Publications

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La, TM;Yamada, H;Seiriki, S;Li, SA;Fujise, K;Katsumi, N;Abe, T;Watanabe, M;Takei, K;
Cell Struct Funct,
45
(2)
, 121-130
(2020)
The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic ?-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in ?-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse ?-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic ?-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.Key words: endocytosis, GluR2, AMPA, cortactin, MIN6.
Limatola, N;Chun, JT;Santella, L;
Biol Bull,
239
(1)
, 13-23
(2020)
AbstractFertilization and early development are usually the most vulnerable stages in the life of marine animals, and the biological processes during this period are highly sensitive to the environment. In nature, sea urchin gametes are shed in seawater, where they undergo external fertilization and embryonic development. In a laboratory, it is possible to follow the exact morphological and biochemical changes taking place in the fertilized eggs and the developing embryos. Thus, observation of successful fertilization and the subsequent embryonic development of sea urchin eggs can be used as a convenient biosensor to assess the quality of the marine environment. In this paper, we have examined how salinity and pH changes affect the normal fertilization process and the following development of Paracentrotus lividus. The results of our studies using confocal microscopy, scanning and transmission electron microscopy, and time-lapse Ca2+ image recording indicated that both dilution and acidification of seawater have subtle but detrimental effects on many aspects of the fertilization process. They include Ca2+ signaling and coordinated actin cytoskeletal changes, leading to a significantly reduced rate of successful fertilization and, eventually, to abnormal or delayed embryonic development.
Logan, G;McCartney, B;
Cytoskeleton (Hoboken),
77
(5-6)
, 229-237
(2020)
Drosophila oogenesis is an excellent in vivo model for investigating cytoskeletal dynamics because of the rapid cytoskeletal remodeling that occurs at the end of stage 10; however, there are few robust tools for detecting microtubules in live complex tissues. The recent development of membrane permeable taxol-based fluorescent probes to label microtubules is significant technical progress, but the effectiveness of these probes and the potential stabilizing effects of the taxol derivative have not been well characterized in vivo. Here, we compared three commercially available taxol-derived microtubule labels to determine their efficacy and potential artifacts. We found that all three probes labeled microtubules with differences in permeability, brightness, and signal to noise ratio. Like taxol, however, all of the probes disrupted the F-actin cytoskeleton at higher concentrations. We also found that the efflux pump inhibitor, verapamil, increased the intensity of the label and modestly increased the severity of the F-actin defects. Of the three probes, Tubulin Tracker (ThermoScientific) was the most permeable and was brightest, with the highest signal to noise ratio. Furthermore, washing out the probe after a 30-min incubation significantly reduced the F-actin artifacts without compromising signal brightness.
Nasteska, D;Fine, N;Ashford, F;Cuozzo, F;Viloria, K;Smith, G;Dahir, A;Dawson, P;Lai, Y;Bastidas-Ponce, A;Bakhti, M;Rutter, G;Fiancette, R;Nano, R;Piemonti, L;Lickert, H;Zhou, Q;Akerman, I;Hodson, D;
Research Square,
106
(35)
, 14872-7
(2020)
Transcriptionally mature and immature β-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that differences in β-cell maturity, defined using PDX1 and MAFA expression, are required for proper islet operation. Functional mapping of rodent and human islets containing proportionally more mature β-cells revealed defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of mature ?-cells led to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, the islet signalling network was found to contribute to differences in maturity across ?-cells. During metabolic stress, islet function could be restored by redressing the balance between immature and mature β-cells. Thus, preserving a balance between immature and mature ?-cells might be important for islet engineering efforts and more broadly the treatment of type 1 and type 2 diabetes.
