C. elegans screen identifies autophagy genes specific to multicellular organisms.

The molecular understanding of autophagy has originated almost exclusively from yeast genetic studies. Little is known about essential autophagy components specific to higher eukaryotes. Here we perform genetic screens in C. elegans and identify four metazoan-specific autophagy genes, named epg-2, -3, -4, and -5. Genetic analysis reveals that epg-2, -3, -4, and -5 define discrete genetic steps of the autophagy pathway. epg-2 encodes a coiled-coil protein that functions in specific autophagic cargo recognition. Mammalian homologs of EPG-3/VMP1, EPG-4/EI24, and EPG-5/mEPG5 are essential for starvation-induced autophagy. VMP1 regulates autophagosome formation by controlling the duration of omegasomes. EI24 and mEPG5 are required for formation of degradative autolysosomes. This study establishes C. elegans as a multicellular genetic model to delineate the autophagy pathway and provides mechanistic insights into the metazoan-specific autophagic process.

Sperm-Leucylaminopeptidases are required for male fertility as structural components of mitochondrial paracrystalline material in Drosophila melanogaster sperm.

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 S-Lap proteins we show the characterization of classical mutants and RNAi lines affecting of the S-Lap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the S-Lap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that S-Lap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that S-Lap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.

Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion.

The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process.

Anaerobacillus alkaliphilus sp. nov., a novel alkaliphilic and moderately halophilic bacterium.

Two alkaliphilic and moderately halophilic bacterial strains B16-10T and Z23-18 characterized by optimal growth at pH 9.0-10.0 and 5 % (w/v) NaCl, were isolated from the rhizosphere soil of the bayonet grass (Bolboschoenus maritimus) in the Kiskunság National Park, Hungary. Cells of both strains stained Gram-positive, were motile straight rods, and formed terminal, ellipsoidal endospores with swollen sporangia. The isolates were facultative anaerobic, catalase positive, oxidase negative. Both strains contained meso-diaminopimelic acid as diagnostic diaminoacid of the peptidoglycan. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone. Anteiso-C15 : 0, C16 : 1ω11c and iso-C14 : 0 were the major cellular fatty acids. The DNA G+C content of both strains was 35.8 mol%. The 16S rRNA gene based phylogenetic analysis revealed that the facultative anaerobic strains B16-10T and Z23-18 showed the highest similarities to the type strains of anaerobic Anaerobacillus isosaccharinicus NB2006T (98.7 and 99.1 %), A. macyae JMM-4T (98.2 and 98.4 %), A. alkalidiazotrophicus MS 6T (97.7 and 98.4 %), A. alkalilacustris Z-0521T (97.5 and 98.3 %) and A. arseniciselenatis DSM 15340T (97.5 and 98.2 %). However, the distinctive phenotypic and genetic results of this study confirmed that strains B16-10T and Z23-18 represent a novel species, for which the name Anaerobacillus alkaliphilus sp. nov. is proposed. The type strain is B16-10T (=DSM 29790T=NCAIM B 02608T).

Bacillus kiskunsagensis sp. nov., a novel alkaliphilic and moderately halophilic bacterium isolated from soda soil.

An alkaliphilic and moderately halophilic strain characterized by optimal growth at pH 9.0-10.0 and 7 % (w/v) NaCl, and designated B16-24T, was isolated from the rhizosphere soil of the bayonet grass Bolboschoenus maritimus at a soda pond in the Kiskunság National Park, Hungary. Cells of the strain were Gram-staining-positive, non-motile, straight rods, and formed central, ellipsoidal endospores with slightly swollen sporangia. The isolate was facultative anaerobic, catalase positive, oxidase negative, and contained a peptidoglycan of type A1γ based on meso-diaminopimelic acid. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone, and anteiso-C15 : 0 the major cellular fatty acid. The DNA G+C content of strain B16-24T was 36.6 mol%. The 16S rRNA gene-based phylogenetic analysis revealed that the novel isolate had the greatest similarities to the type strains of Bacillus okhensis Kh10-101T (97.8 %), B. akibai 1139T (97.4 %), B. alkalisediminis K1-25T (97.3 %) and B. wakoensis N-1T (97.1 %). The DNA-DNA relatedness of strain B16-24T and the closely related Bacillus species ranged between 24±6 % and 35±3 %. The distinctive phenotypic and genetic results of this study confirmed that strain B16-24T represents a novel species within the genus Bacillus, for which the name Bacillus kiskunsagensis sp. nov. is proposed. The type strain is B16-24T (=DSM 29791T=NCAIM B.02610T).

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).

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.

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.

Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids.

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, we showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. Here, we used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, we used RNA sequencing to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.

Bacillus alkalisediminis sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from sediment of extremely shallow soda ponds.

