PtdIns4P is required for the autophagosomal recruitment of STX17 (syntaxin 17) to promote lysosomal fusion.

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.

Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes.

Post-transcriptional gene silencing using double-stranded RNA has revolutionized the field of functional genetics, allowing fast and easy disruption of gene function in various organisms. In Drosophila, many transgenic RNAi lines have been generated in large-scale efforts, including the Drosophila Transgenic RNAi Project (TRiP), to facilitate in vivo knockdown of virtually any Drosophila gene with spatial and temporal resolution. The available transgenic RNAi lines represent a fundamental resource for the fly community, providing an unprecedented opportunity to address a vast range of biological questions relevant to basic and biomedical research fields. However, caution should be applied regarding the efficiency and specificity of the RNAi approach. Here, we demonstrate that pVALIUM10-based RNAi lines, representing ∼13% of the total TRiP collection (1,808 of 13,410 pVALIUM TRiP-based RNAi lines), cause unintended off-target silencing of transgenes expressed from Gateway destination vectors. The silencing is mediated by targeting attB1 and attB2 sequences generated via site-specific recombination and included in the transcribed mRNA. Deleting these attB sites from the Gateway expression vector prevents silencing and restores expected transgene expression.

Peeling Back the Layers of Lymph Gland Structure and Regulation.

During the past 60 years, the fruit fly, Drosophila melanogaster, has proven to be an excellent model to study the regulation of hematopoiesis. This is not only due to the evolutionarily conserved signalling pathways and transcription factors contributing to blood cell fate, but also to convergent evolution that led to functional similarities in distinct species. An example of convergence is the compartmentalization of blood cells, which ensures the quiescence of hematopoietic stem cells and allows for the rapid reaction of the immune system upon challenges. The lymph gland, a widely studied hematopoietic organ of the Drosophila larva, represents a microenvironment with similar features and functions to classical hematopoietic stem cell niches of vertebrates. Lymph gland studies were effectively supported by the unparalleled toolkit developed in Drosophila, which enabled the high-resolution investigation of the cellular composition and regulatory interaction networks of the lymph gland. In this review, we summarize how our understanding of lymph gland structure and hematopoietic cell-to-cell communication evolved during the past decades and compare their analogous features to those of the vertebrate hematopoietic stem cell niche.

Selective autophagy and Golgi quality control in <i>Drosophila</i>.

The LIR motif-docking site (LDS) of Atg8/LC3 proteins is essential for the binding of LC3-interacting region (LIR)-containing proteins and their subsequent degradation by macroautophagy/autophagy. In our recent study, we created a mutated LDS site in Atg8a, the <i>Drosophila</i> homolog of Atg8/LC3 and found that LDS mutants accumulate known autophagy substrates and have reduced lifespan. We also conducted quantitative proteomics analyses and identified several proteins that are enriched in the LDS mutants, including Gmap (Golgi microtubule-associated protein). Gmap contains a LIR motif and accumulates in LDS mutants. We showed that Gmap and Atg8a interact in a LIR-LDS dependent manner and that the Golgi size and morphology are altered in Atg8a-LDS and Gmap-LIR motif mutants. Our findings highlight a role for Gmap in the regulation of Golgiphagy.

GMAP is an Atg8a-interacting protein that regulates Golgi turnover in Drosophila.

Selective autophagy receptors and adapters contain short linear motifs called LIR motifs (LC3-interacting region), which are required for the interaction with the Atg8-family proteins. LIR motifs bind to the hydrophobic pockets of the LIR motif docking site (LDS) of the respective Atg8-family proteins. The physiological significance of LDS docking sites has not been clarified in vivo. Here, we show that Atg8a-LDS mutant Drosophila flies accumulate autophagy substrates and have reduced lifespan. Using quantitative proteomics to identify the proteins that accumulate in Atg8a-LDS mutants, we identify the cis-Golgi protein GMAP (Golgi microtubule-associated protein) as a LIR motif-containing protein that interacts with Atg8a. GMAP LIR mutant flies exhibit accumulation of Golgi markers and elongated Golgi morphology. Our data suggest that GMAP mediates the turnover of Golgi by selective autophagy to regulate its morphology and size via its LIR motif-mediated interaction with Atg8a.

The mechanosensor Filamin A/Cheerio promotes tumourigenesis via specific interactions with components of the cell cortex.

