Fig 4. Drosophila Homolog of KPC1 (dKPC1) is required for sperm storage and Amo Localization.

(A) Domain structure of dKPC1 showing an N-terminal SPRY domain and a C-terminal Ring-finger domain. dKPC1 has 1332 amino acids and is predicted to be ~151 kDa in size. (B) Phylogenetic tree demonstrates homology between dKPC1 and related proteins. (C) Map of the CG6752/dKpc1 locus showing the MiMIC transposon insertion (dKpc1⁵⁸⁵⁴⁹) in Exon 4. Black arrow indicates the direction of transcription. (D-E) Western blots probed with antisera to dKPC1. The antibody detects a single band of ~151 kDa, indicated by the arrowhead, in wild type testes and sperm. There is no dKPC1 detectable in testes dissected from dKpc1⁵⁸⁵⁴⁹ males, from males with RNAi mediated knock-down of dKpc1, in females or in males lacking testes. (F) Amo-GFP sperm stained with antisera to dKPC1: red and to GFP: green. DIC and fluorescence images are merged showing that the two proteins partially co-localize at the tip of the sperm tail. Scale bars: 5 μm. (G) DIC images of wild type seminal receptacles dissected after mating with males of the indicated genotypes. Receptacles are empty after mating with dKpc1⁵⁸⁵⁴⁹ males and males with RNAi-mediated knock-down of dKpc1. Sperm storage is rescued after precise excision of the transposon (dKpc1^PE). Scale bars: 20 μm. (H) Fertility tests with males of the indicated genotypes mated to wild type females. dKpc1⁵⁸⁵⁴⁹ males and males with RNAi-mediated knock down of dKpc1 are sterile. Fertility is rescued by precise excision of the MiMIC transposon in dKpc1 (dKpc1^PE). The number of tests per genotype is denoted above the bars. ***p< 0.001. N.S. = not significant. (I) Wild type and dKpc1⁵⁸⁵⁴⁹ sperm stained with anti Amo: green, concanavalin A: red and DAPI: blue. Amo is missing in dKpc1⁵⁸⁵⁴⁹ sperm tails but localization is rescued by precise excision of the MiMIC transposon (dKpc1^PE). Scale bars: 5 μm.