37 results on '"Julia Ast"'
Search Results
2. Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding
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Alice Adriaenssens, Johannes Broichhagen, Anne de Bray, Julia Ast, Annie Hasib, Ben Jones, Alejandra Tomas, Natalie Figueredo Burgos, Orla Woodward, Jo Lewis, Elisabeth O’Flaherty, Kimberley El, Canqi Cui, Norio Harada, Nobuya Inagaki, Jonathan Campbell, Daniel Brierley, David J. Hodson, Ricardo Samms, Fiona Gribble, and Frank Reimann
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Metabolism ,Neuroscience ,Medicine - Abstract
Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics’ ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) — brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways.
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- 2023
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3. Two Pex5 Proteins With Different Cargo Specificity Are Critical for Peroxisome Function in Ustilago maydis
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Julia Ast, Nils Bäcker, Elena Bittner, Domenica Martorana, Humda Ahmad, Michael Bölker, and Johannes Freitag
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PEX5 ,PEX7 ,beta oxidation ,peroxisome ,targeting signal ,Ustilago maydis ,Biology (General) ,QH301-705.5 - Abstract
Peroxisomes are dynamic multipurpose organelles with a major function in fatty acid oxidation and breakdown of hydrogen peroxide. Many proteins destined for the peroxisomal matrix contain a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by tetratricopeptide repeat (TPR) proteins of the Pex5 family. Various species express at least two different Pex5 proteins, but how this contributes to protein import and organelle function is not fully understood. Here, we analyzed truncated and chimeric variants of two Pex5 proteins, Pex5a and Pex5b, from the fungus Ustilago maydis. Both proteins are required for optimal growth on oleic acid-containing medium. The N-terminal domain (NTD) of Pex5b is critical for import of all investigated peroxisomal matrix proteins including PTS2 proteins and at least one protein without a canonical PTS. In contrast, the NTD of Pex5a is not sufficient for translocation of peroxisomal matrix proteins. In the presence of Pex5b, however, specific cargo can be imported via this domain of Pex5a. The TPR domains of Pex5a and Pex5b differ in their affinity to variations of the PTS1 motif and thus can mediate import of different subsets of matrix proteins. Together, our data reveal that U. maydis employs versatile targeting modules to control peroxisome function. These findings will promote our understanding of peroxisomal protein import also in other biological systems.
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- 2022
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4. 14-3-3ζ Constrains insulin secretion by regulating mitochondrial function in pancreatic β cells
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Yves Mugabo, Cheng Zhao, Ju Jing Tan, Anindya Ghosh, Scott A. Campbell, Evgenia Fadzeyeva, Frédéric Paré, Siew Siew Pan, Maria Galipeau, Julia Ast, Johannes Broichhagen, David J. Hodson, Erin E. Mulvihill, Sophie Petropoulos, and Gareth E. Lim
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Endocrinology ,Metabolism ,Medicine - Abstract
While critical for neurotransmitter synthesis, 14-3-3 proteins are often assumed to have redundant functions due to their ubiquitous expression, but despite this assumption, various 14-3-3 isoforms have been implicated in regulating metabolism. We previously reported contributions of 14-3-3ζ in β cell function, but these studies were performed in tumor-derived MIN6 cells and systemic KO mice. To further characterize the regulatory roles of 14-3-3ζ in β cell function, we generated β cell–specific 14-3-3ζ–KO mice. Although no effects on β cell mass were detected, potentiated glucose-stimulated insulin secretion (GSIS), mitochondrial function, and ATP synthesis were observed. Deletion of 14-3-3ζ also altered the β cell transcriptome, as genes associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute 14-3-3 protein inhibition in mouse and human islets recapitulated the enhancements in GSIS and mitochondrial function, suggesting that 14-3-3ζ is the critical isoform in β cells. In dysfunctional db/db islets and human islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretion, and pan–14-3-3 protein inhibition led to enhanced GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ in the regulation of β cell function and provides a deeper understanding of how insulin secretion is controlled in β cells.
