(A) Map of the Fhos genomic locus and transcripts (after Flybase, (Attrill et al., 2016). Shown are the nine known fhos transcripts (designated Fhos-RA--Fhos-RI), which are divided into two groups (RA-RG and RH-RI). The two groups share a nearly identical set of 3’ exons (red dashed rectangle), which encode a conventional FHOD-family formin, but are expressed via distinct regulatory regions, and possess distinct sets of 5’ exons, including 5’ coding exons (blue and purple dashed rectangles) that encode different N-terminal domains. The two transcript variants, RH and RA, used to generate, respectively, the long and short transgenic UAS-Fhos constructs are indicated by orange arrows. The insertion positions of three MiMIC elements, MI04231 (inserted downstream of long isoform initiation sites), MI01421 (inserted downstream of all transcript initiation sites), and MI09324 (used to produce the Fhos-GFP 'protein trap') are indicated by inverted triangles. Positions of two dsRNA target sequences used, one common to all fhos isoforms (red bar) and the other specific to the long forms (blue bar) are shown above the transcript map. The CRISPR/Cas9-generated deletion of the guanine residue at position 99 of the short isoform transcript and its adjacent sequence are indicated. (B) Schematic representation of three representative Fhos protein isoforms. The canonical formin domains common to all forms are colored red, while the alternative N-terminal domains are in blue (long forms) and purple (short form). Canonical domains indicated include the GTPase binding domain (GBD), formin homology (FH) domains 1/2 and 3, and the diaphanous autoregulatory domain (DAD). The positions of the I966A point mutation in the FH2 domain and the premature stop codon, generate by the frameshift mutation ΔG99 in the Fhos-PA N-terminal domain are indicated. (C–D’) Zasp (red) and phalloidin (blue and gray) stainings demonstrate the severe, null-like disruption of myofibril and sarcomere microfilament organization in hemizygous FhosMI01421/Df(3L)BSC612 pharate adult flies (C,C’), similar to that observed in fhosΔ1 hemizygotes. No rescue is observed following expression of UAS-GFP-Fhos-PA (green) driven by arm-Gal4 in this background (D,D’). (E–F) α-actinin (red) and phalloidin (blue and gray) stainings demonstrate the severe, null-like phenotypes following specific RNAi mediated knockdown of the Fhos long-isoforms (E,E’) and in hemizygous FhosMI04231/Df(3L)BSC612 (F) pharate adult flies. (G) Zasp (red) and phalloidin (blue) stainings demonstrate normal myofibril and sarcomeric structure of Fhos ΔG99/Df(3L)BSC612 hemizygotes, in which the short Fhos isoforms are not expressed. (H–L’’) Fhos localization in myofibrils, as monitored at two distinct pupal developmental time points, 45 hr APF (H–I’), and 65 hr APF (J–J’’), via a GFP 'exon trap' engineered at the insertion site of the MiMIC transposon MI09324 (green triangle in A). The GFP-tagged Fhos proteins (all isoforms) generated in this manner are visualized with anti-GFP (green or gray), Z-discs are visualized with anti-α-actinin or anti-Zasp (red), thin filament pointed ends visualized by anti-Tmod (blue) and microfilaments with phalloidin (blue). The diffuse/punctate initial localization of Fhos-GFP overlying broad portions of the growing myofibrils (H), in some cases shows an adjacent localization to the nascent Z-disc (I white arrowhead) or to array pointed ends (I’ red arrowhead). The initial punctate localization gives way to a striated pattern restricted to the vicinities of both the barbed (Z) and pointed (M) ends of the thin-filament arrays (J–J’’). (K–K”) Localization of the short isoform of Fhos in IFMs from a young adult fly, visualized by expression of UAS-GFP-Fhos-PA using the mef2-GAL4 driver (anti-GFP, green or gray). Z-discs are visualized with anti-α-actinin (red), and microfilaments with phalloidin (blue). GFP-Fhos-PA localizes to the vicinity of the pointed ends of the arrays (M). (L–L”) Localization of the long isoforms of Fhos in IFMs from a young adult fly, visualized with anti-HA (green and gray), following expression of UAS-HA-Fhos-PH using the mef2-GAL4 driver. Fhos-PH-PA localizes to the vicinity of the barbed ends of the arrays (Z), where it overlaps with the general Fhos distribution to both the barbed and pointed ends (M) of the arrays (visualized with anti-Fhos [red]). Microfilaments visualized with phalloidin (blue). Scale bars in all panels correspond to 5 μm.