Thylakoid membrane stacking controls electron transport mode during the dark-to-light transition by adjusting the distances between PSI and PSII.

The balance between linear electron transport (LET) and cyclic electron transport (CET) plays an essential role in plant adaptation and protection against photo-induced damage. This balance is largely maintained by phosphorylation-driven alterations in the PSII-LHCII assembly and thylakoid membrane stacking. During the dark-to-light transition, plants shift this balance from CET, which prevails to prevent overreduction of the electron transport chain and consequent photo-induced damage, towards LET, which enables efficient CO2 assimilation and biomass production. Using freeze-fracture cryo-scanning electron microscopy and transmission electron microscopy of Arabidopsis leaves, we reveal unique membrane regions possessing characteristics of both stacked and unstacked regions of the thylakoid network that form during this transition. A notable consequence of the morphological attributes of these regions, which we refer to as 'stacked thylakoid doublets', is an overall increase in the proximity and connectivity of the two photosystems (PSI and PSII) that drive LET. This, in turn, reduces diffusion distances and barriers for the mobile carriers that transfer electrons between the two PSs, thereby maximizing LET and optimizing the plant's ability to utilize light energy. The mechanics described here for the shift between CET and LET during the dark-to-light transition are probably also used during chromatic adaptation mediated by state transitions.

MTCH2 cooperates with MFN2 and lysophosphatidic acid synthesis to sustain mitochondrial fusion.

Fusion of the outer mitochondrial membrane (OMM) is regulated by mitofusin 1 (MFN1) and 2 (MFN2), yet the differential contribution of each of these proteins is less understood. Mitochondrial carrier homolog 2 (MTCH2) also plays a role in mitochondrial fusion, but its exact function remains unresolved. MTCH2 overexpression enforces MFN2-independent mitochondrial fusion, proposedly by modulating the phospholipid lysophosphatidic acid (LPA), which is synthesized by glycerol-phosphate acyl transferases (GPATs) in the endoplasmic reticulum (ER) and the OMM. Here we report that MTCH2 requires MFN1 to enforce mitochondrial fusion and that fragmentation caused by loss of MTCH2 can be specifically counterbalanced by overexpression of MFN2 but not MFN1, partially independent of its GTPase activity and mitochondrial localization. Pharmacological inhibition of GPATs (GPATi) or silencing ER-resident GPATs suppresses MFN2's ability to compensate for the loss of MTCH2. Loss of either MTCH2, MFN2, or GPATi does not impair stress-induced mitochondrial fusion, whereas the combined loss of MTCH2 and GPATi or the combined loss of MTCH2 and MFN2 does. Taken together, we unmask two cooperative mechanisms that sustain mitochondrial fusion.

Parallel evolution of cannabinoid biosynthesis.

Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.

Cellular and metabolic characteristics of pre-leukemic hematopoietic progenitors with GATA2 haploinsufficiency.

Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as Gata2 heterozygote mice do not develop hematologic malignancies. Here we show that two different germline Gata2 mutations (TgErg/Gata2het and TgErg/Gata2L359V) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of Erg/Gata2het fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring GATA2 aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline GATA2 mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline Gata2 genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with GATA2 germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.

Neonatal Enthesis Healing Involves Noninflammatory Acellular Scar Formation through Extracellular Matrix Secretion by Resident Cells.

Wound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

Epiblast fragmentation by shedding-a novel mechanism to eliminate cells in post-implantation mouse embryos.

The role of programmed cell death during embryonic development has been described previously, but its specific contribution to peri- and post-implantation stages is still debatable. Here, we used transmission electron microscopy and immunostaining of E5.5-7.5 mouse embryos to investigate death processes during these stages of development. We report that in addition to canonical apoptosis observed in E5.5-E7.5 embryos, a novel type of cell elimination occurs in E7.5 embryos among the epiblasts at the apical side, in which cells shed membrane-enclosed fragments of cytosol and organelles into the lumen, leaving behind small, enucleated cell remnants at the apical surface. This process is caspase-independent as it occurred in Apaf1 knockout embryos. We suggest that this novel mechanism controls epiblast cell numbers. Altogether, this work documents the activation of two distinct programs driving irreversible terminal states of epiblast cells in the post-implantation mouse embryo.

Toxicity of Water- and Organic-Soluble Wood Tar Fractions from Biomass Burning in Lung Epithelial Cells.