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- 2022
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5. Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics
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Julia Ast, Anastasia Arvaniti, Nicholas H. F. Fine, Daniela Nasteska, Fiona B. Ashford, Zania Stamataki, Zsombor Koszegi, Andrea Bacon, Ben J. Jones, Maria A. Lucey, Shugo Sasaki, Daniel I. Brierley, Benoit Hastoy, Alejandra Tomas, Giuseppe D’Agostino, Frank Reimann, Francis C. Lynn, Christopher A. Reissaus, Amelia K. Linnemann, Elisa D’Este, Davide Calebiro, Stefan Trapp, Kai Johnsson, Tom Podewin, Johannes Broichhagen, and David J. Hodson
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Science - Abstract
Glucagon-like peptide-1 receptor is an important regulator of appetite and glucose homeostasis. Here the authors describe super-resolution microscopy and in vivo imaging compatible fluorescent probes, which reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics in islets and brain.
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- 2020
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6. Reagents and models for detecting endogenous GLP1R and GIPR
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Julia Ast, Johannes Broichhagen, and David J. Hodson
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GLP1R ,GIPR ,GLP1 ,GIP ,incretin-mimetics ,diabetes ,Medicine ,Medicine (General) ,R5-920 - Abstract
Glucagon-like peptide-1 receptor (GLP1R) agonists target the GLP1R, whereas dual GLP1R/ gastric inhibitory polypeptide receptor (GIPR) agonists target both the GLP1R and GIPR. Despite the importance of these drug classes for the treatment of diabetes and obesity, still very little is known about the localization of GLP1R and GIPR themselves. Complicating matters is the low abundance of GLP1R and GIPR mRNA/protein, as well as a lack of specific and validated reagents for their detection. Without knowing where GLP1R and GIPR are located, it is difficult to propose mechanisms of action in the various target organs, and whether this is indirect or direct. In the current review, we will explain the steps needed to properly validate reagents for endogenous GLP1R/GIPR detection, describe the available approaches to visualize GLP1R/GIPR, and provide an update on the state-of-art. The overall aim is to provide a reference resource for researchers interested in GLP1R and GIPR signaling.
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- 2021
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7. SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells
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Oladapo E. Olaniru, Jordan Cheng, Julia Ast, Anastasia Arvaniti, Patricio Atanes, Guo C. Huang, Aileen J.F. King, Peter M. Jones, Johannes Broichhagen, David J. Hodson, and Shanta J. Persaud
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GPR56 ,SNAP-tag ,Trafficking ,Islets ,Apoptosis ,CRISPR-Cas9 ,Internal medicine ,RC31-1245 - Abstract
Objective: Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear. Methods: Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 β-cells and in MIN6 β-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and β-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 β-cells and human islets. Results: SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while the deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 β-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect. Conclusions: These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in β-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect β-cells against apoptosis, offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes.
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- 2021
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8. Conditional and Reversible Activation of Class A and B G Protein-Coupled Receptors Using Tethered Pharmacology
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Tom Podewin, Julia Ast, Johannes Broichhagen, Nicholas H. F. Fine, Daniela Nasteska, Philipp Leippe, Manuel Gailer, Teresa Buenaventura, Nisha Kanda, Ben J. Jones, Celine M’Kadmi, Jean-Louis Baneres, Jacky Marie, Alejandra Tomas, Dirk Trauner, Anja Hoffmann-Röder, and David J. Hodson
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Chemistry ,QD1-999 - Published
- 2018
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9. Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics
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Julia Ast, Anastasia Arvaniti, Nicholas H. F. Fine, Daniela Nasteska, Fiona B. Ashford, Zania Stamataki, Zsombor Koszegi, Andrea Bacon, Ben J. Jones, Maria A. Lucey, Shugo Sasaki, Daniel I. Brierley, Benoit Hastoy, Alejandra Tomas, Giuseppe D’Agostino, Frank Reimann, Francis C. Lynn, Christopher A. Reissaus, Amelia K. Linnemann, Elisa D’Este, Davide Calebiro, Stefan Trapp, Kai Johnsson, Tom Podewin, Johannes Broichhagen, and David J. Hodson
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Science - Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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- 2020
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10. Non-human primate and rodent embryonic stem cells are differentially sensitive to embryotoxic compounds
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Lauren Walker, Laura Baumgartner, Kevin C. Keller, Julia Ast, Susanne Trettner, and Nicole I. zur Nieden
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Osteogenesis ,Embryonic stem cell test ,Marmoset ,Rhesus ,Sensitivity ,Embryotoxicity ,Toxicology. Poisons ,RA1190-1270 - Abstract
