[3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro].

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

A patient-specific lung cancer assembloid model with heterogeneous tumor microenvironments.

Cancer models play critical roles in basic cancer research and precision medicine. However, current in vitro cancer models are limited by their inability to mimic the three-dimensional architecture and heterogeneous tumor microenvironments (TME) of in vivo tumors. Here, we develop an innovative patient-specific lung cancer assembloid (LCA) model by using droplet microfluidic technology based on a microinjection strategy. This method enables precise manipulation of clinical microsamples and rapid generation of LCAs with good intra-batch consistency in size and cell composition by evenly encapsulating patient tumor-derived TME cells and lung cancer organoids inside microgels. LCAs recapitulate the inter- and intratumoral heterogeneity, TME cellular diversity, and genomic and transcriptomic landscape of their parental tumors. LCA model could reconstruct the functional heterogeneity of cancer-associated fibroblasts and reflect the influence of TME on drug responses compared to cancer organoids. Notably, LCAs accurately replicate the clinical outcomes of patients, suggesting the potential of the LCA model to predict personalized treatments. Collectively, our studies provide a valuable method for precisely fabricating cancer assembloids and a promising LCA model for cancer research and personalized medicine.

Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish.

BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.

Serum CTRP9 and high-molecular weight adiponectin are associated with ischemic stroke.

BACKGROUND: C1q/TNF-related protein 9 (CTRP9) and adiponectin (APN) have beneficial metabolic regulatory and vasoprotective effects. This study explored alteration of CTRP9 and APN multimers during onset of ischemic stroke and development, to provide novel clinical and experimental basis for recognition and prevention of ischemic stroke. METHODS: There were 269 patients with ischemic stroke and 182 control subjects included in this study. Serum levels of CTRP9 and APN multimers in different disease stages were measured. RESULTS: Serum CTRP9, total APN (tAPN), and high-molecular weight (HMW) APN decreased gradually in stage I (acute stage, within 72 h of onset) of ischemic stroke and increased during stage III (11th day to one month) and stage IV (1 month after), compared to control. In the non-hyperlipidemia group, serum CTRP9, tAPN, and HMW were decreased in ischemic stroke patients compared to control (P < 0.05). Serum CTRP9 is closely related to serum tAPN and HMW (r = 0.992, 0.991). Serum CTRP9 are protective against ischemic stroke (OR = 0.400, 95% CI 0.197-0.810, P < 0.05). CONCLUSIONS: Lower serum CTRP9, tAPN, LMW, and HMW are significantly associated with increased ischemic stroke risk in non-hyperlipidemia subjects. CTRP9, tAPN, and HMW isoforms may be valuable clinical indicators for patients with ischemic stroke.

Effective delivery of osteopontin small interference RNA using exosomes suppresses liver fibrosis via TGF-ß1 signaling.

Objective and aims: Osteopontin (OPN), an oxidant stress sensitive cytokine, plays a central role in liver fibrosis. While OPN expression can be reduced by small interfering RNA (siRNA), the challenge to deliver siRNA safely and effectively into liver remains unresolved. Exosomes are promising natural nanocarriers for drug delivery that are able to enter cells with different biological barriers efficiently. In this study, we used exosomes as a delivery vehicle to target OPN in liver fibrosis. Methods: Exosomes selectively home to fibrotic liver according to small animal imaging system. Electroporation technique was used to engineer exosomes to carry siRNA targeting OPN (ExosiRNA-OPN). Primary hepatic stellate cells (HSCs) were isolated and treated with ExosiRNA-OPN to assess the effect on activated HSCs (aHSCs). Immunofluorescence for α-SMA, an aHSCs marker, and sirius red staining were performed to assess ECM deposition. Finally, plasma OPN from patients with liver fibrosis was identified by ELISA assay. Results: Exosome-mediated siRNA delivery systems show high uptake and low toxicity. Besides, ExosiRNA-OPN suppressed HSCs activation and ECM deposition and more efficiently improved liver function when compared to naked siRNA-OPN. Moreover, ExosiRNA-OPN was assumed inhibiting TGF-ß1 signaling activation, along with other fibrotic-related genes based on a GEO datasheet of liver fibrosis samples for correlation analyzes. ExosiRNA-OPN inhibited TGF-ß1 signaling by decreasing high-mobility group box-1 (HMGB1). Plasma proteins from chronic HBV-induced fibrosis patients were identified that patients with high OPN expression correlates with more advanced fibrosis progression. Discussion: This study shows that exosome-mediated siRNA-OPN delivery may be an effective option for the treatment of liver fibrosis.