Cancer development has been linked to aberrant sensing and interpretation of mechanical cues and force-generating properties. Here, we show that upregulation of the actin crosslinking protein Cheerio (Cher), the fly ortholog of Filamin A (FLNA), and the conformation of its mechanosensitive region (MSR) are instrumental to the malignancy of polarity-deficient, Ras-driven tumours in Drosophila epithelia. We demonstrate that impaired growth and cytoskeletal contractility of tumours devoid of cher can be rescued by stimulating myosin activity. Profiling the Cher interactome in tumour-bearing imaginal discs identified several components of the cell cortex, including the ß-heavy Spectrin Karst (Kst), the scaffolding protein Big bang (Bbg), and 14-3-3ε. We show that Cher binds Bbg through the MSR while the interaction with 14-3-3ε and Kst is MSR-independent. Importantly, our genetic studies define Bbg, Kst, and 14-3-3ε as tumour suppressors. The tumour-promoting function of Cher thus relies on its capacity to control the contractile state of the cytoskeleton through interactions with myosin and specific components of the cell cortex.

A Novel Method for Primary Blood Cell Culturing and Selection in Drosophila melanogaster.

The blood cells of the fruit fly Drosophila melanogaster show many similarities to their vertebrate counterparts, both in their functions and their differentiation. In the past decades, a wide palette of immunological and transgenic tools and methods have been developed to study hematopoiesis in the Drosophila larva. However, the in vivo observation of blood cells is technically restricted by the limited transparency of the body and the difficulty in keeping the organism alive during imaging. Here we describe an improved ex vivo culturing method that allows effective visualization and selection of live blood cells in primary cultures derived from Drosophila larvae. Our results show that cultured hemocytes accurately represent morphological and functional changes following immune challenges and in case of genetic alterations. Since cell culturing has hugely contributed to the understanding of the physiological properties of vertebrate blood cells, this method provides a versatile tool for studying Drosophila hemocyte differentiation and functions ex vivo.

Ion Channels and Pumps in Autophagy: A Reciprocal Relationship.

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP3Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps-including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases-in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases.

There and back again: The mechanisms of differentiation and transdifferentiation in Drosophila blood cells.

Transdifferentiation is a conversion of an already differentiated cell type into another cell type without the involvement of stem cells. This transition is well described in the case of vertebrate immune cells, as well as in Drosophila melanogaster, which therefore serves as a suitable model to study the process in detail. In the Drosophila larva, the latest single-cell sequencing methods enabled the clusterization of the phagocytic blood cells, the plasmatocytes, which are capable of transdifferentiation into encapsulating cells, the lamellocytes. Here we summarize the available data of the past years on the plasmatocyte-lamellocyte transition, and make an attempt to harmonize them with transcriptome-based blood cell clustering to better understand the underlying mechanisms of transdifferentiation in Drosophila, and in general.

Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments.

Blood development in multicellular organisms relies on specific tissue microenvironments that nurture hematopoietic precursors and promote their self-renewal, proliferation, and differentiation. The mechanisms driving blood cell homing and their interactions with hematopoietic microenvironments remain poorly understood. Here, we use the Drosophila melanogaster model to reveal a pivotal role for basement membrane composition in the formation of hematopoietic compartments. We demonstrate that by modulating extracellular matrix components, the fly blood cells known as hemocytes can be relocated to tissue surfaces where they function similarly to their natural hematopoietic environment. We establish that the Collagen XV/XVIII ortholog Multiplexin in the tissue-basement membranes and the phagocytosis receptor Eater on the hemocytes physically interact and are necessary and sufficient to induce immune cell-tissue association. These results highlight the cooperation of Multiplexin and Eater as an integral part of a homing mechanism that specifies and maintains hematopoietic sites in Drosophila.

Identification of reference markers for characterizing honey bee (Apis mellifera) hemocyte classes.

Cell mediated immunity of the honey bee (Apis mellifera) involves the activity of several hemocyte populations, currently defined by morphological features and lectin binding characteristics. The objective of the present study was to identify molecular markers capable of characterizing subsets of honey bee hemocytes. We developed and employed monoclonal antibodies with restricted reactions to functionally distinct hemocyte subpopulations. Melanizing cells, known as oenocytoids, were defined by an antibody to prophenoloxidase, aggregating cells were identified by the expression of Hemolectin, and phagocytic cells were identified by a marker expressed on granulocytes. We anticipate that this combination of antibodies not only allows for the detection of functionally distinct hemocyte subtypes, but will help to further the exploration of hematopoietic compartments, as well as reveal details of the honey bee cellular immune defense against parasites and microbes.

Headcase is a Repressor of Lamellocyte Fate in Drosophila melanogaster.

Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.

JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress.