Many industrial chemicals and their respective by-products need to be comprehensively evaluated for toxicity using reliable and efficient assays. In terms of teratogenicity evaluations, the murine-based embryonic stem cell test (EST) offers a promising solution to screen for multiple tissue endpoints. However, use of a mouse model in the EST can yield only a limited understanding of human development, anatomy, and physiology. Non-human primate or human in vitro models have been suggested to be a pharmacologically and pathophysiologically desirable alternative to murine in vitro models. Here, we comparatively evaluated the sensitivity of embryonic stem cells (ESCs) of a non-human primate to skeletal teratogens with mouse ESCs hypothesizing that inclusion of non-human primate cells in in vitro tests would increase the reliability of safety predictions for humans. First, osteogenic capacity was compared between ESCs from the mouse and a New World monkey, the common marmoset. Then, cells were treated with compounds that have been previously reported to induce bone teratogenicity. Calcification and MTT assays evaluated effects on osteogenesis and cell viability, respectively. Our data indicated that marmoset ESCs responded differently than mouse ESCs in such embryotoxicity screens with no obvious dependency on chemical or compound classes and thus suggest that embryotoxicity screening results could be affected by species-driven response variation. In addition, ESCs derived from rhesus monkey, an Old World monkey, and phylogenetically closer to humans than the marmoset, were observed to respond differently to test compounds than marmoset ESCs. Together these results indicate that there are significant differences in the responses of non-human primate and mouse ESC to embryotoxic agents.
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- 2015
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11. Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals.
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Alina C Stiebler, Johannes Freitag, Kay O Schink, Thorsten Stehlik, Britta A M Tillmann, Julia Ast, and Michael Bölker
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Genetics ,QH426-470 - Abstract
Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.
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- 2014
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12. Journal Club
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Andreas Peschel, Andreas Diepold, Thilo M. Fuchs, Julia Ast, Marion Lemoine, Bernhard Schink, Kürşad Turgay, Bärbel Stecher, Kai Thormann, Remy Colin, Johannes Sander, Petra Neumann-Staubitz, Khadija Aichane, and Daniela Kruck
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Molecular Biology ,Biotechnology - Published
- 2022
13. Enzyme self-label-bound ATTO700 in single-molecule and super-resolution microscopy
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Michael Trumpp, Anna Oliveras, Hannes Gonschior, Julia Ast, David J. Hodson, Petra Knaus, Martin Lehmann, Melissa Birol, and Johannes Broichhagen
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Microscopy ,near-infrared ATTO700 ,Metals and Alloys ,Proteins ,General Chemistry ,acceptor ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Fluorescence Resonance Energy Transfer ,Förster Resonance Energy Transfer ,Materials Chemistry ,Ceramics and Composites ,Nanotechnology ,Function and Dysfunction of the Nervous System ,500 Naturwissenschaften und Mathematik::540 Chemie::540 Chemie und zugeordnete Wissenschaften - Abstract
Herein, we evaluate near-infrared ATTO700 as an acceptor in SNAP- and Halo-tag protein labelling for Förster Resonance Energy Transfer (FRET) by ensemble and single molecule measurements. Microscopy of cell surface proteins in live cells is perfomed including super-resolution stimulated emission by depletion (STED) nanoscopy.
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- 2022
14. GC-globulin/vitamin D-binding protein is required for pancreatic α cell adaptation to metabolic stress
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Katrina Viloria, Daniela Nasteska, Julia Ast, Annie Hasib, Federica Cuozzo, Silke Heising, Linford J.B. Briant, Martin Hewison, and David J. Hodson
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Endocrinology, Diabetes and Metabolism ,Internal Medicine - Abstract
GC-globulin (GC), or vitamin D-binding protein, is a multifunctional protein involved in transport of circulating vitamin 25(OH)D and fatty acids, as well as actin-scavenging. In the pancreatic islets, the gene encoding GC, GC, is highly-localized to glucagon-secreting α cells. Despite this, the role of GC in α cell function is poorly understood. We previously showed that GC is essential for α cell morphology, electrical activity and glucagon secretion. We now show that loss of GC exacerbates α cell failure during metabolic stress. High fat diet-fed GC-/- mice have basal hyperglucagonemia, which is associated with decreased α cell size, impaired glucagon secretion and Ca2+ fluxes, and changes in glucose-dependent F-actin remodelling. Impairments in glucagon secretion can be rescued using exogenous GC to replenish α cell GC levels, increase glucagon granule area and restore the F-actin cytoskeleton. Lastly, GC levels decrease in α cells of donors with type 2 diabetes, which is associated with changes in α cell mass, morphology and glucagon expression. Together, these data demonstrate an important role for GC in α cell adaptation to metabolic stress.
