (d-f) and wsp-1(RNAi) (g-i) embryos. (C) ARX-1 localization during ventral enclosure. Immunostaining with an anti-ARX-1 antibody (a,e,i) and an anti-actin antibody (b,f,j) with merged images shown (c,g,k) for wild-type (a-d), arx-1(RNAi) (e-h) and wsp-1(RNAi) (i-l) embryos. Magnified images of hypodermal cells are shown (d,h,l) for an anti-ARX-1 antibody (left), an anti-actin antibody (center) and merged images (right). (D) Expression of AJM-1 fused with GFP during ventral enclosure showing hypodermal cell shapes shortly after the time of cell arrest in embryos depleted of the Arp2/3 complex. The two extreme anterior pairs of cells are already fused. (E) ARX-7 localization during early embryogenesis. Immunofluorescent staining with an anti-ARX-7 antibody (a,d,g) and an anti-actin antibody (b,e,h) with merged images shown (c,f,i) for wild-type (a-c), arx-7(RNAi) (d-f) and wsp-1(RNAi) (g-i) embryos. (F) Immunofluorescence staining with an anti-ARX-7 antibody (a,e,i) and an anti-actin antibody (b,f,j) and merged images (c,g,k) of wild-type (a-d), arx-7(RNAi) (e-h) and wsp-1(RNAi) (i-l) embryos. Magnified images of hypodermal cells are shown (d,h,l) for the anti-ARX-7 antibody (left), the anti-actin antibody (center) and merged images (right). In C,D,F, the anterior is positioned to the left, posterior to the right and the ventral is towards the bottom. The leading edges of the migrating hypodermal cells are indicated with arrows.

 


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  		Fig. 7C. Localization of the Arp2/3 complex. (A) Western blotting of total protein lysates from mixed-stage wild-type embryos with an anti-ARX-1 antibody (left), an anti-ARX-7 antibody (center) and a monoclonal antibody against actin (right). The band corresponding to each protein is indicated with an arrowhead. ARX-1 is at 48 kDa, ARX-7 is at 16 kDa and actin is at 45 kDa. (B) ARX-1 localization during early embryogenesis. Immunostaining with an anti-ARX-1 antibody (a,d,g) and an anti-actin antibody (b,e,h) with merged images shown (c,f,i) for wild-type (a-c), arx-1(RNAi) (d-f) and wsp-1(RNAi) (g-i) embryos. (C) ARX-1 localization during ventral enclosure. Immunostaining with an anti-ARX-1 antibody (a,e,i) and an anti-actin antibody (b,f,j) with merged images shown (c,g,k) for wild-type (a-d), arx-1(RNAi) (e-h) and wsp-1(RNAi) (i-l) embryos. Magnified images of hypodermal cells are shown (d,h,l) for an anti-ARX-1 antibody (left), an anti-actin antibody (center) and merged images (right). (D) Expression of AJM-1 fused with GFP during ventral enclosure showing hypodermal cell shapes shortly after the time of cell arrest in embryos depleted of the Arp2/3 complex. The two extreme anterior pairs of cells are already fused. (E) ARX-7 localization during early embryogenesis. Immunofluorescent staining with an anti-ARX-7 antibody (a,d,g) and an anti-actin antibody (b,e,h) with merged images shown (c,f,i) for wild-type (a-c), arx-7(RNAi) (d-f) and wsp-1(RNAi) (g-i) embryos. (F) Immunofluorescence staining with an anti-ARX-7 antibody (a,e,i) and an anti-actin antibody (b,f,j) and merged images (c,g,k) of wild-type (a-d), arx-7(RNAi) (e-h) and wsp-1(RNAi) (i-l) embryos. Magnified images of hypodermal cells are shown (d,h,l) for the anti-ARX-7 antibody (left), the anti-actin antibody (center) and merged images (right). In C,D,F, the anterior is positioned to the left, posterior to the right and the ventral is towards the bottom. The leading edges of the migrating hypodermal cells are indicated with arrows.

 


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  		Fig. 8. WSP-1 localizes at the periphery of cells. (A) Immunostaining of WSP-1 (a,d,g,j,n,r) and actin (b,e,h,k,o,s) in wild-type (a-c,j-m), as well as wsp-1(RNAi) (d-f,n-q) and arx-2(RNAi) (g-i,r-u) treated cells. Merged images are shown in c,f,i,l,p,t. Magnified images of migrating hypodermal cells are shown (m,q,u) for the anti-WSP-1 antibody (left), the anti-actin antibody (center) and for the merged images (right). WSP-1 localizes to the periphery of cells from early development to morphogenesis (a,j). Localization of WSP-1 is partially destabilized and observed in nuclei in arx-2(RNAi) (g,i,r,t,u) embryos. In j-u, anterior is left, posterior is right and ventral is bottom. The leading edges of the migrating hypodermal cells are indicated with arrows. (B) Western blotting of untreated and arx-1(RNAi)-treated embryo lysates with an anti-ARX-1 antibody (upper lane), anti-WSP-1 antibody (middle lane) and anti-actin antibody (bottom lane). In arx-1(RNAi) embryos, WSP-1 isoform1 is increased by four-fold, and the upper band that corresponds with WSP-1 isoform2 was detected.

 


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  		Fig. 9. Immunostaining by the anti-WSP-1 and anti-ARX-1 antibodies. (A) Anti-WSP-1 antibody conjugated with Alexa Fluor 546, (B) anti-ARX-1 antibody conjugated with Alexa Fluor 633, (C) expression of AJM-1::GFP and (D) the merged image are shown. WSP-1 is red, ARX-1 is blue and AJM-1 is green. The magnified image around the leading edge is shown in E, from left WSP-1, ARX-1, AJM-1 and the merged image. All images show the ventral view. The leading edge of the most anterior hypodermal cell is indicated with an arrowhead (A-E) and with a dotted line (E).

 


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  		Fig. 10. Cell polarities are properly orientated in wsp-1(RNAi)-treated embryos. (A-C) Time-lapse recording of the three-cell to four-cell stage by DIC microscopy. The P blastomere on the right side is dividing. (A) Wild-type, (B) wsp-1(RNAi)- and (C) cdc42(RNAi)-treated embryos. In cdc42(RNAi) embryos, the spindle of the P blastomere is oriented longitudinally resulting in the same direction of cell division as in the AB blastomere, as reported previously (Kay and Hunter, 2001Go; Gotta et al., 2001Go). In wsp-1(RNAi)-treated embryos showing ventral enclosure abnormalities, normal positioning of the spindle followed by proper directional P cell division was still observed. The direction of the spindle is indicated with double-headed arrows. In A-C, the anterior is positioned to the left and the posterior to the right.

 

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© The Company of Biologists Ltd 2003