Figure 1.
LUMP encodes a dsRNA binding domain protein.
A. The LUMP protein has two putative dsRNA binding domains (dsRBD). B. Genomic structure of the lump locus. The triangle represents location of the P element insertion in CG10630KG00804 in the 5′ UTR of lump. The arrows show the direction of transcription of lump and flanking genes. The genomic DNA used to drive expression in transgenic animals is depicted (genomic rescue) C, D. The P-element insertion causes a reduction of lump mRNA and protein levels in lump1 mutants.
Figure 2.
LUMP is not required for RNA interference or miRNA biogenesis.
Top Panels. Ago2414 mutants defective for RNAi and carrying one copy of pGMR-GAL4, UAS-whiteRNAi fail to suppress the white locus (left panel). RNAi is not blocked in lump1 mutants carrying one copy of pGMR-GAL4, UAS-whiteRNAi (center panel). pGMR-GAL4, UAS-whiteRNAi in a wild type background (right panel). Lower Panels. Northern blots for bantam and mir-7 miRNAs reveal no differences in quantity of mature miRNA in lump1 mutant flies. 2S rRNA is a 30 nucleotide ribosomal RNA used as a loading control.
Figure 3.
lump1 mutants are male sterile.
A. Number of progeny produced by males crossed to females of the depicted genotypes. w1118 control males crossed to lump1 mutant females produce similar numbers of progeny as wild type controls. lump1 mutant males crossed to w1118 control females are sterile. lump1 males carrying a transgenic wild type copy of the lump gene (rescue) have fertility restored to wild type levels. All data are presented as ± SEM. For each data set statistical significance was tested using ANOVA for independent observations. *** represents significant differences at P < 0.001. B–D. TOTO-3 DNA staining of spermatid nuclei. B. TOTO-3 staining of sperm nuclei reveals w1118 male seminal vesicles contain mature sperm. C. Seminal vesicles from lump1 mutant males are devoid of intact spermatid nuclei. D. Seminal vesicles from lump1 mutant males carrying a wild type transgenic copy of the lump gene contain mature sperm. E. GFP-LUMP expression in the testes. LUMP-GFP is expressed in spermatocyte nuclei of primary spermatocytes, in the nuclei and cytoplasm of onion stage spermatocytes and begins to degrade in elongating spermatids.
Figure 4.
lump mutants are defective for spermatid individualization.
(A, B) Testes squashes from 3-day old w1118 and lump1 mutant males showing primary spermatocytes with normal morphology. (C, D) Testes squashes from 3-day old onion stage spermatocytes from w1118 and lump1 mutant males have a single nucleus (arrow) and nebenkern (arrowhead) indicating no defects in meiosis. E. Testes squash from 3-day old w1118 male showing mature motile sperm. F. Testes squashes from 3-day old lump1 male showing clustered spermatids with elongated ‘lumps’. Spermatids are unable to individualize and have prominent blebbing (arrows).
Figure 5.
Spermatid individualization Defects in lump1.
A. A cartoon of the actin-based individualization complex (illustrated by red triangles) drives the expulsion of unwanted cellular material from a syncytium of 64 closely associated spermatids. The cellular waste is collected in the cystic bulge (CB). At the end of the tail, the CB pinches off to release individual spermatozoa and form the waste bag (WB), which is subsequently degraded. w1118 TOTO-3 DNA staining and protamine-GFP staining shows regular, aligned spermatid nuclei. lump1 mutant TOTO-3 staining shows condensed spermatid nuclei, but with individual spermatids out of alignment with the others in the bundle. B. Quantification of the number of intact DNA bundles in lump mutant males compared to w1118 and lump1 heterozygotes. All data are presented as ± SEM. For each data set statistical significance was tested using ANOVA for independent observations. *** represents significant differences at P<0.0005. Testes stained with Alexa-Fluor-phalloidin (red) showing actin cones of the individualization complexes (ICs). Similar alignment defects are observed in lump1 The IC in lump1 mutants carrying a wild-type transgenic copy is restored. C. The number of intact actin cones in lump1 mutant males is significantly reduced compared to w1118 and lump1 mutants carrying a rescuing transgene. All data are presented as ± SEM. For each data set statistical significance was tested using ANOVA for independent observations. *** represents significant differences at P<0.0001.
Figure 6.
LUMP dsRBD-1 is required for LUMP male fertility function.
A. The consensus dsRBD motifs for LUMP double-stranded RNA binding domain (dsRBD) are shown. Conserved hydrophobic alanine or leucine residues were replaced with basic lysines residues (K) in the first dsRBD, the second dsRBD, or both. B. The ability of the mutated LUMP constructs to rescue lump1 male fertility was analyzed with fertility assays. lump1 mutants carrying rescuing constructs with lesions in dsRBD1 or dsRBD1+2 are sterile. lump1 mutants carrying rescuing constructs with lesions in dsRBD2 alone or GFP-LUMP are fertile. All data are presented as ± SEM. For each data set statistical significance was tested using ANOVA for independent observations. *** significant differences at P<0.0004.