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- 2022
15. Journal Club
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Larsen Vornholz, Johannes Sander, Sabine Keuter, Kirsten Küsel, Julia Ast, Carmen E. Wurzbacher, Muriel C. F. van Teeseling, Khadija Aichane, Miriam Herbert, and Daniela Kruck
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Molecular Biology ,Biotechnology - Published
- 2021
16. Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics
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Shugo Sasaki, Julia Ast, Daniela Nasteska, Fiona B. Ashford, Andrea Bacon, Zania Stamataki, Tom Podewin, Alejandra Tomas, Nicholas H. F. Fine, David J. Hodson, Giuseppe D'Agostino, Maria Lucey, Elisa D’Este, Christopher A. Reissaus, Amelia K. Linnemann, Stefan Trapp, Zsombor Koszegi, Johannes Broichhagen, Ben Jones, Kai Johnsson, Daniel I. Brierley, Francis C. Lynn, Benoit Hastoy, Anastasia Arvaniti, Frank Reimann, Davide Calebiro, Ast, Julia [0000-0002-0039-4762], Fine, Nicholas H F [0000-0003-2343-8534], Nasteska, Daniela [0000-0002-8996-5102], Stamataki, Zania [0000-0003-3823-4497], Sasaki, Shugo [0000-0002-3696-7809], Brierley, Daniel I [0000-0002-4360-2648], Hastoy, Benoit [0000-0003-1244-7857], D'Agostino, Giuseppe [0000-0002-3502-4251], Reimann, Frank [0000-0001-9399-6377], Lynn, Francis C [0000-0001-9318-1063], Linnemann, Amelia K [0000-0001-7356-4876], Calebiro, Davide [0000-0002-3811-1553], Trapp, Stefan [0000-0003-0665-4948], Johnsson, Kai [0000-0002-8002-1981], Podewin, Tom [0000-0002-1632-5104], Broichhagen, Johannes [0000-0003-3084-6595], Hodson, David J [0000-0002-8641-8568], Apollo - University of Cambridge Repository, Medical Research Council (MRC), Fine, Nicholas H. F. [0000-0003-2343-8534], Brierley, Daniel I. [0000-0002-4360-2648], D’Agostino, Giuseppe [0000-0002-3502-4251], Lynn, Francis C. [0000-0001-9318-1063], Linnemann, Amelia K. [0000-0001-7356-4876], Hodson, David J. [0000-0002-8641-8568], and Fine, Nicholas HF [0000-0003-2343-8534]
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0301 basic medicine ,Models, Molecular ,genetic structures ,Lydia Becker Institute ,123 ,Human Embryonic Stem Cells ,96 ,General Physics and Astronomy ,96/33 ,MOUSE ,14 ,59 ,Optical imaging ,631/92/96 ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Tissue Distribution ,631/80/2373/2238 ,14/19 ,Super-resolution microscopy ,EXPRESSING CELLS ,Receptor ,lcsh:Science ,Peptide sequence ,PHARMACOLOGY ,Mice, Knockout ,Multidisciplinary ,Molecular Structure ,Endocrine system and metabolic diseases ,Brain ,3. Good health ,Multidisciplinary Sciences ,631/1647/245/2226 ,Science & Technology - Other Topics ,64/60 ,Signal transduction ,psychological phenomena and processes ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction ,endocrine system ,Fluorophore ,Science ,BETA ,BIOLOGY ,behavioral disciplines and activities ,General Biochemistry, Genetics and Molecular Biology ,Article ,Glucagon-Like Peptide-1 Receptor ,38 ,Cell Line ,03 medical and health sciences ,Islets of Langerhans ,14/34 ,ResearchInstitutes_Networks_Beacons/lydia_becker_institute_of_immunology_and_inflammation ,GLP-1 RECEPTOR ,Animals ,Humans ,Amino Acid Sequence ,Glucagon-like peptide 1 receptor ,G protein-coupled receptor ,Fluorescent Dyes ,RELEASE ,Science & Technology ,45 ,FLUOROGENIC PROBES ,HEK 293 cells ,General Chemistry ,Peptide Fragments ,96/100 ,ALPHA ,030104 developmental biology ,HEK293 Cells ,Microscopy, Fluorescence, Multiphoton ,nervous system ,chemistry ,692/163/2743 ,TISSUE ,13/51 ,14/63 ,Biophysics ,lcsh:Q ,14/69 ,Chemical tools ,030217 neurology & neurosurgery - Abstract
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo., Glucagon-like peptide-1 receptor is an important regulator of appetite and glucose homeostasis. Here the authors describe super-resolution microscopy and in vivo imaging compatible fluorescent probes, which reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics in islets and brain.