Salvioni, L;Zuppone, S;Andreata, F;Monieri, M;Mazzucchelli, S;Di Carlo, C;Morelli, L;Cordiglieri, C;Donnici, L;De Francesco, R;Corsi, F;Prosperi, D;Vago, R;Colombo, M;
Adv. Therap.,
3
(8)
, 2000007
(2020)
Systemic chemotherapy has not significantly reduced clinical demand for triple negative breast cancer (TNBC) treatments. To address the need for more effective therapy, the use of nonviral nanoparticles is explored to deliver suicide gene therapy as valuable alternative to protect nucleic acids in the bloodstream and improve their tumor uptake. Biocompatible cationic lipid nanoparticles are developed as a novel delivery system of a suicide plasmid gene encoding saporin. Active targeting is accomplished by taking advantage of nanoparticle functionalization with U11 peptide, designed to be directed toward urokinase plasminogen activator receptor, limiting off?target toxicity. The antitumor effect of U11?lipid?protamine?DNA (U11?LPD) nanoparticles are tested in TBNC cells, showing a strong prevalence of targeted versus nontargeted nanoparticles in terms of uptake kinetics and proliferation inhibition. Transfection of green fluorescent protein (GFP) plasmid in MDA?MB?231 cells is demonstrated. U11?LPD nanoparticles administered by retro bulbar injection exhibit excellent tumor tropism in TNBC orthotopic xenograft mice and effectively transfect TNBC cells with saporin plasmid resulting in tumor mass reduction. No systemic toxicity or organ damage is discovered after repeated treatments with nanoparticles. The findings suggest that systemic administration of targeted LPD nanoparticles to deliver saporin safely allows for active inhibition of cancer progression even in the absence of specific promoter gene sequences.
Deng, T;Stempor, P;Appert, A;Daube, M;Ahringer, J;Hajnal, A;Lattmann, E;
PLoS Genet,
16
(3)
, e1008470
(2020)
Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.
Moreno-Andrés, D;Yokoyama, H;Scheufen, A;Holzer, G;Lue, H;Schellhaus, AK;Weberruss, M;Takagi, M;Antonin, W;
Cells,
9
(7)
, 1702
(2020)
The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.
Cao, T;Sujkowski, A;Cobb, T;Wessells, RJ;Jin, JP;
J Biol Chem,
295
(12)
, 3794-3807
(2020)
The troponin complex regulates the Ca2+ activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid-rich C-terminal extension of ?70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+ buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.
De, A;Beligala, DH;Sharma, VP;Burgos, CA;Lee, AM;Geusz, ME;
Clin Exp Metastasis,
37
(5)
, 617-635
(2020)
Epithelial-mesenchymal transition (EMT) is a key event preceding tumor cell metastasis that increases cell invasiveness and cancer stem cell (CSC) populations. Studies suggest that genes used in generating circadian rhythms also serve in regulating EMT. To test the role of circadian clocks in cellular EMT events two cancer cell lines were compared, one that has a well-established circadian clock, C6 from rat glioma, and one that does not, MCF-7 from human breast tumor. MCF-7 tumorsphere cultures were tested for evidence of circadian rhythms because of previously reported circadian rhythm enhancement in C6 tumorspheres shown by elevated rhythm amplitude and increased expression of circadian clock gene Per2. Bioluminescence imaging of Per2 gene expression in MCF-7 tumorspheres revealed a previously unconfirmed circadian clock in this important cancer research model. Inducing CSC generation through EMT in C6 and MCF-7 monolayer cultures revealed circadian oscillations in the size of the post-EMT CSC population, confirming that circadian rhythms are additional processes controlling this stage of cancer progression. EMT was verified by distinct cellular morphological changes and expression of stem cell proteins OCT4, nestin, MSI1, and CD133 along with EMT-related proteins ZEB1, vimentin, and TWIST. Quantifying single-cell events and behaviors through time-lapse imaging indicated the post-EMT population size was determined largely by circadian rhythms in epithelial-like cancer cells undergoing EMT. We then identified a specific phase of the circadian rhythm in Per2 gene activation as a potential target for therapeutic treatments that may suppress EMT, minimize CSCs, and limit metastasis.
