Spermitin: A Novel Mitochondrial Protein in Drosophila Spermatids (original) (raw)
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PLOS Genetics
Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of SLap proteins we show the characterization of classical mutants and RNAi lines affecting of the SLap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the SLap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that SLap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that SLap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.
Testis-Specific Bb8 Is Essential in the Development of Spermatid Mitochondria
Mitochondria are essential organelles of developing spermatids in Drosophila, which undergo dramatic changes in size and shape after meiotic division, where mitochondria localized in the cytoplasm, migrate near the nucleus, aggregate, fuse and create the Nebenkern. During spermatid elongation the two similar mitochondrial derivatives of the Nebenkern start to elongate parallel to the axoneme. One of the elongated mitochondrial derivatives starts to lose volume and becomes the minor mitochondrial derivative, while the other one accumulates paracrystalline and becomes the major mitochondrial derivative. Proteins and intracellular environment that are responsible for cyst elongation and paracrys-talline formation in the major mitochondrial derivative need to be identified. In this work we investigate the function of the testis specific big bubble 8 (bb8) gene during spermato-genesis. We show that a Minos element insertion in bb8 gene, a predicted glutamate dehy-drogenase, causes recessive male sterility. We demonstrate bb8 mRNA enrichment in spermatids and the mitochondrial localisation of Bb8 protein during spermatogenesis. We report that megamitochondria develop in the homozygous mutant testes, in elongating sper-matids. Ultrastructural analysis of the cross section of elongated spermatids shows enlarged mitochondria and the production of paracrystalline in both major and minor mito-chondrial derivatives. Our results suggest that the Bb8 protein and presumably glutamate metabolism has a crucial role in the normal development and establishment of the identity of the mitochondrial derivatives during spermatid elongation.
BMC cell biology, 2017
In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known...
Investigating spermatogenesis in Drosophila melanogaster
Methods, 2014
The process of spermatogenesis in Drosophila melanogaster provides a powerful model system to probe a variety of developmental and cell biological questions, such as the characterization of mechanisms that regulate stem cell behavior, cytokinesis, meiosis, and mitochondrial dynamics. Classical genetic approaches, together with binary expression systems, FRT-mediated recombination, and novel imaging systems to capture single cell behavior, are rapidly expanding our knowledge of the molecular mechanisms regulating all aspects of spermatogenesis. This methods chapter provides a detailed description of the system, a review of key questions chapter that have been addressed or remain unanswered thus far, and an introduction to tools and techniques available to probe each stage of spermatogenesis.
BMC Developmental Biology, 2010
Background: Mammals and Drosophila melanogaster share some striking similarities in spermatogenesis. Mitochondria in spermatids undergo dramatic morphological changes and syncytial spermatids are stripped from their cytoplasm and then individually wrapped by single membranes in an individualization process. In mammalian and fruit fly testis, components of the mitochondrial iron metabolism are expressed, but so far their function during spermatogenesis is unknown. Here we investigate the role of Drosophila mitoferrin (dmfrn), which is a mitochondrial carrier protein with an established role in the mitochondrial iron metabolism, during spermatogenesis.
Mitochondrial regulation in spermatogenesis
Reproduction
The classic roles of mitochondria in energy production, metabolism, and apoptosis have been well defined. However, a growing body of evidence suggests that mitochondria are also active players in regulating stem cell fate decision and lineage commitment via signaling transduction, protein modification, and epigenetic modulations. This is particularly interesting for spermatogenesis, during which germ cells demonstrate changing metabolic requirements across various stages of development. It is increasingly recognized that proper male fertility depends on exquisitely controlled plasticity of mitochondrial features, activities, and functional states. The unique role of mitochondria in germ cell ncRNA processing further adds another layer of complexity to mitochondrial regulation during spermatogenesis. In this review, we will discuss potential regulatory mechanisms of how mitochondria swiftly reshape their features, activities, and functions to support critical germ cell fate transitio...
Scientific Reports, 2021
The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. Here we present a previously unknown tissue-specific role of Ago in spermatid differentiation. We identified a classical mutant of ago which is semi-lethal and male-sterile. During the characterization of ago function in testis, we found that ago plays role in spermatid development, following meiosis. We confirmed spermatogenesis defects by silencing ago by RNAi in testes. The ago mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, we determined the subcellular localization of Ago protein with mCherry-Ago transgene in spermatids. Our findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology. Drosophila melanogaster testis is a particularly suitable model to follow the process of spermatogenesis 1,2. During Drosophila spermatogenesis from a single spermatogonia sixty-four sperms are being produced, through mitotic and meiotic cell divisions and intensive cellular remodeling. After each division, the daughter cells remain in connection with plasma bridges, which allows them to develop simultaneously in a syncytial cyst. The early transitamplifying mitotic divisions result in sixteen spermatocytes. Spermatocytes undergo a maturing process, they have high transcriptional activity until meiosis and the accumulated transcripts contribute to the development of the transcriptionally mostly inactive post-meiotic stages 3. After meiosis the mitochondria aggregate and fuse to establish the nebenkern structure, which consists of two mitochondrial derivatives. Next, the spermatids start to elongate to reach a full length of ~ 1.8 mm, which approximately 150 times longer than their initial diameter. The two mitochondrial derivatives run along the axoneme in the spermatid's tail and progressively differentiate. One of them becomes the major derivative with paracrystalline material accumulation, while the other one loses its volume and becomes the minor mitochondrial derivative. As part of the spermatid maturation, the nuclei also elongate while their volume decrease as the chromatin structure condensates and histones are replaced by protamines. Individualization, the process which establishes the individual sperms starts with the formation of the individualization complex (IC), which contains the F-actin-rich cone-shaped investment cones that are forming around the elongated nuclei. The IC migrates from the nuclei towards the tail end of the cyst, during its progression the majority of the cytosol and its content are displaced. An individual membrane starts to develop around each spermatid and a cystic bulge emerges. A non-apoptotic caspase activity facilitates the process of individualization 4. The caspase activity is mainly restricted to the cystic bulges and later in the waste bags at the end of the individualization 4,5. Remodeling of the round spermatocytes to functioning sperms is requiring strict regulation, selective protein degradation, and sufficient proteasome activity 6. There are multiple ubiquitin ligases known to have a role in the different steps of spermatogenesis (e.g.: Parkin, Ntc/Cul1/SkpA-SCF Ntc) 7,8 , and testis-specific proteasome subunits are emphasizing the ubiquitin-proteasome system's (UPS) role 9. E1, E2, and E3 orchestrate the ubiquitination of proteins inducing their degradation. While there are only a few types of E1 and E2 enzymes, many E3 enzymes were identified 10,11. E3 ligases can mark proteins for time and tissue-specific degradation therefore they have a role in many different cellular processes such as cell cycle, epigenetic regulation, mitophagy, etc. 12. Two E3 ligase complexes are necessary to activate the non-apoptotic caspase
PLoS ONE, 2011
In animals, male fertility requires the successful development of motile sperm. During Drosophila melanogaster spermatogenesis, 64 interconnected spermatids descended from a single germline stem cell are resolved into motile sperm in a process termed individualization. Here we identify a putative double-stranded RNA binding protein LUMP that is required for male fertility. lump 1 mutants are male-sterile and lack motile sperm due to defects in sperm individualization. We show that one dsRNA binding domains (dsRBD) is essential for LUMP function in male fertility. These findings reveal LUMP is a novel factor required for late stages of male germline differentiation.