Fig 1.
Strong abundance of proteins in the insoluble pellet of wild-type sperm extract.
(A) Coomassie Brilliant Blue stained gel and immunoblot analysis of the SDS-soluble supernatant (sn) and the SDS-resistant pellet (p) fractions isolated from wild-type sperm lysates by SDS extraction. *β-Tubulin is mainly present in the supernatant fraction of the lysate. (B) List of proteins identified only in the 50-60 kDa fraction of SDS-resistant pellet of sperm extract. Green highlights represent the Sperm-Leucylaminopeptidases.
Fig 2.
Phenotypes of the mutant alleles of S-Lap genes.
(A) Phenotypes of classical mutants (S-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848) and RNAi lines (S-Lap7KK105511, S-Lap8KK106866) driven by bam-Gal4 testis-specific driver. (B) Diagrams show the average progeny yield of the S-Lap alleles in wild type (green), heterozygotes (blue) and in the transheterozygous (orange) combination. S-Lap1Δ7 allele shows a stronger reduction in progeny number among heterozygotes and gives no more than 40 offspring in average in genetic combinations with other S-Lap alleles. Error bars indicate mean +s.e.m., n = 15 **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test).
Fig 3.
Paracrystalline material formation defects in the S-Lap mutants.
(A) Wild-type spermatids show synchronized development of their mitochondrial derivatives in the cysts, where paracrystalline material starts to form at the attachment site of the axoneme (red dashed line) and the major mitochondrial derivative (yellow dashed line) (arrows in A), then accumulate in the growing mitochondria, as a contiguous dense material (arrows in A’), finally it fills the inner space in regularly arranged structure by the end of individualization (arrows in A”), while the minor mitochondrial derivative (green dashed line) regresses (A”). (B-E) The S-Lap mutants (S-Lap1Δ7, S-Lap2MI14597, S-Lap6MI06848) show unsynchronized mitochondrial development with unstructured electron dense paracrystalline material in the major mitochondrial derivatives of spermatids (arrows in B, C, D, E). Scale bars: 0.5 µm. (E-G) Transmission electron micrographs of the longitudinal sections of elongated spermatids in wild type (F), S-Lap1Δ7 (G) and S-Lap2MI14597 (H) mutant testes. The paracrystalline material is solid and unified inside the major mitochondrial derivative (yellow dashed line) in wild type (F), but scattered in the S-Lap mutants (G, H, arrowheads). Scale bars: 0.5 μm.
Fig 4.
S-Lap1 is localized to the paracrystalline material of the elongated mitochondria.
(A) Expression of the genomic reporter construct S-Lap160aa-GFP (green) shows localization in elongated mitochondria (arrow) by staining with Mitotracker (red). Scale bar: 50 μm. The bottom schematic diagram shows the structure of the S-Lap160aa-GFP construct with the 5’ genomic region, the mitochondrial targeting sequence coding 60 amino acids and the eGFP sequence. (B) Localization of S-Lap1 protein in elongated mitochondria stained with S-Lap1 antibody (green) and Mitotracker (red) in the squash preparation of wild-type testis. Scale bar: 10 μm. (C) Coomassie Brilliant Blue stained gel and immunoblots of the SDS-soluble supernatant (sn) and the SDS-resistant pellet (p) fraction of wild-type sperm lysates. S-Lap1 is present in the insoluble pellet fraction and β-Tubulin mainly in the soluble fraction of the extract. (D) Representative immuno-electron micrograph of a cross-section of wild-type elongated spermatids (brown dashed line) shows the presence of S-Lap1 protein inside the major mitochondrial derivative (yellow dashed line), represented by gold particles (black dots, arrowheads). Axoneme (red dashed line) and the minor mitochondrial derivative (green dashed line) are also shown. Scale bar: 0.5 μm.
Fig 5.
S-Lap1-HAint localizes to the elongated mitochondria.
(A) S-Lap1-HAint protein (green) co-localizes (arrow) with the elongated mitochondria of spermatids, but not with the mitochondria of the elongating cyst (arrowhead) (stained with Mitotracker (red)). Scale bar: 10 μm. (B) Immunoblots of the SDS-soluble supernatant (sn) and the SDS-resistant pellet (p) fraction of the transgenic S-Lap1-HAint sperm lysates using HA antibody. S-Lap1-HAint is mainly present in the insoluble fraction of sperm lysate. (C) Immuno-electron micrograph of a cross-section of transgenic S-Lap1-HAint elongated spermatids (brown dashed line) shows the occurrence of S-Lap1-HAint protein inside the major mitochondrial derivative (yellow dashed line), represented by gold particles (black dots, arrowheads). Axoneme (red dashed line) and the minor mitochondrial derivative (green dashed line) are also shown. Scale bar: 0.5 μm. (D-E) Cross-section (D) and longitudinal section (E) of immuno-labeled micrographs show S-Lap1-HAint protein accumulation inside the major mitochondrial derivative (yellow dashed line) in spermatids from homozygous S-Lap1-HAint; S-Lap1Δ7 testes, represented by gold particles (black dots, arrowheads). Spermatids (brown dashed line) and axoneme (red dashed line) are labeled. Scale bars: 0.5 μm. (F) S-Lap1-HAint localizes to the elongated mitochondria in testis of S-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848 mutants. Scale bar: 200 μm. (G) Immunoblots of total lysate, (H) the SDS-soluble supernatant (sn) and the SDS-resistant pellet (p) fraction of testis expressing the homozygous S-Lap1-HAint transgene in the classical mutant backgrounds (S-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848) using HA antibody. One lane represents 1 testis equivalent protein extract.
Fig 6.
Schematic structure of the Drosophila melanogaster sperm.