Angelini, G;Castagneto-Gissey, L;Casella-Mariolo, J;Caristo, ME;Russo, MF;Lembo, E;Verrastro, O;Stefanizzi, G;Marini, PL;Casella, G;Bornstein, SR;Rubino, F;Mingrone, G;
Am J Physiol Gastrointest Liver Physiol,
319
(4)
, G502-G511
(2020)
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver-related mortality. NAFLD is associated with obesity, hepatic fat accumulation, and insulin resistance, all of which contribute to its pathophysiology. Weight-loss is the main therapy for NAFLD, and metabolic surgery is the most effective treatment for morbid obesity and its metabolic comorbidities. Although has been reported that Roux-en-Y gastric bypass can reverse NAFLD, it is unclear whether such effects result from reduced weight, from a lower calorie-intake, or from the direct influence of surgery on mechanisms contributing to NAFLD. We aimed to investigate whether gastrointestinal (GI) bypass surgery could induce direct effects on hepatic fat accumulation and insulin resistance, independently of weight reduction. Twenty Wistar rats on a high-fat diet underwent duodenal-jejunal-bypass (DJB) or sham operation and were pair fed (PF) for 15 wk after surgery to obtain a matched weight. Outcome measures include ectopic fat deposition, expression of genes and proteins involved in fat metabolism, insulin-signaling, and gluconeogenesis in liver and muscle. Despite no differences in body weight and calorie intake, DJB showed lower ectopic fat accumulation, improved peripheral and hepatic insulin sensitivity, and enhanced lipid droplet degradation. In both tissues, DJB increased insulin signaling, whereas hepatic key enzymes involved in gluconeogenesis and de novo lipogenesis were decreased. These findings suggest that DJB can reverse, independently of weight loss, ectopic fat deposition and insulin resistance, two features of NAFLD that share a mutual pathway, in which perilipin-2 (PLIN2) seems to be the main player, supporting further investigation into strategies that target the gut to treat metabolic liver diseases.NEW & NOTEWORTHY Our findings suggest that duodenal-jejunal bypass can reverse, independently of weight loss, ectopic fat deposition and insulin resistance, two features of nonalcoholic fatty liver disease that share a mutual pathway, in which perilipin-2 seems to be the main player. Our study supports further investigation into the role of proximal small intestine exclusion in the pathophysiology of nonalcoholic fatty liver disease to uncover less invasive treatments that mimic the effects of metabolic surgery and aims to prevent and treat metabolic liver disease.
Kroeger, H;Grandjean, J;Chiang, W;Bindels, D;Mastey, R;Okalova, J;Nguyen, A;Powers, E;Kelly, J;Grimsey, N;Michaelides, M;Carroll, J;Wiseman, R;Lin, J;
bioRxiv,
(2020)
Dysregulation of the endoplasmic reticulum (ER) Unfolded Protein Response (UPR) is implicated in the pathology of many human diseases associated with ER stress. Inactivating genetic variants in the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital heritable vision loss in patients by an unknown pathomechanism. To investigate this, we generated retinal organoids from patient iPSCs carrying ATF6 disease-causing variants and ATF6 null hESCs generated by CRISPR. Interestingly, we found that cone photoreceptor cells in ATF6 mutant retinal organoids lacked inner and outer segments concomitant with absence of cone phototransduction gene expression; while rod photoreceptors developed normally. Adaptive optics retinal imaging of patients with disease-causing variants in ATF6 also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the phenotypes observed in our retinal organoids. These results reveal that ATF6 is essential for the formation of human cone photoreceptors, and associated absence of cone phototransduction components explains the severe visual impairment in patients with ATF6 -associated retinopathy. Moreover, we show that a selective small molecule ATF6 activator compound restores the transcriptional activity of ATF6 disease-causing variants and stimulates the growth of cone photoreceptors in patient retinal organoids, demonstrating that pharmacologic targeting of ATF6 signaling is a therapeutic strategy that needs to be further explored for blinding retinal diseases.
