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First published online 24 July 2008
doi: 10.1242/jcs.034215

Journal of Cell Science 121, 2662-2670 (2008)
Published by The Company of Biologists 2008
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Essential role of ADF/cofilin for assembly of contractile actin networks in the C. elegans somatic gonad

Kanako Ono, Sawako Yamashiro and Shoichiro Ono*

Department of Pathology, Emory University, Atlanta, GA 30322, USA


Figure 1
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  		Fig. 1. Requirement of UNC-60A but not UNC-60B for assembly of organized actin networks in the myoepithelial sheath of the somatic gonad. Dissected hermaphroditic gonads from wild-type (A-C), unc-60B(su158) (D-F), control RNAi (G-I) or unc-60A(RNAi) (J-L) worms were stained for F-actin by tetramethylrhodamine-phalloidin (A,D,G,J) and for DNA by DAPI (B,E,H,K). (C,F,I,L) Merged images are shown (F-actin in red and DNA in blue). Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Expression and localization of UNC-60A in myoepithelial-sheath cells. (A-D) A wild-type somatic gonad was stained for UNC-60A (A), MyoA myosin heavy chain (B) and DNA (C). (D) A merged image is shown (UNC-60A in red, MyoA in green and DNA in blue). UNC-60A was strongly expressed in oocytes (asterisks) and weakly in the myoepithelial sheath (arrows). Brightness and contrast in A are adjusted for weak UNC-60A staining in the myoepithelial sheath, so the strong signals in oocytes are saturated. (E-G) Colocalization of UNC-60A (E) and actin (F) in the myoepithelial sheath was detected when oocytes were not permeabilized for anti-UNC-60A antibody. (G) A merged image (UNC-60A in red and actin in green) shows that puncta of UNC-60A localize to actin filaments. Scale bars: 20 µm.

 

Figure 3
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  		Fig. 3. Somatic requirement of UNC-60A for organized assembly of actin networks in the myoepithelial sheath of the somatic gonad. rrf-1(pk1417) (RNAi-defective in soma) worms were treated with control RNAi (A-C) or unc-60A(RNAi) (D-F), and the dissected gonads were stained for F-actin (A,D) and DNA (B,E). (C,F) Merged images are shown (F-actin in red and DNA in blue). unc-60A(RNAi) resulted in the formation of abnormal clumps of oocytes but did not affect actin-filament organization in the myoepithelial sheath. Note that the micrographs in A-C are composites of multiple micrographs of the same gonad. Scale bar: 20 µm.

 

Figure 4
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  		Fig. 4. Rescue of the unc-60A(RNAi) phenotype in the myoepithelial sheath by transgenic expression of UNC-60B mutants with defective severing activity. Wild-type worms with no transgene (A-F), Plet-502::GFP-UNC-60B(WT) (G-L), Plet-502::GFP-UNC-60B({Delta}150) (M-R) or Plet-502::GFP-UNC-60B({Delta}152) (S-X) were treated with control RNAi or unc-60A(RNAi) as indicated, and the dissected gonads were stained for F-actin (left-hand column) and DNA (shown in merged images only, blue). GFP localization is shown in the middle column. Merged images are shown in the right-hand column (F-actin in red, GFP in green and DNA in blue). Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5. Different actin-filament-severing activities of the UNC-60A and UNC-60B variants. Fluorescently labeled actin filaments were tethered to a glass coverslip and treated with 10 nM to 10 µM of UNC-60A (A,B), UNC-60B(WT) (C,D), UNC-60B({Delta}150) (E,F) or UNC-60B({Delta}152) (G,H). The filaments were observed before (A,C,E,G) or 2 minutes after (B,D,F,H) the incubation, and the same fields are shown for each treatment. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Rescue of the unc-60B(null) phenotype in the body-wall muscle by transgenic expression of UNC-60B. Actin-filament organization in the body-wall muscle of wild-type (A), unc-60B(su158) (D), unc-60B(su158); Pmyo-3::GFP-UNC-60B(WT) (G), unc-60B(su158); Pmyo-3::GFP-UNC-60B({Delta}150) (J) and unc-60B(su158); Pmyo-3::GFP-UNC-60B({Delta}152) (M) worms was visualized by staining with tetramethylrhodamine-phalloidin. The GFP fluorescence is shown in the middle column [wild-type and unc-60B(su158) do not have a transgene expressing GFP]. Merged images are shown to the right (F-actin in red and GFP in green). The arrow in G indicates an actin aggregate in a cell that did not express GFP-UNC-60B(WT). Scale bar: 20 µm.

 

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