Widespread smoke from wildfires and biomass burning contributes to air pollution and the deterioration of air quality and human health. A common and major emission of biomass burning, often found in collected smoke particles, is spherical wood tar particles, also known as "tar balls". However, the toxicity of wood tar particles and the mechanisms that govern their health impacts and the impact of their complicated chemical matrix are not fully elucidated. To address these questions, we generated wood tar material from wood pyrolysis and isolated two main subfractions: water-soluble and organic-soluble fractions. The chemical characteristics as well as the cytotoxicity, oxidative damage, and DNA damage mechanisms were investigated after exposure of A549 and BEAS-2B lung epithelial cells to wood tar. Our results suggest that both wood tar subfractions reduce cell viability in exposed lung cells; however, these fractions have different modes of action that are related to their physicochemical properties. Exposure to the water-soluble wood tar fraction increased total reactive oxygen species production in the cells, decreased mitochondrial membrane potential (MMP), and induced oxidative damage and cell death, probably through apoptosis. Exposure to the organic-soluble fraction increased superoxide anion production, with a sharp decrease in MMP. DNA damage is a significant process that may explain the course of toxicity of the organic-soluble fraction. For both subfractions, exposure caused cell cycle alterations in the G2/M phase that were induced by upregulation of p21 and p16. Collectively, both subfractions of wood tar are toxic. The water-soluble fraction contains chemicals (such as phenolic compounds) that induce a strong oxidative stress response and penetrate living cells more easily. The organic-soluble fraction contained more polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs and induced genotoxic processes, such as DNA damage.

Identification of bacteria-derived HLA-bound peptides in melanoma.

A variety of species of bacteria are known to colonize human tumours1-11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12-14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

Mechanisms of lung toxicity induced by biomass burning aerosols.

BACKGROUND: Carbonaceous aerosols emitted from indoor and outdoor biomass burning are major risk factors contributing to the global burden of disease. Wood tar aerosols, namely, tar ball particles, compose a substantial fraction of carbonaceous emissions, especially from biomass smoldering. However, their health-related impacts and toxicity are still not well known. This study investigated the toxicity of the water-soluble fraction of pyrolyzed wood tar aerosols in exposed mice and lung epithelial cells. RESULTS: Mice exposed to water-soluble wood tar aerosols showed increased inflammatory and oxidative stress responses. Bronchial epithelial cells exposed to the same water-soluble wood tar aerosols showed increased cell death with apoptotic characteristics. Alterations in oxidative status, including changes in reactive oxygen species (ROS) levels and reductions in the expression of antioxidant genes related to the transcription factor Nrf2, were observed and were confirmed by increased levels of MDA, a lipid peroxidation adduct. Damage to mitochondria was observed as an early event responsible for the aforementioned changes. CONCLUSIONS: The toxicity and health effect-related mechanisms of water-soluble wood tar were investigated for the first time in the context of biomass burning. Wood tar particles may account for major responses such as cell death, oxidative stress, supression of protection mechnaisms and mitochondrial damaged cause by expsoure to biomass burning aerosols.

In situ electron microscopy characterization of intracellular ion pools in mineral forming microalgae.

The formation of coccoliths, intricate calcium carbonate scales that cover the cells of unicellular marine microalgae, is a highly regulated biological process. For decades, scientists have tried to elucidate the cellular, chemical, and structural mechanisms that control the precise mineralogy and shape of the inorganic crystals. Transmission electron microscopy was pivotal in characterizing some of the organelles that orchestrate this process. However, due to the difficulties in preserving soluble inorganic phases during sample preparation, only recently, new intracellular ion-pools were detected using state-of-the-art cryo X-ray and electron microscopy techniques. Here, we combine a completely non-aqueous sample preparation procedure and room temperature electron microscopy, to investigate the presence, cellular location, and composition, of mineral phases inside mineral forming microalga species. This methodology, which fully preserves the forming coccoliths and the recently identified Ca-P-rich bodies, allowed us to identify a new class of ion-rich compartments that have complex internal structure. In addition, we show that when carefully choosing heavy metal stains, elemental analysis of the mineral phases can give accurate chemical signatures of the inorganic phases. Applying this approach to mineral forming microalgae will bridge the gap between the low-preservation power for inorganic phases of conventional chemical-fixation based electron microscopy, and the low-yield of advanced cryo techniques.

Energy Sources of the Depth-Generalist Mixotrophic Coral Stylophora pistillata.