Muñoz-Castañeda, R;Zingg, B;Matho, K;Wang, Q;Chen, X;Foster, N;Narasimhan, A;Li, A;Hirokawa, K;Huo, B;Bannerjee, S;Korobkova, L;Park, C;Park, Y;Bienkowski, M;Chon, U;Wheeler, D;Li, X;Wang, Y;Kelly, K;An, X;Attili, S;Bowman, I;Bludova, A;Cetin, A;Ding, L;Drewes, R;D’Orazi, F;Elowsky, C;Fischer, S;Galbavy, W;Gao, L;Gillis, J;Groblewski, P;Gou, L;Hahn, J;Hatfield, J;Hintiryan, H;Huang, J;Kondo, H;Kuang, X;Lesnar, P;Li, X;Li, Y;Lin, M;Liu, L;Lo, D;Mizrachi, J;Mok, S;Naeemi, M;Nicovich, P;Palaniswamy, R;Palmer, J;Qi, X;Shen, E;Sun, Y;Tao, H;Wakemen, W;Wang, Y;Xie, P;Yao, S;Yuan, J;Zhu, M;Ng, L;Zhang, L;Lim, B;Hawrylycz, M;Gong, H;Gee, J;Kim, Y;Peng, H;Chuang, K;Yang, X;Luo, Q;Mitra, P;Zador, A;Zeng, H;Ascoli, G;Huang, Z;Osten, P;Harris, J;Dong, H;
bioRxiv,
(2020)
An essential step toward understanding brain function is to establish a cellular-resolution structural framework upon which multi-scale and multi-modal information spanning molecules, cells, circuits and systems can be integrated and interpreted. Here, through a collaborative effort from the Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based description of one brain structure – the primary motor cortex upper limb area (MOp-ul) of the mouse. Applying state-of-the-art labeling, imaging, computational, and neuroinformatics tools, we delineated the MOp-ul within the Mouse Brain 3D Common Coordinate Framework (CCF). We defined over two dozen MOp-ul projection neuron (PN) types by their anterograde targets; the spatial distribution of their somata defines 11 cortical sublayers, a significant refinement of the classic notion of cortical laminar organization. We further combine multiple complementary tracing methods (classic tract tracing, cell type-based anterograde, retrograde, and transsynaptic viral tracing, high-throughput BARseq, and complete single cell reconstruction) to systematically chart cell type-based MOp input-output streams. As PNs link distant brain regions at synapses as well as host cellular gene expression, our construction of a PN type resolution MOp-ul wiring diagram will facilitate an integrated analysis of motor control circuitry across the molecular, cellular, and systems levels. This work further provides a roadmap towards a cellular resolution description of mammalian brain architecture.
Logan, G;McCartney, B;
bioRxiv,
(2020)
Cells reposition their nuclei for a diversity of specialized functions through a wide variety of cytoskeletal mechanisms. To complete oogenesis, Drosophila nurse cells employ novel actin cable arrays to reposition their nuclei. During oogenesis, 15 nurse cells connected by ring canals contract to “dump” their cytoplasmic contents into the oocyte. Just prior to dumping, actin cables initiate from the nurse cell cortex and elongate toward their nuclei, pushing them away from the ring canals to prevent obstruction. How the actin cable arrays generate directional nuclear movement is not known. We found regional differences in the actin cable growth rate that are dependent on the differential localization of the actin assembly factors Enabled (Ena) and Diaphanous (Dia). Mislocalization of Ena resulted in actin cable arrays with a uniform growth rate. In the absence of growth rate asymmetry, nuclear relocation was significantly altered and cytoplasmic dumping was incomplete. This novel mechanism for nuclear repositioning relies on the regulated cortical localization of Dia and Ena producing asymmetric actin cable arrays that push the nuclei away from the ring canals, enabling successful oogenesis.Summary statementThis work demonstrates that an asymmetric actin cable array regulated by the differential localization of Diaphanous and Enabled is necessary to reposition nurse cell nuclei and complete oogenesis in Drosophila.