Alkaliphilic strains characterized by optimal growth at pH 9.0 and 5 % (w/v) NaCl designated K1-25(T) and H3-93 were isolated from extremely shallow soda ponds located in Hungary. Cells of both strains were Gram-stain-positive, non-motile, straight rods and formed central, ellipsoidal endospores with swollen sporangia. The isolates were aerobic, catalase-positive, oxidase-negative and contained a peptidoglycan of type A1γ based on meso-diaminopimelic acid. In both strains, menaquinone-7 (MK-7) was the predominant isoprenoid quinone and the major cellular fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0). The DNA G+C contents of strains K1-25(T) and H3-93 were 39.0 and 36.3 mol%, respectively. 16S rRNA gene sequence-based phylogenetic analysis revealed 99.2 % similarity between strains K1-25(T) and H3-93 and the novel isolates had the highest similarities to Bacillus akibai 1139(T) (97.8 and 98.3 %, respectively), Bacillus wakoensis N-1(T) (97.0 and 97.4 %), Bacillus okhensis Kh10-101(T) (97.1 and 97.4 %) and Bacillus krulwichiae AM31D(T) (96.9 and 97.1 %). DNA-DNA hybridization between our strains and the type strains of closely related Bacillus species was lower than 70 %. Although DNA-DNA hybridization between strains K1-25(T) and H3-93 was 27 %, the phenotypic and chemotaxonomic data did not support the differentiation of these two strains into separate species. Therefore, they represent genomovars of a novel species, for which the name Bacillus alkalisediminis sp. nov. is proposed. The type strain is K1-25(T) ( = DSM 21670(T)  = NCAIM B02301(T)).

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.

Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles.

Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility.

Langmuir and Langmuir-Blodgett films of bidisperse silica nanoparticles.

We present the studies on the structure and optical properties of bidisperse Stöber silica nanoparticulate Langmuir films prepared at the air/water interface in a Wilhelmy film balance and transferred onto glass slides using the Langmuir-Blodgett technique. Three different compositions (covered area ratios: 4:1; 1:1, and 1:4) of two bidisperse systems were used in the experiments. Bidisperse samples (B1 and B2) were prepared by mixing the appropriate amount of monodisperse sols of particles with 61 and 100 nm diameters (B1) and those with 37 and 100 nm diameters (B2). By surface pressure-area isotherms and (transmission and scanning) electron microscopy images we provide information about the structure of the films. Optical properties of the supported films were measured with UV-vis spectroscopy and the transmittance spectra were evaluated in terms of an optical model which allows monotonous in-depth variation of the refractive index across the film. (1) We have found that the refractive index decreased from the substrate-layer interface toward the air-layer interface when the smaller particles were in majority, and increased otherwise. That would suggest that the smaller particles of each bidisperse system can be positioned at the air side of the film if they are in minority in the sample and they can be situated on the substrate if they are in majority. The scanning electron microscope images of bidisperse films supported the in-depth film structure suggested by optical studies.

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 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.

Drosophila Arl8 is a general positive regulator of lysosomal fusion events.

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. Here we show that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, we demonstrate that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor.

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.

Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans.

Here we show that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number. Furthermore, loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutant animals with aberrant insulin-like growth factor-1 (insulin/IGF-1) or transforming growth factor-beta (TGF-beta) signaling. This function for unc-51 and bec-1 in cell size control and their interaction with these two growth modulatory pathways may represent a link between the hormonal and nutritional regulation of cell growth.

Effects of sex and insulin/insulin-like growth factor-1 signaling on performance in an associative learning paradigm in Caenorhabditis elegans.

Learning is an adaptive change in behavior in response to environmental stimuli. In mammals, there is a distinct female bias to learn skills that is still unprecedented in other animal taxa. Here we have investigated the biological determinants of performance in an associative learning paradigm in the nematode Caenorhabditis elegans. Using an assay of chemotactic reactions associated with food deprivation, wild-type male worms show inferior learning ability relative to hermaphrodites. Sex-based learning difference is therefore an ancient evolutionary feature appearing even in relatively simple animals. C. elegans mutants with reduced insulin/IGF-1 signaling also exhibit a greatly reduced learning ability in this assay. In addition, hyperactivation of insulin/IGF-1 signaling through loss-of-function mutations in the PTEN phosphatase daf-18, a negative regulator of insulin/IGF-1 signaling, enhances learning ability beyond that of wild type. According to our epistasis analysis, the effect of DAF-2 on learning acts via phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production, but not the DAF-16 FOXO transcription factor. This implies that the signaling pathway from DAF-2 affecting this learning paradigm branches between PIP(3) production and DAF-16. However, learning capacity of nematodes is lowered by loss-of-function mutations in daf-16, suggesting involvement of noninsulin/IGF-1 signaling-dependent DAF-16 activation in learning. Potentially, sex and insulin/IGF-1 signaling affect performance in this learning assay via effects on the neurobiology of learning.