Phosphorylation at Ser724 of the ER stress sensor IRE1α governs its activation state and limits ER stress-induced hepatosteatosis.

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1S724A/S724A) and investigated the importance of phosphorylation at Ser724 within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser724 exacerbated ER stress-induced hepatic steatosis in tunicamycin-treated Ern1S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer-binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid ß-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser724 of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.

Hyperlipidemic plasma molecules bind and inhibit adiponectin activity.

INTRODUCTION: Adiponectin is a potent vascular protective molecule. Recent findings have suggested adiponectin resistance during early diabetes. However, the molecular mechanisms responsible remain unidentified. Here, we took an unbiased approach to identify whether hyperlipidemic plasma molecules exist that bind and inhibit adiponectin function, contributing to adiponectin resistance and diabetic vascular injury. METHODS: Adult rats were randomly assigned to receive either a normal or a high-fat diet for 8 weeks. Plasma was co-immunoprecipitated with anti-APN antibody and analyzed by mass spectrometry. The APN binding molecules and their effect upon APN biological activity were determined. RESULTS: As expected, the high-fat-diet increased plasma triglyceride, total cholesterol, and low-density lipoprotein. Importantly, the circulating APN level was significantly increased at this time point. Mass spectrometry identified 18 proteins with increased APN binding in hyperlipidemic plasma, among which four proteins critical in lipid metabolism, including apolipoprotein A1 (APOA1), APOA4, APOC1, and paraoxonase 1, were further investigated. Incubating recombinant APN with APOA1 markedly (P < 0.01), and incubating with APOC1 significantly (P < 0.05), inhibited APN activity as evidenced by the reduced AMPK activation in HUVECs. APOA4 and paraoxonase 1 incubation had no effect upon APN activity. Finally, plasma APOA1 was significantly increased (P < 0.05) in hyperlipidemic plasma compared with the control plasma. CONCLUSIONS: It was demonstrated for the first time that increased APOA1 and APOC1 in hyperlipidemic plasma binds and inhibits APN activity. This result not only identifies a novel molecular mechanism responsible for adiponectin resistance during early stage diabetes, but also provides additional new insight into the diverse/controversial (protective and harmful) functions of high-density lipoprotein.

Atypical laminin spots and pull-generated microtubule-actin projections mediate Drosophila wing adhesion.

During Drosophila metamorphosis, dorsal and ventral wing surfaces adhere, separate, and reappose in a paradoxical process involving cell-matrix adhesion, matrix production and degradation, and long cellular projections. The identity of the intervening matrix, the logic behind the adhesion-reapposition cycle, and the role of projections are unknown. We find that laminin matrix spots devoid of other main basement membrane components mediate wing adhesion. Through live imaging, we show that long microtubule-actin cables grow from those adhesion spots because of hydrostatic pressure that pushes wing surfaces apart. Formation of cables resistant to pressure requires spectraplakin, Patronin, septins, and Sdb, a SAXO1/2 microtubule stabilizer expressed under control of wing intervein-selector SRF. Silkworms and dead-leaf butterflies display similar dorso-ventral projections and expression of Sdb in intervein SRF-like patterns. Our study supports the morphogenetic importance of atypical basement-membrane-related matrices and dissects matrix-cytoskeleton coordination in a process of great evolutionary significance.

Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity.

Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR-confirmed, SARS-CoV-2-hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD-binding antibodies exhibited neutralizing capacity, but only 74% of N-protein-positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein-binding antibodies does not always correlate with presence of S-RBD-neutralizing antibodies and caution against the extensive use of N-protein-based serology testing for determination of potential COVID-19 immunity.

Enacting national social distancing policies corresponds with dramatic reduction in COVID19 infection rates.