Energy sources of corals, ultimately sunlight and plankton availability, change dramatically from shallow to mesophotic (30-150 m) reefs. Depth-generalist corals, those that occupy both of these two distinct ecosystems, are adapted to cope with such extremely diverse conditions. In this study, we investigated the trophic strategy of the depth-generalist hermatypic coral Stylophora pistillata and the ability of mesophotic colonies to adapt to shallow reefs. We compared symbiont genera composition, photosynthetic traits and the holobiont trophic position and carbon sources, calculated from amino acids compound-specific stable isotope analysis (AA-CSIA), of shallow, mesophotic and translocated corals. This species harbors different Symbiodiniaceae genera at the two depths: Cladocopium goreaui (dominant in mesophotic colonies) and Symbiodinium microadriaticum (dominant in shallow colonies) with a limited change after transplantation. This allowed us to determine which traits stem from hosting different symbiont species compositions across the depth gradient. Calculation of holobiont trophic position based on amino acid δ15N revealed that heterotrophy represents the same portion of the total energy budget in both depths, in contrast to the dogma that predation is higher in corals growing in low light conditions. Photosynthesis is the major carbon source to corals growing at both depths, but the photosynthetic rate is higher in the shallow reef corals, implicating both higher energy consumption and higher predation rate in the shallow habitat. In the corals transplanted from deep to shallow reef, we observed extensive photo-acclimation by the Symbiodiniaceae cells, including substantial cellular morphological modifications, increased cellular chlorophyll a, lower antennae to photosystems ratios and carbon signature similar to the local shallow colonies. In contrast, non-photochemical quenching remains low and does not increase to cope with the high light regime of the shallow reef. Furthermore, host acclimation is much slower in these deep-to-shallow transplanted corals as evident from the lower trophic position and tissue density compared to the shallow-water corals, even after long-term transplantation (18 months). Our results suggest that while mesophotic reefs could serve as a potential refuge for shallow corals, the transition is complex, as even after a year and a half the acclimation is only partial.

The mitochondrial carrier Citrin plays a role in regulating cellular energy during carcinogenesis.

Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.

Chlorophyll catabolism precedes changes in chloroplast structure and proteome during leaf senescence.

The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll-binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra-plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.

Mutant p53-dependent mitochondrial metabolic alterations in a mesenchymal stem cell-based model of progressive malignancy.

It is well accepted that malignant transformation is associated with unique metabolism. Malignant transformation involves a variety of cellular pathways that are associated with initiation and progression of the malignant process that remain to be deciphered still. Here we used a mouse model of mutant p53 that presents a stepwise progressive transformation of adult Mesenchymal Stem Cells (MSCs). While the established parental p53Mut-MSCs induce tumors, the parental p53WT-MSCs that were established in parallel, did not. Furthermore, tumor lines derived from the parental p53Mut-MSCs (p53Mut-MSC-TLs), exhibited yet a more aggressive transformed phenotype, suggesting exacerbation in tumorigenesis. Metabolic tracing of these various cell types, indicated that while malignant transformation is echoed by a direct augmentation in glycolysis, the more aggressive p53Mut-MSC-TLs demonstrate increased mitochondrial oxidation that correlates with morphological changes in mitochondria mass and function. Finally, we show that these changes are p53Mut-dependent. Computational transcriptional analysis identified a mitochondrial gene signature specifically downregulated upon knock/out of p53Mut in MSC-TLs. Our results suggest that stem cells exhibiting different state of malignancy are also associated with a different quantitative and qualitative metabolic profile in a p53Mut-dependent manner. This may provide important insights for cancer prognosis and the use of specific metabolic inhibitors in a personalized designed cancer therapy.

Colocalization and Disposition of Cellulosomes in Clostridium clariflavum as Revealed by Correlative Superresolution Imaging.

Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies in C. clariflavum and demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome's most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D) superresolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface.IMPORTANCE Conversion of plant biomass into soluble sugars is of high interest for production of fermentable industrial materials, such as biofuels. Biofuels are a very attractive alternative to fossil fuels, both for recycling of agricultural wastes and as a source of sustainable energy. Cellulosomes are among the most efficient enzymatic degraders of biomass known to date, due to the incorporation of a multiplicity of enzymes into a potent, multifunctional nanomachine. The intimate association with the bacterial cell surface is inherent in its efficient action on lignocellulosic substrates, although this property has not been properly addressed experimentally. The dramatic increase in cellulosome performance on recalcitrant feedstocks is critical for the design of cost-effective processes for efficient biomass degradation.

SNARE priming is essential for maturation of autophagosomes but not for their formation.

Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. αSNAP, the adaptor molecule for the SNARE-priming enzyme N-ethylmaleimide-sensitive factor (NSF) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of αSNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of αSNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal-lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss.

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death, is necessary for developmental cell death in Drosophila midgut.

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported 1 on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.