Immune challenge induces N-terminal cleavage of the Drosophila serpin Necrotic

The Drosophila Necrotic protein is a serine proteinase inhibitor, which regulates the Toll-mediated innate immune response. Necrotic specifically inhibits an extracellular serine proteinase cascade leading to activation of the Toll ligand, Spätzle. Necrotic carries a polyglutamine extension amino-terminal to the core serpin structure. We show here that cleavage of this N-terminal extension occurs following immune challenge. This modification is blocked in PGRP-SA^semmelweiss mutants after Gram-positive bacterial challenge and in persephone mutants after fungal or Gram-positive bacterial challenge, indicating that activation of either of the Toll pathway upstream branches induces N-terminal cleavage of the serpin. The absolute requirement of persephone gene product for this cleavage indicates that Gram-positive bacteria activate a redundant set of proteinases upstream of Toll. Both full-length Necrotic and the core serpin are active inhibitors of a range of serine proteinases: the highest affinity being for cathepsin G and elastases. We found a 13-fold increase in the specificity of the core serpin over that of full-length Necrotic for one of the tested proteinases (porcine pancreatic elastase). This finding indicates that cleavage of the Necrotic amino-terminal extension might modulate Toll activation following the initial immune response.     

93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade

Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35–50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.     

Directed expression of the HIV-1 accessory protein Vpu in Drosophila fat-body cells inhibits Toll-dependent immune responses

Human immunodeficiency virus 1 (HIV-1) expresses several accessory proteins that manipulate various host-cell processes to achieve optimum replicative efficiency. One of them, viral protein U (Vpu), has been shown to interfere with the cellular degradation machinery through interaction with SCF^β-TrCP complexes. To learn more about Vpu function in vivo, we used the genetically tractable fruit fly, Drosophila melanogaster. Our results show that the directed expression of Vpu, but not the non-phosphorylated form, Vpu2/6, in fat-body cells affects Drosophila antimicrobial responses. In flies, the Toll and Imd pathways regulate antimicrobial-peptide gene expression. We show that Vpu specifically affects Toll pathway activation by inhibiting Cactus degradation. Given the conservation of the Toll/nuclear factor-κB (NF-κB) signalling pathways between flies and mammals, our results suggest a function for Vpu in the inhibition of host NF-κB-mediated innate immune defences and provide a powerful genetic approach for studying Vpu inhibition of NF-κB signalling in vivo.     

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.     

Persephone/Sp?tzle Pathogen Sensors Mediate the Activation of Toll Receptor Signaling in Response to Endogenous Danger Signals in Apoptosis-deficient Drosophila

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as damage-associated molecular patterns (DAMPs) and a noninfectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. In this study, we found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spätzle (Spz). We speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. We demonstrated that Toll signaling activation required the action of Persephone, a CLIP domain serine protease that usually reacts to microbial proteolytic activities. Our findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants.     

Protein degradation in silkworm peritracheal athrocytes and its physiological role in metamorphosis

The major functions of silkworm peritracheal athrocytes (nephrocytes) include endocytosis. Although athrocytes are also believed to function in protein degradation, there is limited experimental evidence for this. In this study, we detected the uptake and degradation of foreign proteins in peritracheal athrocytes by immunohistochemical, Western blot, and ex vivo analyses. IgG-FITC was detected in the athrocytes of silkworm larvae following injection, and LysoTracker analysis showed endosomal and lysosomal colocalizations. Athrocytes from larvae injected with IgG were incubated in Grace's medium for 2 days before being analyzed for the degradation of IgG by Western blotting. The level of incorporated IgG decreased and degradation products appeared following ex vivo culture. The highest level of IgG incorporation and degradation in the athrocytes was observed at the early pupal stage. The athrocytes also incorporated arylphorin, a major larval haemolymph protein and storage protein in silkworms. At the early pupal stage, arylphorin was actively degraded in the athrocytes. These results indicate that, in cooperation with the fat body, peritracheal athrocytes may function in the digestion of arylphorin during silkworm metamorphosis.     

Insects Can Count: Sensory Basis of Host Discrimination in Parasitoid Wasps Revealed

The solitary parasitoid Leptopilina heterotoma is one of the best studied organisms concerning the ecology, behaviour and physiology of host discrimination. Behavioural evidence shows that L. heterotoma uses its ovipositor to discriminate not only between parasitized and unparasitized Drosophila melanogaster larvae, but also to discriminate between hosts with different numbers of parasitoid eggs. The existing knowledge about how and when the parasitoid marks the host motivated us to unravel the chemosensory basis of host discrimination by L. heterotoma that allows it to choose the “best” host available. In this paper we report on electrophysiological recordings of multi-neural responses from the single taste sensillum on the tip of the unpaired ovipositor valve. We stimulated this sensillum with haemolymph of unparasitized, one-time-parasitized and two-times-parasitized Drosophila larvae. We demonstrate for the first time that quantitative characteristics of the neural responses to these haemolymph samples differed significantly, implying that host discrimination is encoded by taste receptor neurons in the multi-neuron coeloconic ovipositor sensillum. The activity of three of the six neurons present in the sensillum suffices for host discrimination and support the hypothesis that L. heterotoma females employ an ensemble code of parasitization status of the host.     

Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.     

CEP290 is essential for the initiation of ciliary transition zone assembly

Cilia play critical roles during embryonic development and adult homeostasis. Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. The evolutionarily conserved TZ component centrosomal protein 290 (CEP290) is the most frequently mutated human ciliopathy gene, but its roles in ciliogenesis are not completely understood. Here, we report that CEP290 plays an essential role in the initiation of TZ assembly in Drosophila. Mechanistically, the N-terminus of CEP290 directly recruits DAZ interacting zinc finger protein 1 (DZIP1), which then recruits Chibby (CBY) and Rab8 to promote early ciliary membrane formation. Complete deletion of CEP290 blocks ciliogenesis at the initiation stage of TZ assembly, which can be mimicked by DZIP1 deletion mutants. Remarkably, expression of the N-terminus of CEP290 alone restores the TZ localization of DZIP1 and subsequently ameliorates the defects in TZ assembly initiation in cep290 mutants. Our results link CEP290 to DZIP1-CBY/Rab8 module and uncover a previously uncharacterized important function of CEP290 in the coordination of early ciliary membrane formation and TZ assembly.

Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. A study in Drosophila reveals that the cilia TZ core protein CEP290 coordinates early ciliary membrane formation and TZ assembly; the N-terminus of CEP290 recruits DZIP1, which in turn recruits Rab8 and CBY to promote early ciliary membrane formation.     

Glycolipid Trafficking in Drosophila Undergoes Pathway Switching in Response to Aberrant Cholesterol Levels

In lipid storage diseases, the intracellular trafficking of sphingolipids is altered by conditions of aberrant cholesterol accumulation. Drosophila has been used recently to model lipid storage diseases, but the effects of sterol accumulation on sphingolipid trafficking are not known in the fly, and the trafficking of sphingolipids in general has not been studied in this model organism. Here, we examined the uptake and intracellular distribution of a fluorescent glycolipid analog, BODIPY-lactosyl-ceramide, in Drosophila neurons. The uptake mechanism and intracellular trafficking route of this simple glycolipid are largely conserved. Our principle finding is that cholesterol steers trafficking of the glycolipid between Golgi, lysosome, and recycling compartments. Our analyses support the idea that cholesterol storage in Drosophila triggers a switch in glycolipid trafficking from the biosynthetic to the degradative endolysosomal pathway, whereas cholesterol depletion eliminates recycling of the glycolipid. Unexpectedly, we observe a novel phenomenon we term “hijacking,” whereby lactosyl-ceramide diverts the trafficking pathway of an endocytic cargo, dextran, completely away from its lysosomal target. This work establishes that glycolipid trafficking in Drosophila undergoes changes similar to those seen in mammalian cells under conditions of cholesterol storage and therefore validates Drosophila as a suitable model organism in which to study lipid storage diseases.     

Pericardial cell ultrastructure in the tobacco hornworm: Manduca sexta L. (Lepidoptera: Sphingidae)

The ultrastructure of the pericardial athrocytes of fifth instar tobacco hornworm Manduca sexta L. (Lepidoptera: Sphingidae) was examined by transmission electron microscopy. These cells are highly specialized for the maintenance of hemolymph homeostasis by synthesis and secretion of some proteins, and uptake and degradation of others. We observed a striking radial zonation with numerous labyrinthine channels extending into the cell providing a large surface area for enhanced efficiency of endocytosis. Granular material was imported into the endosomal compartment by fusion of endocytotic vesicles from the basal region. Anatomical evidence supports the hypothesis that proteins are transported from the endosome to the lysosome, by maturation of the early endosome to form the late endosome/lysosome, as opposed to vesicular transport. The presence of catalase in athrocyte lysosomes is reported for the first time. Endocytosis in the athrocytes of insects is compared with endocytosis in vertebrate systems.     