Garone, M;Birsa, N;Rosito, M;Salaris, F;Mochi, M;de Turris, V;Nair, R;Cunningham, T;Fisher, E;Fratta, P;Rosa, A;
bioRxiv,
(2020)
Mutations in RNA-binding proteins (RBPs) have been genetically associated with the motoneuron disease Amyotrophic Lateral Sclerosis (ALS). Using both human induced Pluripotent Stem Cells and mouse models, we found that FUS-ALS causative mutations have a profound impact on a network of RBPs, including two relevant factors with important roles in neuronal RNA metabolism: HuD and FMRP. Mechanistically, cytoplasmic localization of mutant FUS leads to upregulation of HuD levels through competition with FMRP for HuD 3’UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show altered axon branching and growth upon injury. Abnormal axon branching and regrowth in FUS mutant motoneurons could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, aberrant axonal growth and branching might represent broad early events in the pathogenesis of ALS.
Sun, Y;Chen, X;Fischer, S;Lu, S;Gillis, J;Zador, A;
bioRxiv,
(2020)
Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Here, we introduce BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing. We determined the expression of cadherins and cell-type markers in 29,933 cells, and the projections of 3,164 cells in both the mouse motor cortex and auditory cortex. Associating gene expression and projections in 1,349 neurons revealed shared cadherin signatures of homologous projections across the two cortical areas. These cadherins were enriched across multiple branches of the transcriptomic taxonomy. By correlating multi-gene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a path to uncovering the molecular logic underlying neuronal circuits.
Dolat, L;Valdivia, R;
bioRxiv,
(2020)
Our understanding of how the obligate intracellular bacterium Chlamydia trachomatis reprograms the cell biology of host cells in the upper genital tract is largely based on observations made in cell culture with transformed epithelial cell lines. Here we describe a primary spherical organoid system derived from endometrial tissue to recapitulate epithelial cell diversity, polarity, and ensuing responses to Chlamydia infection. Using high-resolution and time-lapse microscopy, we catalogue the infection process in organoids from invasion to egress, including the reorganization of the cytoskeleton and positioning of intracellular organelles. We show this model is amenable to screening C. trachomatis mutants for defects in the fusion of pathogenic vacuoles, the recruitment of intracellular organelles, and inhibition of cell death. Moreover, we reconstructed a primary immune cell response by co-culturing infected organoids with neutrophils, and determined that the effector TepP limits the recruitment of neutrophils to infected organoids. Collectively, our model details a system to study the cell biology of Chlamydia infections in three dimensional structures that better reflect the diversity of cell types and polarity encountered by Chlamydia upon infection of their animal hosts.Summary statement3D endometrial organoids to model Chlamydia infection and the role of secreted virulence factors in reprogramming host epithelial cells and immune cell recruitment
Keith, S;Bishop, C;Fallacaro, S;McCartney, B;
bioRxiv,
(2020)
Perturbations to animal-associated microbial communities (the microbiota) have deleterious effects on various aspects of host fitness, including dysregulated energy metabolism. However, the molecular processes underlying these microbial impacts on the host are poorly understood. In this study, we identify a novel connection between the microbiota and the neuronal factor Arc1 that affects metabolism and development in Drosophila. We find that Arc1 exhibits tissue-specific microbiota-dependent expression changes, and that flies bearing a null mutation of Arc1 complete larval development at a dramatically slowed rate compared to wild-type animals. In contrast, monoassociation with a single Acetobacter sp. isolate was sufficient to enable Arc1 mutants to develop at a wild-type rate. These developmental phenotypes are highly sensitive to composition of the larval diet, suggesting the growth rate defects of GF flies lacking Arc1 reflect metabolic dysregulation. Additionally, we show that pre-conditioning the larval diet with Acetobacter sp. partially accelerates Arc1 mutant development, but live bacteria are required for the full growth rate promoting effect. Finally, GF Arc1 mutants display multiple traits consistent with reduced insulin signaling activity that are reverted by association with Acetobacter sp., suggesting a potential mechanism underlying the microbe-dependent developmental phenotypes. Our results reveal a novel role for Arc1 in modulating insulin signaling, metabolic homeostasis, and growth rate that is specific to the host’s microbial and nutritional environment.SUMMARYDrosophila Arc1 exhibits microbiota-dependent, tissue-specific differential expression, mitigates the impacts of germ-free rearing on insulin signaling and growth rate, but is dispensable for metabolic homeostasis in Acetobacter-colonized flies.
