The tumor suppressor archipelago E3 ligase is required for spermatid differentiation in Drosophila testis.

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.

The SDHB Arg230His mutation causing familial paraganglioma alters glycolysis in a new Caenorhabditis elegans model.

The conserved B-subunit of succinate dehydrogenase (SDH) participates in the tricarboxylic acid cycle (TCA) cycle and mitochondrial electron transport. The Arg230His mutation in SDHB causes heritable pheochromocytoma/paraganglioma (PPGL). In Caenorhabditiselegans, we generated an in vivo PPGL model (SDHB-1 Arg244His; equivalent to human Arg230His), which manifests delayed development, shortened lifespan, attenuated ATP production and reduced mitochondrial number. Although succinate is elevated in both missense and null sdhb-1(gk165) mutants, transcriptomic comparison suggests very different causal mechanisms that are supported by metabolic analysis, whereby only Arg244His (not null) worms demonstrate elevated lactate/pyruvate levels, pointing to a missense-induced, Warburg-like aberrant glycolysis. In silico predictions of the SDHA-B dimer structure demonstrate that Arg230His modifies the catalytic cleft despite the latter's remoteness from the mutation site. We hypothesize that the Arg230His SDHB mutation rewires metabolism, reminiscent of metabolic reprogramming in cancer. Our tractable model provides a novel tool to investigate the metastatic propensity of this familial cancer and our approach could illuminate wider SDH pathology.This article has an associated First Person interview with the first author of the paper.

Cellular Immune Response Involving Multinucleated Giant Hemocytes with Two-Step Genome Amplification in the Drosophilid Zaprionus indianus.

Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

Deinococcus budaensis sp. nov., a mesophilic species isolated from a biofilm sample of a hydrothermal spring cave.

Following the exposure of a biofilm sample from a hydrothermal spring cave (Gellért Hill, Budapest, Hungary) to gamma radiation, a strain designated FeSTC15-38T was isolated and studied by polyphasic taxonomic methods. The spherical-shaped cells stained Gram-negative, and were aerobic and non-motile. The pH range for growth was pH 6.0-9.0, with an optimum at pH 7.0. The temperature range for growth was 20-37 °C, with an optimum at 28 °C. Phylogenetic analysis based on the 16S rRNA gene sequence of the isolate indicated that the organism belongs to the genus Deinococcus. The highest sequence similarities appeared with Deinococcus hopiensis KR-140T (94.1 %), Deinococcus aquaticus PB314T (93.3 %) and Deinococcus aerophilus 5516T-11T (92.7 %). The DNA G+C content of the novel strain was 68.2 mol%. The predominant fatty acids (>10 %) were iso-C16 : 0 and C16 : 1ω7c, and the cell-wall peptidoglycan type was A3ß l-Orn-Gly2-3, corroborating the assignment of the strain to the genus Deinococcus. Strain FeSTC15-38T contained MK-8 as the major menaquinone and several unidentified phospholipids, glycolipids and phosphoglycolipids. Resistance to gamma radiation (D10) of strain FeSTC15-38T was <3.0 kGy. According to phenotypic and genotypic data, strain FeSTC15-38T represents a novel species for which the name Deinococcus budaensis sp. nov. is proposed. The type strain is FeSTC15-38T (=NCAIM B.02630T=DSM 101791T).

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 paracrystalline 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 spermatogenesis. We show that a Minos element insertion in bb8 gene, a predicted glutamate dehydrogenase, 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 spermatids. Ultrastructural analysis of the cross section of elongated spermatids shows enlarged mitochondria and the production of paracrystalline in both major and minor mitochondrial 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.

Reduced expression of CDP-DAG synthase changes lipid composition and leads to male sterility in Drosophila.

Drosophila spermatogenesis is an ideal system to study the effects of changes in lipid composition, because spermatid elongation and individualization requires extensive membrane biosynthesis and remodelling. The bulk of transcriptional activity is completed with the entry of cysts into meiotic division, which makes post-meiotic stages of spermatogenesis very sensitive to even a small reduction in gene products. In this study, we describe the effect of changes in lipid composition during spermatogenesis using a hypomorphic male sterile allele of the Drosophila CDP-DAG synthase (CdsA) gene. We find that the CdsA mutant shows defects in spermatid individualization and enlargement of mitochondria and the axonemal sheath of the spermatids. Furthermore, we could genetically rescue the male sterile phenotype by overexpressing Phosphatidylinositol synthase (dPIS) in a CdsA mutant background. The results of lipidomic and genetic analyses of the CdsA mutant highlight the importance of correct lipid composition during sperm development and show that phosphatidic acid levels are crucial in late stages of spermatogenesis.

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects.

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.

How and why to study autophagy in Drosophila: it's more than just a garbage chute.

During the catabolic process of autophagy, cytoplasmic material is transported to the lysosome for degradation and recycling. This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles. Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival. Misregulation of autophagy is likely involved in numerous human pathologies including aging, cancer, infections and neurodegeneration, so its biomedical relevance explains the still growing interest in this field. Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila. We show examples for results obtained in different assays, explain how to interpret these with regard to autophagic activity, and how to find out which step of autophagy a given gene product is involved in.