The outbreak the SARS-CoV-2 (CoV-2) virus has resulted in over 6.5 million cases of COVID19, greatly stressing global healthcare infrastructure. Lacking medical prophylactic measures to combat disease spread, many nations have adopted social distancing policies in order to mitigate transmission of CoV-2. While mathematical models have suggested the efficacy of social distancing to curb the spread of CoV-2, there is a lack of systematic studies to quantify the real-world efficacy of these approaches. Here, we first demonstrate that implementation of social distancing policies in US states corresponded with a reduction in COVID19 spread rates, and that the reduction in spread rate is proportional to the average change in mobility. We validate this observation on a worldwide scale by analyzing COVID19 spread rate in 134 nations with varying social distancing policies. Globally, we find that social distancing policies significantly reduced the COVID19 spread rate, with resulting in an estimated 65% reduction (95% CI = 39-80%) in new COVID19 cases over a two week time period. These data suggest that social distancing policies may be a powerful tool to prevent spread of COVID19 in real-world scenarios.

Spectraplakin Shot Maintains Perinuclear Microtubule Organization in Drosophila Polyploid Cells.

Polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis as part of their differentiation programs. Upon cell-cycle exit and differentiation, non-centrosomal sites govern microtubule distribution in most cells. Little is known on how polyploid cells, differentiated but cycling, organize their microtubules. We show that microtubules in Drosophila adipocytes and other polyploid tissues form a dense perinuclear cortex responsible for nuclear size and position. Confirming a relation between this perinuclear cortex and the polyploid endocycle, polyploidization of normally diploid cells was sufficient for cortex formation. A critical component of the perinuclear microtubule organizer (pnMTOC) is Shot, absence of which caused collapse of the perinuclear network into a condensed organizer through kinesin-dependent microtubule sliding. Furthermore, this ectopic organizer was capable of directing partial assembly of a deeply disruptive cytokinesis furrow. In all, our study revealed the importance of perinuclear microtubule organization for stability of endocycling Drosophila cells.

Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly.

Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen in addition to the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg in morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from cultured embryonic tissue experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements.

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4αEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.

Dual role for inositol-requiring enzyme 1α in promoting the development of hepatocellular carcinoma during diet-induced obesity in mice.

Obesity is associated with both endoplasmic reticulum (ER) stress and chronic metabolic inflammation. ER stress activates the unfolded protein response (UPR) and has been implicated in a variety of cancers, including hepatocellular carcinoma (HCC). It is unclear whether individual UPR pathways are mechanistically linked to HCC development, however. Here we report a dual role for inositol-requiring enzyme 1α (IRE1α), the ER-localized UPR signal transducer, in obesity-promoted HCC development. We found that genetic ablation of IRE1α in hepatocytes not only markedly reduced the occurrence of diethylnitrosamine (DEN)-induced HCC in liver-specific IRE1α knockout (LKO) mice when fed a normal chow (NC) diet, but also protected against the acceleration of HCC progression during high-fat diet (HFD) feeding. Irrespective of their adiposity states, LKO mice showed decreased hepatocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation, even in the face of increased hepatic apoptosis. Furthermore, IRE1α abrogation blunted obesity-associated activation of hepatic inhibitor of nuclear factor kappa B kinase subunit beta (IKKß)-nuclear factor kappa B (NF-κB) pathway, leading to reduced production of the tumor-promoting inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). Importantly, higher IRE1α expression along with elevated STAT3 phosphorylation was also observed in the tumor tissues from human HCC patients, correlating with their poorer survival rate. CONCLUSION: IRE1α acts in a feed-forward loop during obesity-induced metabolic inflammation to promote HCC development through STAT3-mediated hepatocyte proliferation. (Hepatology 2018).

Hepatic regulation of VLDL receptor by PPARß/δ and FGF21 modulates non-alcoholic fatty liver disease.

OBJECTIVE: The very low-density lipoprotein receptor (VLDLR) plays an important role in the development of hepatic steatosis. In this study, we investigated the role of Peroxisome Proliferator-Activated Receptor (PPAR)ß/δ and fibroblast growth factor 21 (FGF21) in hepatic VLDLR regulation. METHODS: Studies were conducted in wild-type and Pparß/δ-null mice, primary mouse hepatocytes, human Huh-7 hepatocytes, and liver biopsies from control subjects and patients with moderate and severe hepatic steatosis. RESULTS: Increased VLDLR levels were observed in liver of Pparß/δ-null mice and in Pparß/δ-knocked down mouse primary hepatocytes through mechanisms involving the heme-regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI), activating transcription factor (ATF) 4 and the oxidative stress-induced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. Moreover, by using a neutralizing antibody against FGF21, Fgf21-null mice and by treating mice with recombinant FGF21, we show that FGF21 may protect against hepatic steatosis by attenuating endoplasmic reticulum (ER) stress-induced VLDLR upregulation. Finally, in liver biopsies from patients with moderate and severe hepatic steatosis, we observed an increase in VLDLR levels that was accompanied by a reduction in PPARß/δ mRNA abundance and DNA-binding activity compared with control subjects. CONCLUSIONS: Overall, these findings provide new mechanisms by which PPARß/δ and FGF21 regulate VLDLR levels and influence hepatic steatosis development.

