Bsg25D and Ens physically interact and colocalize in myotubes in vivo. (A) Top: Map of Bsg25D indicating coiled–coil (CC) domain–rich regions and regions that bind Ens (this work), MTs (Kowanda et al., 2016), and γ-Tubulin (predicted from alignment to mouse Ninein; Delgehyr et al., 2005). In Bsg25D−, the entire coding region has been removed and replaced by a mini-white (mini w+) reporter cassette. Bottom: Coimmunoprecipitation of overexpressed Ens-GFP and Bsg25D-HA from S2 cells. The largest Bsg25D-HA band is the main protein species, and smaller bands likely represent cleavage products. IP, immunoprecipitation. (B) Bsg25D expression in the stage 16 embryo. Left and right panels are the same image with and without Tropomyosin, a muscle marker. Left: Bsg25D, gray. Right: Bsg25D, magenta; Tropomyosin, green. Red box indicates primordial germ cells. (C) Left: Extended focus projection of one stage 16 hemisegment. Right: Orthogonal (YZ) section of indicated (red box) lateral transverse myotube. Crosshairs identify a Bsg25D+ punctum inside myotube. Bsg25D, magenta; Tropomyosin, green. (D) Immunofluorescent staining of stage 16 myotube for Bsg25D and Plp. Image is a single slice from a Z-stack. Bsg25D, magenta; Plp, green; colocalization, white (see red arrows). (E) Immunofluorescent staining of stage 16 myotube for Bsg25D and Ens. Image is a single slice from a Z-stack. In the merged image, Bsg25D, magenta; Ens, green; colocalization, white (highlighted by arrows). In D and E, images in bottom row are higher magnification views of boxed regions, and dashed lines outline lateral transverse muscles. In all images, scale bars = 5 µm.