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

Fig 4. Dia regulates actin and Arp2/3 activity during myoblast fusion.

Fig 4

A. In stage 15 embryos expressing 2xUAS-diaDN::GFP, myoblasts are stained with phalloidin and antibodies against DiaDN::GFP. Immunostaining for Sns and Duf was used to examine cell adhesion. Sns and Duf localize correctly at the fusion site, confirming adhesion between FCMs and FC/myotube. B. Stage 15 embryos expressing 2xUAS-diaDN::GFP with 2xDMef2-Gal4. Myoblasts are stained with phalloidin and antibodies against GFP. Arrowhead points to a myoblast adhering to the myotube but failing to generate a F-actin focus. C. Percentage of fusion sites with and without actin focus. Embryos expressing 2x UAS-diaDN::GFP were stained with phalloidin, and the formation of actin focus was quantified in these embryos. Actin focus forms in 60% of fusion sites and is absent in 40%. D. Time-lapse imaging of DiaDN fusion block. Three copies of UAS-diaDN::GFP and one copy of UAS-moesin::mCherry were driven by two copies of Dmef2-Gal4. F-actin dynamics were visualized by moesin::mCherry. Still images from the time-lapse sequence show 2–3 myoblasts adhered to a myotube (dashed lines), but unable to fuse. No F-actin focus was detected at the fusion site (arrow) (S1 Movie; S3I Fig). E-F. Comparison of SCAR and WASp localization in DiaDN-expressing FCMs. In embryos expressing high levels of DiaDN, immunostaining was used to examine the localization of SCAR and WASp. At a fusion site in which an actin focus forms (upper panels), SCAR and WASp both correctly localize to the actin focus (arrow; phalloidin). When actin focus formation is disrupted by DiaDN (lower panels), SCAR and WASp no longer accumulate at the fusion site (asterisk). G. Dia localization in sltr s1946 ; kette J4-48 double mutant. In this double mutant, phalloidin was used to label F-actin, and Dia antibody to detect the localization of Dia. Fusion is blocked in double mutants, with no actin focus forming. Dia still accumulates at the fusion site. H. Dia localization in myoblasts expressing UAS-PH plcγ::GFP. Stage 15 embryos expressing 2xPH plcγ::GFP with 2xDMef2-Gal4. Myoblasts are labeled with antibodies against GFP. PH plcγ sequesters PI(4,5)P2, generating a small actin focus and blocking myoblast fusion[54]. In an FCM that adheres to a FC, immunostaining reveals that Dia accumulates at the fusion site. Scale bar: 2.5μM.