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila.

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.

A novel actin binding site of myosin required for effective muscle contraction.

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Shared developmental roles and transcriptional control of autophagy and apoptosis in Caenorhabditis elegans.

Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells that, depending on the cellular milieu, can either promote survival or act as an alternative mechanism of programmed cell death (PCD) in terminally differentiated cells. Despite the important developmental and medical implications of autophagy and the main form of PCD, apoptosis, orchestration of their regulation remains poorly understood. Here, we show in the nematode Caenorhabditis elegans, that various genetic and pharmacological interventions causing embryonic lethality trigger a massive cell death response that has both autophagic and apoptotic features. The two degradation processes are also redundantly required for normal development and viability in this organism. Furthermore, the CES-2-like basic region leucine-zipper (bZip) transcription factor ATF-2, an upstream modulator of the core apoptotic cell death pathway, is able to directly regulate the expression of at least two key autophagy-related genes, bec-1/ATG6 and lgg-1/ATG8. Thus, the two cell death mechanisms share a common method of transcriptional regulation. Together, these results imply that under certain physiological and pathological conditions, autophagy and apoptosis are co-regulated to ensure the proper morphogenesis and survival of the developing organism. The identification of apoptosis and autophagy as compensatory cellular pathways in C. elegans might help us to understand how dysregulated PCD in humans can lead to diverse pathologies, including cancer, neurodegeneration and diabetes.

Bacillus aurantiacus sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from Hungarian soda lakes.

Three alkaliphilic and moderately halophilic strains designated K1-5T, K1-10 and B1-1, characterized by optimal growth at pH 9.0-10.0 and at 3-7 % (w/v) NaCl, were isolated from extremely shallow, alkaline soda lakes located in Hungary. Cells of the strains are Gram-positive, straight rods and form a central to subterminal, ellipsoidal endospore. The isolates are strictly aerobic, catalase-positive, oxidase-negative and contain a peptidoglycan of type A1 gamma based on meso-diaminopimelic acid. In strain K1-5T, menaquinone-7 (MK-7) is the predominant isoprenoid quinone and anteiso-C15 : 0 is the major cellular fatty acid. The DNA G+C content of strain K1-5T is 42.9 mol%. 16S rRNA gene-based phylogenetic analysis revealed that the strains exhibit levels of sequence similarity of less than 95.8 % to known Bacillus species. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus aurantiacus sp. nov. is proposed. The type strain is K1-5T (=DSM 18675T =CCM 7447T =NCAIM B002265T).

Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans.

Programmed cell death (PCD) is an essential and highly orchestrated process that plays a major role in morphogenesis and tissue homeostasis during development. In humans, defects in regulation or execution of cell death lead to diabetes, neurodegenerative disorders, and cancer. Two major types of PCD have been distinguished: the caspase-mediated process of apoptosis and the caspase-independent process involving autophagy. Although apoptosis and autophagy are often activated together in response to stress, the molecular mechanisms underlying their interplay remain unclear. Here we show that BEC-1, the C. elegans ortholog of the yeast and mammalian autophagy proteins Atg6/Vps30 and Beclin 1, is essential for development. We demonstrate that BEC-1 is necessary for the function of the class III PI3 kinase LET-512/Vps34, an essential protein required for autophagy, membrane trafficking, and endocytosis. Furthermore, BEC-1 forms a complex with the antiapoptotic protein CED-9/Bcl-2, and its depletion triggers CED-3/Caspase-dependent PCD. Based on our results, we propose that bec-1 represents a link between autophagy and apoptosis, thus supporting the view that the two processes act in concerted manner in the cell death machinery.

Pannonibacter phragmitetus gen. nov., sp. nov., a novel alkalitolerant bacterium isolated from decomposing reed rhizomes in a Hungarian soda lake.

Three alkalitolerant bacterial strains were isolated from the surface of decomposing rhizomes of reed [Phragmites australia (Cav.) Trin. et Steudel] in Lake Fertö (Hungary). Cells of the novel isolates were Gram-negative, motile rods and formed star-shaped aggregates. They were facultatively anaerobic and chemo-organotrophic. Bacteriochlorophyll a was not synthesized under aerobic conditions. The strains were catalase and oxidase positive, produced acid from D-glucose under aerobic and anaerobic conditions and reduced nitrate to nitrogen. They tolerated pH values from 7.0 to 11.0 and grew in the absence of NaCl as well as in up to 5% (w/v) NaCl. The G + C content of the DNA was 64.6 mol% and the major isoprenoid quinone was Q-10. The dominant cellular fatty acid was C18 : 1omega7c. The cell membrane contained phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl serine and one unknown phospholipid as polar lipids. Polyphasic taxonomic characterization revealed that strain C6/19T is most closely related to the Stappia-Roseibium cluster in the alpha-subclass of the Proteobacteria (showing 95.8-93.6% 16S rDNA sequence similarity). According to the phylogenetic and phenotypic evidence presented, a new genus and species is proposed, Pannonibacter phragmitetus gen. nov., sp. nov. The type strain is C6/19T (=DSM 14782T =NCAIM B02025T).