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation.

Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture.

Interplay between apicobasal cell polarity modules and the cytoskeleton is critical for differentiation and integrity of epithelia. However, this coordination is poorly understood at the level of gene regulation by transcription factors. Here, we establish the Drosophila activating transcription factor 3 (atf3) as a cell polarity response gene acting downstream of the membrane-associated Scribble polarity complex. Loss of the tumor suppressors Scribble or Dlg1 induces atf3 expression via aPKC but independent of Jun-N-terminal kinase (JNK) signaling. Strikingly, removal of Atf3 from Dlg1 deficient cells restores polarized cytoarchitecture, levels and distribution of endosomal trafficking machinery, and differentiation. Conversely, excess Atf3 alters microtubule network, vesicular trafficking and the partition of polarity proteins along the apicobasal axis. Genomic and genetic approaches implicate Atf3 as a regulator of cytoskeleton organization and function, and identify Lamin C as one of its bona fide target genes. By affecting structural features and cell morphology, Atf3 functions in a manner distinct from other transcription factors operating downstream of disrupted cell polarity.

Hemolectin expression reveals functional heterogeneity in honey bee (Apis mellifera) hemocytes.

The identification of molecular markers considerably facilitated the classification and functional analysis of blood cell types. Apis mellifera hemocytes have been classified by morphological criteria and lectin binding properties; however, the use of molecular markers has been minimal. Here we describe a monoclonal antibody to a non-phagocytic subpopulation of A. mellifera hemocytes and to a constituent of the hemolymph clot. We demonstrate that the antibody identifies the A. mellifera hemolectin, a protein carrying human von Willebrand factor homology domains, characteristic of proteins involved in blood coagulation and platelet aggregation in mammals. Hemolectin expressing A. mellifera hemocytes contain the protein as cytoplasmic granules and contribute to the formation of a protein matrix, building up around foreign particles. Consequently, hemolectin as a marker molecule reveals a clear functional heterogeneity of hemocytes, allowing for the analytical separation of hemocyte classes, and could promote the molecular identification of hemocyte lineages in A. mellifera.

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila.

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.

The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster.

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.

Multinucleated Giant Hemocytes Are Effector Cells in Cell-Mediated Immune Responses of Drosophila.

We identified and characterized a so far unrecognized cell type, dubbed the multinucleated giant hemocyte (MGH), in the ananassae subgroup of Drosophilidae. Here, we describe the functional and ultrastructural characteristics of this novel blood cell type as well as its characterization with a set of discriminative immunological markers. MGHs are encapsulating cells that isolate and kill the parasite without melanization. They share some properties with but differ considerably from lamellocytes, the encapsulating cells of Drosophila melanogaster, the broadly used model organism in studies of innate immunity. MGHs are nonproliferative effector cells that are derived from phagocytic cells of the sessile tissue and the circulation, but do not exhibit phagocytic activity. In contrast to lamellocytes, MGHs are gigantic cells with filamentous projections and contain many nuclei, which are the result of the fusion of several cells. Although the structure of lamellocytes and MGHs differ remarkably, their function in the elimination of parasites is similar, which is potentially the result of the convergent evolution of interactions between hosts and parasites in different geographic regions. MGHs are highly motile and share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation.

In vivo immunostaining of hemocyte compartments in Drosophila for live imaging.

In recent years, Drosophila melanogaster has become an attractive model organism in which to study the structure and development of the cellular immune components. The emergence of immunological markers greatly accelerated the identification of the immune cells (hemocytes), while the creation of genetic reporter constructs allowed unique insight into the structural organization of hematopoietic tissues. However, investigation of the hemocyte compartments by the means of immunological markers requires dissection and fixation, which regularly disrupt the delicate structure and hamper the microanatomical characterization. Moreover, the investigation of transgenic reporters alone can be misleading as their expression often differs from the native expression pattern of their respective genes. We describe here a method that combines the reporter constructs and the immunological tools in live imaging, thereby allowing use of the array of available immunological markers while retaining the structural integrity of the hematopoietic compartments. The procedure allows the reversible immobilization of Drosophila larvae for high-resolution confocal imaging and the time-lapse video analysis of in vivo reporters. When combined with our antibody injection-based in situ immunostaining assay, the resulting double labeling of the hemocyte compartments can provide new information on the microanatomy and functional properties of the hematopoietic tissues in an intact state. Although this method was developed to study the immune system of Drosophila melanogaster, we anticipate that such a combination of genetic and immunological markers could become a versatile technique for in vivo studies in other biological systems too.