Molecular Mechanism That Induces Activation of Sp?tzle,the Ligand for the Drosophila Toll Receptor

The Drosophila Toll receptor is activated by an endogenous cytokine ligand Spätzle. Active ligand is generated in response to positional cues in embryonic dorso-ventral patterning and microbial pathogens in the insect immune response. Spätzle is secreted as a pro-protein and is processed into an active form by the serine endoproteases Easter and Spätzle-processing enzyme during dorso-ventral patterning and infection, respectively. Here, we provide evidence for the molecular mechanism of this activation process. We show that the Spätzle prodomain masks a predominantly hydrophobic region of Spätzle and that proteolysis causes a conformational change that exposes determinants that are critical for binding to the Toll receptor. We also gather that a conserved sequence motif in the prodomain presents features of an amphipathic helix likely to bind a hydrophobic cleft in Spätzle thereby occluding the putative Toll binding region. This mechanism of activation has a striking similarity to that of coagulogen, a clotting factor of the horseshoe crab, an invertebrate that has changed little in 400 million years. Taken together, our findings demonstrate that an ancient passive defense system has been adapted during evolution and converted for use in a critical pathway of innate immune signaling and embryonic morphogenesis.     

Dual comprehensive approach to decipher the Drosophila Toll pathway, ex vivo RNAi screenings and immunoprecipitation-mass spectrometry

The Drosophila Toll pathway is involved in embryonic development, innate immunity, and cell-cell interactions. However, compared to the mammalian Toll-like receptor innate immune pathway, its intracellular signaling mechanisms are not fully understood. We have previously performed a series of ex vivo genome-wide RNAi screenings to identify genes required for the activation of the Toll pathway. In this study, we have conducted an additional genome-wide RNAi screening using the overexpression of Tube, an adapter molecule in the Toll pathway, and have performed a co-immunoprecipitation assay to identify components present in the dMyd88-Tube complex. Based on the results of these assays, we have performed a bioinformatic analysis, and describe candidate molecules and post-translational modifications that could be involved in Drosophila Toll signaling.     

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the “short loop” recycling marker Rab4, and little co-localization with the marker of “long loop” recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the β₂-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.     

Rab GTPases regulating receptor trafficking at the late endosome–lysosome membranes

Lysosomes serve key degradative functions for the turnover of membrane lipids and protein components. Its biogenesis is principally dependent on exocytic traffic from the late endosome via the trans‐Golgi network, and it also receives cargo to be degraded from the endocytic pathway. Membrane trafficking to the late endosome–lysosome is tightly regulated to maintain the amplitude of signalling events and cellular homeostasis. Key coordinators of lysosomal traffic include members of the Rab small GTPase family. Amongst these, Rab7, Rab9 and the more recently studied Rab22B/31 have all been reported to regulate membrane trafficking processed at the late endosome–lysosome system. We discuss what is known about the roles of these Rab proteins and their interacting partners on the regulation of traffic of important receptor proteins such as the epidermal growth factor receptor (EGFR) and the mannose 6‐phosphate receptor (M6PR), in association with the late endosome–lysosome system. Better knowledge of EGFR and M6PR traffic in this regard may aid in understanding the pathological processes, such as oncogenic transformations associated with these receptors. Copyright © 2012 John Wiley & Sons, Ltd.     

Delipidation of insect lipoprotein,lipophorin, affects its binding to the lipophorin receptor,LpR: Implications for the role of LpR-mediated endocytosis

The insect lipophorin receptor (LpR), an LDL receptor (LDLR) homologue that is expressed during restricted periods of insect development, binds and endocytoses high-density lipophorin (HDLp). However, in contrast to LDL, HDLp is not lysosomally degraded, but recycled in a transferrin-like manner, leaving a function of receptor-mediated uptake of HDLp to be uncovered. Since a hallmark of circulatory HDLp is its ability to function as a reusable shuttle that selectively loads and unloads lipids at target tissues without being endocytosed or degraded, circulatory HDLp can exist in several forms with respect to lipid loading. To investigate whether lipid content of the lipoprotein affects binding and subsequent endocytosis by LpR, HDLp was partially delipidated in vitro by incubation with α-cyclodextrin, yielding a particle of buoyant density 1.17 g/mL (HDLp-1.17). Binding experiments demonstrated that LpR bound HDLp-1.17 with a substantially higher affinity than HDLp both in LpR-transfected Chinese hamster ovary (CHO) cells and isolated insect fat body tissue endogenously expressing LpR. Similar to HDLp, HDLp-1.17 was targeted to the endocytic recycling compartment after endocytosis in CHO(LpR) cells. The complex of HDLp-1.17 and LpR appeared to be resistant to endosomal pH, as was recently demonstrated for the LpR–HDLp complex, corroborating that HDLp-1.17 is recycled similar to HDLp. This conclusion was further supported by the observation of a significant decrease with time of HDLp-1.17-containing vesicles after endocytosis of HDLp-1.17 in LpR-expressing insect fat body tissue. Collectively, our results indicate that LpR favors the binding and subsequent endocytosis of HDLp-1.17 over HDLp, suggesting a physiological role for LpR in selective endocytosis of relatively lipid-unloaded HDLp particles, while lipid reloading during their intracellular itinerary might result in decreased affinity for LpR and thus allows recycling.