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- 2020
17. GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition
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Audrys G. Pauza, Pratik Thakkar, Tatjana Tasic, Igor Felippe, Paul Bishop, Michael P. Greenwood, Kristina Rysevaite-Kyguoliene, Julia Ast, Johannes Broichhagen, David J. Hodson, Helio C. Salgado, Dainius H. Pauza, Nina Japundzic-Zigon, Julian F.R. Paton, and David Murphy
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Carotid Body ,Glucose ,Physiology ,Rats, Inbred SHR ,Hypertension ,Animals ,Blood Pressure ,Cardiology and Cardiovascular Medicine ,Article ,Rats - Abstract
Background: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. Methods: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. Results: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. Conclusions: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.
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- 2022
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18. Reagents and models for detecting endogenous GLP1R and GIPR
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Johannes Broichhagen, David Hodson, and Julia Ast
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Medicine (General) ,brain ,Review ,General Biochemistry, Genetics and Molecular Biology ,Glucagon-Like Peptide-1 Receptor ,Receptors, Gastrointestinal Hormone ,03 medical and health sciences ,R5-920 ,0302 clinical medicine ,Animals ,Humans ,GLP1R ,pancreas ,GIPR ,030304 developmental biology ,GLP-1R ,0303 health sciences ,GIP ,diabetes ,General Medicine ,3. Good health ,Microscopy, Electron ,Medicine ,incretin-mimetics ,GLP1 ,GLP-1 ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Glucagon-like peptide-1 receptor (GLP1R) agonists target the GLP1R, whereas dual GLP1R/ gastric inhibitory polypeptide receptor (GIPR) agonists target both the GLP1R and GIPR. Despite the importance of these drug classes for the treatment of diabetes and obesity, still very little is known about the localization of GLP1R and GIPR themselves. Complicating matters is the low abundance of GLP1R and GIPR mRNA/protein, as well as a lack of specific and validated reagents for their detection. Without knowing where GLP1R and GIPR are located, it is difficult to propose mechanisms of action in the various target organs, and whether this is indirect or direct. In the current review, we will explain the steps needed to properly validate reagents for endogenous GLP1R/GIPR detection, describe the available approaches to visualize GLP1R/GIPR, and provide an update on the state-of-art. The overall aim is to provide a reference resource for researchers interested in GLP1R and GIPR signaling.
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- 2021
19. 14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells
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M. Galipeau, Ju Jing Tan, Erin E. Mulvihill, Asmita Ghosh, C. Zhao, Ge Lim, E. Fadzeyeva, Johannes Broichhagen, S. S. Pan, F. Pare, Sophie Petropoulos, Julia Ast, Scott A. Campbell, Yves Mugabo, and David J. Hodson
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Gene isoform ,Programmed cell death ,Cell type ,Insulin ,medicine.medical_treatment ,General Medicine ,Biology ,Mitochondria ,Cell biology ,Transcriptome ,Mice ,Glucose ,14-3-3 Proteins ,Downregulation and upregulation ,Insulin-Secreting Cells ,Insulin Secretion ,Knockout mouse ,YWHAZ ,medicine ,Animals - Abstract
While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3ζ in adipocytes, but whether 14-3-3ζ has additional metabolic roles in other cell types, such as the pancreatic β-cell, is unclear. We previously documented a pro-survival role of 14-3-3ζ in MIN6 insulinoma cells, as depletion of 14-3-3ζ induced cell death, but paradoxically, whole-body deletion of 14-3-3ζ in mice resulted in significantly enlarged β-cell area with no effects on insulin secretion. To better understand the role of 14-3-3ζ in β-cells, we generated β-cell-specific 14-3-3ζ knockout (β14-3-3ζKO) mice, and while no differences in β-cell mass were observed, β14-3-3ζKO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3ζ led to profound changes to the β-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3ζ is a critical isoform in β-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ and its related isoforms in insulin secretion and mitochondrial function in β-cells. A deeper understanding of how 14-3-3ζ influences β-cell function will further advance our knowledge of how insulin secretion from β-cells is regulated.
