2R11 alleles map to CG15609, the fly homologue of Eps15 homology domain containing protein-binding protein 1. (a) Diagram of the genomic locus of CG15609 (red arrow), the genomic rescue construct (red box), and the exon–intron structure of CG15609 isoform B (red bars signify coding exons, black bars signify non coding exons), where the molecular lesions of 2R11 alleles are shown. ΔEHBP1^ex24 is a deletion caused by imprecise excision of P{lacW}l(2)k09837 (indicated as P allele). (b) Schematic representation of dEHBP1 protein structure. Percentages indicate identity/similarity between the fly and mouse homologues. C2 represents the lipid-binding domain and is colored in green, CH stands for calponin homology actin-binding domain and is colored in blue, coiled coil protein interaction domain is colored in red, and CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif is shown as a triangle at the end of the protein sequence. a.a., amino acid. (c) The predicted structure of the dEHBP1 protein, in different mutant 2R11 alleles. (d) Lethal phase analysis of different mutations. NR, not rescued lethality; R, lethality rescued by genomic rescue; r, lethality rescued by cDNA, expressed ubiquitously by tubGal4 driver. (e–e′′′) Anti-dEHBP1 fails to recognize the majority of the protein in pupal thoracic homozygous clones of CG15609^O4. Sections at both XY and XZ levels (indicated by yellow dashed line in e) are shown. Single-channel representations are shown in (e′) for GFP (wild-type region), (e′′) for dEHBP1, and (e′′′) for Rab11, which marks the recycling endosome. (f–g′) Anti-dEHBP1specifically recognizes dEHBP1 in embryonic CNS of control, balanced embryos (f–f′′) in comparison to CNS from homozygous mutant siblings (g–g′′). Bars, 10 µm.