Inter-adipocyte Adhesion and Signaling by Collagen IV Intercellular Concentrations in Drosophila.

Sheet-forming Collagen IV is the main component of basement membranes, which are planar polymers of extracellular matrix underlying epithelia and surrounding organs in all animals. Adipocytes in both insects and mammals are mesodermal in origin and often classified as mesenchymal. However, they form true tissues where cells remain compactly associated. Neither the mechanisms providing this tissue-level organization nor its functional significance are known. Here we show that discrete Collagen IV intercellular concentrations (CIVICs), distinct from basement membranes and thicker in section, mediate inter-adipocyte adhesion in Drosophila. Loss of these Collagen-IV-containing structures in the larval fat body caused intercellular gaps and disrupted continuity of the adipose tissue layer. We also found that Integrin and Syndecan matrix receptors attach adipocytes to CIVICs and direct their formation. Finally, we show that Integrin-mediated adhesion to CIVICs promotes normal adipocyte growth and prevents autophagy through Src-Pi3K-Akt signaling. Our results evidence a surprising non-basement membrane role of Collagen IV in non-epithelial tissue morphogenesis while demonstrating adhesion and signaling functions for these structures.

Basement Membrane Manipulation in Drosophila Wing Discs Affects Dpp Retention but Not Growth Mechanoregulation.

Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. In this study, we manipulated BM composition to cause dramatic changes in tissue tension. We found that increased tissue compression when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor ß ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. Our results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment.

Tango1 spatially organizes ER exit sites to control ER export.

Exit of secretory cargo from the endoplasmic reticulum (ER) takes place at specialized domains called ER exit sites (ERESs). In mammals, loss of TANGO1 and other MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family proteins prevents ER exit of large cargoes such as collagen. Here, we show that Drosophila melanogaster Tango1, the only MIA/cTAGE family member in fruit flies, is a critical organizer of the ERES-Golgi interface. Tango1 rings hold COPII (coat protein II) carriers and Golgi in close proximity at their center. Loss of Tango1, present at ERESs in all tissues, reduces ERES size and causes ERES-Golgi uncoupling, which impairs secretion of not only collagen, but also all other cargoes we examined. Further supporting an organizing role of Tango1, its overexpression creates more and larger ERESs. Our results suggest that spatial coordination of ERES, carrier, and Golgi elements through Tango1's multiple interactions increases secretory capacity in Drosophila and allows secretion of large cargo.

The metabolic ER stress sensor IRE1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity.

Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1f/f; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1f/f; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.

Toll pathway modulates TNF-induced JNK-dependent cell death in Drosophila.

Signalling networks that control the life or death of a cell are of central interest in modern biology. While the defined roles of the c-Jun N-terminal kinase (JNK) pathway in regulating cell death have been well-established, additional factors that modulate JNK-mediated cell death have yet to be fully elucidated. To identify novel regulators of JNK-dependent cell death, we performed a dominant-modifier screen in Drosophila and found that the Toll pathway participates in JNK-mediated cell death. Loss of Toll signalling suppresses ectopically and physiologically activated JNK signalling-induced cell death. Our epistasis analysis suggests that the Toll pathway acts as a downstream modulator for JNK-dependent cell death. In addition, gain of JNK signalling results in Toll pathway activation, revealed by stimulated transcription of Drosomycin (Drs) and increased cytoplasm-to-nucleus translocation of Dorsal. Furthermore, the Spätzle (Spz) family ligands for the Toll receptor are transcriptionally upregulated by activated JNK signalling in a non-cell-autonomous manner, providing a molecular mechanism for JNK-induced Toll pathway activation. Finally, gain of Toll signalling exacerbates JNK-mediated cell death and promotes cell death independent of caspases. Thus, we have identified another important function for the evolutionarily conserved Toll pathway, in addition to its well-studied roles in embryonic dorso-ventral patterning and innate immunity.