- Published
- 2021
20. An expanded LUXendin color palette for GLP1R detection and visualization in vitro and in vivo
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Ramona Birke, Kilian Roßmann, Julia Ast, Bettina Mathes, Johannes Broichhagen, Ben Jones, Nicholas H. F. Fine, Jenny Eichhorst, Alissa Novak, T. Podewin, Amelia K. Linnemann, Alejandra Tomas, David J. Hodson, and Martin Lehmann
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medicine.anatomical_structure ,In vivo ,Pancreatic islets ,medicine ,Palette (computing) ,Computational biology ,WHOLE ANIMAL ,Beta cell ,Biology ,In vitro ,Visualization - Abstract
The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissue. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615 and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows non-invasive imaging, highlighting differentially-accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using widefield microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insight into GLP1R biology.
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- 2021
21. Interrogating surface
- Author
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Pascal, Poc, Vanessa A, Gutzeit, Julia, Ast, Joon, Lee, Ben J, Jones, Elisa, D'Este, Bettina, Mathes, Martin, Lehmann, David J, Hodson, Joshua, Levitz, and Johannes, Broichhagen
- Subjects
Chemistry - Abstract
Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down., Impermeable SNAP-tag substrates allow exclusive labelling of receptors on the cell membrane for nanoscopy, SiMPull and in vivo use.
- Published
- 2021
22. Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization
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Volker Haucke, Dorien A. Roosen, Ben Jones, Martin Lehmann, Christiane Huhn, Johannes Broichhagen, Kilian Roßmann, David J. Hodson, Julia Ast, Ramona Birke, and Bettina Mathes
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Xanthene ,Rhodamines ,chemistry.chemical_compound ,Membrane ,chemistry ,Live cell imaging ,HEK 293 cells ,Biophysics ,STED microscopy ,Protein tag ,Induced pluripotent stem cell - Abstract
Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.
- Published
- 2021
23. Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics
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Amelia K. Linnemann, Fiona B. Ashford, Johannes Broichhagen, Maria Lucey, Giuseppe D'Agostino, Shugo Sasaki, Anastasia Arvaniti, Zsombor Koszegi, Stefan Trapp, Ben Jones, Kai Johnsson, Benoit Hastoy, Tom Podewin, Daniel I. Brierley, Alejandra Tomas, David J. Hodson, Daniela Nasteska, Francis C. Lynn, Andrea Bacon, Julia Ast, Christopher A. Reissaus, Frank Reimann, Davide Calebiro, Zania Stamataki, Elisa D’Este, and Nicholas H. F. Fine
- Subjects
Models, Molecular ,Science ,Human Embryonic Stem Cells ,General Physics and Astronomy ,Endogeny ,Optical imaging ,Glucagon-Like Peptide-1 Receptor ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Islets of Langerhans ,Mice ,Animals ,Humans ,Distribution (pharmacology) ,Tissue Distribution ,Amino Acid Sequence ,Super-resolution microscopy ,Author Correction ,lcsh:Science ,Receptor ,Fluorescent Dyes ,Mice, Knockout ,Multidisciplinary ,Molecular Structure ,Chemistry ,Dynamics (mechanics) ,Endocrine system and metabolic diseases ,Brain ,General Chemistry ,Glucagon-like peptide-1 ,Fluorescence ,Peptide Fragments ,HEK293 Cells ,Microscopy, Fluorescence, Multiphoton ,Biophysics ,lcsh:Q ,Chemical tools ,Signal Transduction - Abstract
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
- Published
- 2020
24. Interrogating surfaceversusintracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates
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Julia Ast, Bettina Mathes, Joshua Levitz, David J. Hodson, Johannes Broichhagen, Elisa D’Este, Vanessa A. Gutzeit, Joon Lee, Martin Lehmann, Pascal Poc, and Ben Jones
- Subjects
DYNAMICS ,DIMERIZATION ,Membrane permeability ,Chemistry, Multidisciplinary ,OLIGOMERIZATION ,Protein tag ,010402 general chemistry ,01 natural sciences ,LIVE-CELL ,ACTIVATION ,03 medical and health sciences ,Extracellular ,SURFACE PROTEINS ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,Bioconjugation ,Science & Technology ,Chemistry ,FLUOROGENIC PROBES ,MICROSCOPY ,General Chemistry ,Transmembrane protein ,0104 chemical sciences ,SNAP-tag ,GENERAL-METHOD ,Physical Sciences ,Biophysics ,FLUOROPHORES ,03 Chemical Sciences ,Intracellular - Abstract
Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.
