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. 2015 Aug 21;11(8):e1005381. doi: 10.1371/journal.pgen.1005381

Fig 7. Constitutively active Dia alters the F-actin structure at the fusion site and regulates localization of Arp2/3 regulators.

Fig 7

A-B. Stage 14 embryos stained for F-actin (phalloidin, white) and for Dia::GFP or DiaΔDAD::GFP (GFP antibody, white); FCM (magenta), FC/Myotube (turquoise). Scale bar: 5μM. A. Control DMef2-Gal4>UAS-dia::GFP myoblasts show colocalization of Dia::GFP and the F-actin focus at the fusion site. B. DMef2-Gal4>UAS-diaΔDAD::GFP myoblasts show that F-actin does not form a well-defined focus at the fusion site but appears diffuse. DiaΔDAD::GFP localizes to the plasma membrane and is enriched at cell contact sites. C. Fluorescent intensity curves confirm the distribution of F-actin in embryos expressing Dia::GFP and DiaΔDAD::GFP. D-E. Stage 15 embryos stained for F-actin (phalloidin) and DiaΔDAD::HA (antibodies against HA) showing FCM (magenta) and FC/Myotube (turquoise). Scale bar: 5μM. D. DMef2-Gal4 driven expression of DiaΔDAD::HA in kette J4-48 mutant background. The morphology of the F-actin focus at the fusion site appears similar to DMef2-Gal4>UAS-diaΔDAD::GFP embryos. While F-actin localizes at the cell cortex, it spreads out at the fusion site and does not make a concentrated focus. E. DMef2-Gal4 driven expression of DiaΔDAD::GFP in sltr s1946 mutant background. Similar to expression of DiaCA alone, the F-actin localizes at the cell cortex and spreads out at the fusion site. F-G. Stage 14 embryos stained for F-actin (phalloidin), DiaΔDAD::GFP (GFP antibody) and SCAR or WASp. Scale bar: 5μM. F. SCAR localization in control and DMef2-Gal4>UAS-diaΔDAD::GFP embryos. In control embryos, SCAR accumulates at the fusion site, as confirmed by the fluorescent intensity curves. When expressing DiaΔDAD::GFP, SCAR loses its characteristic concentration at the fusion site and becomes found throughout the cytoplasm. The multiple peaks in the SCAR fluorescent intensity curve confirm SCAR’s change in localization. G. Localization of WASp in control and DMef2-Gal4>UAS-diaΔDAD::GFP embryo. In control embryos, WASp accumulates at the fusion site, as confirmed by the fluorescent intensity curves. When expressing DiaΔDAD::GFP, WASp displays a more diffused localization. Fluorescent intensity curves confirm the broader distribution of WASp signal in relation to the controls.