Hudson, A;Loughran, G;Szabo, N;Wills, N;Atkins, J;Cooley, L;
bioRxiv,
(2020)
Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.
Xu, W;Ashford, F;Bitsi, S;Schiffer, L;Qadir, M;Arlt, W;Tomas, A;Hodson, D;Mauvais-Jarvis, F;
bioRxiv,
(2020)
Male mice with elimination of the androgen receptor (AR) in islet ? cells (?ARKO) exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hypoinsulinemia and hyperglycemia when challenged with a Western diet. Testosterone activation of an extranuclear AR in ? cells potentiates GSIS by amplifying the insulinotropic action of glucagon-like peptide-1 (GLP-1). Here, using a combination of ?ARKO and ? cell-selective GLP-1 receptor knockout mice and their islets, we show that AR activation in ? cells amplifies the insulinotropic effect of islet-derived GLP-1. In ? cell models expressing cAMP sensors, testosterone enhances the ability of GLP-1, but not that of glucose-dependent insulinotropic polypeptide or glucagon, to produce cAMP. Accordingly, testosterone selectively enhances the ability of GLP-1 to potentiate GSIS. Notably, testosterone enhances GLP-1 production of cAMP at the plasma membrane and endosomes. In male mouse and human islets, the insulinotropic effect of testosterone is abolished following inhibition of the membrane and endosomal cAMP-dependent protein kinase A and exchange protein activated by cAMP islet 2 pathways. Thus, membrane localization of AR enhances the ability of the GLP-1 receptor to produce cAMP, thus increasing glucose-stimulated insulin exocytosis.Significance StatementThis study reveals that testosterone, acting on the androgen receptor (AR) in insulin-producing ? cells amplifies the insulinotropic action of glucagon-like peptide-1 (GLP-1) by increasing GLP-1-mediated production of cAMP at the plasma membrane and endosomal compartments, to promote insulin vesicles exocytosis in human ? cells. This study establishes a novel biological paradigm in which membrane location of a steroid nuclear receptor enhances the ability of a G protein-coupled receptor to produce cAMP. It has exceptional clinical significance for targeted delivery of testosterone to ? cells in the large population of aging and androgen-deficient men who are at increased risk of diabetes.
Nasteska, D;Cuozzo, F;Thakker, A;Bakar, R;Westbrook, R;Akerman, I;Cantley, J;Tennant, D;Hodson, D;
bioRxiv,
(2020)
The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a hypoxia-inducible factor target that uses molecular oxygen to hydroxylate proline. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting ?-cells. Here, we show that deletion of PHD3 specifically in ?-cells (?PHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, ?PHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift away from glycolysis. However, ?PHD3KO islets are able to maintain oxidative phosphorylation and insulin secretion by increasing utilization of fatty acids to supply the tricarboxylic acid cycle. This nutrient-sensing switch cannot be sustained and ?PHD3KO islets begin to show signs of failure in response to prolonged metabolic stress, including impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the ?-cell glucose metabolism machinery by suppressing the use of fatty acids as a primary fuel source, under obesogenic and insulin resistant states.SIGNIFICANCE STATEMENTProlyl-4-hydroxylase 3 (PHD3) is involved in the oxygen-dependent regulation of cell phenotype. A number of recent studies have shown that PHD3 might operate at the interface between oxygen availability and metabolism. To understand how PHD3 influences insulin secretion, which depends on intact glucose metabolism, we generated mice lacking PHD3 specifically in pancreatic ?-cells. These mice, termed ?PHD3KO, are apparently normal until fed high fat diet at which point their ?-cells switch to fatty acids as a fuel source. This switch cannot be tolerated and ?-cells in ?PHD3KO mice eventually fail. Thus, PHD3 maintains glucose-stimulated insulin secretion in ?-cells during states of fatty acid excess, such as diabetes and obesity.