- Published
- 2020
25. SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells
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Anastasia Arvaniti, Julia Ast, Oladapo E. Olaniru, Guo Cai Huang, Shanta J. Persaud, David J. Hodson, Peter M. Jones, Aileen King, Jordan Cheng, Patricio Atanes, and Johannes Broichhagen
- Subjects
Agonist ,medicine.drug_class ,chemistry.chemical_element ,Apoptosis ,Caspase 3 ,Calcium ,Calcium in biology ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,0302 clinical medicine ,Insulin-Secreting Cells ,medicine ,Humans ,Receptor ,Internal medicine ,Molecular Biology ,030304 developmental biology ,SNAP-tag ,0303 health sciences ,Microscopy, Confocal ,Trafficking ,Cell Biology ,Microfluorimetry ,16. Peace & justice ,RC31-1245 ,Cell biology ,HEK293 Cells ,GPR56 ,chemistry ,030220 oncology & carcinogenesis ,Original Article ,Islets ,CRISPR-Cas9 ,Signal Transduction - Abstract
Objective Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear. Methods Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 β-cells and in MIN6 β-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and β-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 β-cells and human islets. Results SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while the deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 β-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect. Conclusions These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in β-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect β-cells against apoptosis, offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes., Highlights • GPR56 predominantly underwent constitutive internalisation in β-cells in the absence of exogenous agonist. • The GPR56 agonists, collagen III and P7, showed differential effects on GPR56 trafficking and islet functions. • Constitutive and agonist-dependent GPR56 activation is coupled to protection of β-cells against apoptosis.
- Published
- 2021
26. LUXendins reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics
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Stefan Trapp, Zania Stamataki, T. Podewin, Alejandra Tomas, David J. Hodson, Andrea Bacon, Davide Calebiro, Julia Ast, Fiona B. Ashford, Anastasia Arvaniti, Daniela Nasteska, Johannes Broichhagen, Benoit Hastoy, Elisa D’Este, Amelia K. Linnemann, Christopher A. Reissaus, Nicholas H. F. Fine, Zsombor Koszegi, Ben Jones, and Kai Johnsson
- Subjects
chemistry.chemical_classification ,0303 health sciences ,geography ,geography.geographical_feature_category ,genetic structures ,030209 endocrinology & metabolism ,Peptide ,Endogeny ,Islet ,behavioral disciplines and activities ,Glucagon-like peptide-1 ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Membrane ,nervous system ,chemistry ,Distribution (pharmacology) ,Receptor ,psychological phenomena and processes ,030304 developmental biology ,G protein-coupled receptor - Abstract
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and STED-compatible LUXendin651 we describe islet GLP1R expression patterns, reveal higher-order GLP1R organization including the existence of membrane nanodomains, and track single receptor subpopulations. We furthermore show that different fluorophores can confer agonistic behavior on the LUXendin backbone, with implications for the design of stabilized incretin-mimetics. Thus, our labeling probes possess divergent activation modes, allow visualization of endogenous GLP1R, and provide new insight into class B GPCR distribution and dynamics.
- Published
- 2019
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27. Conditional and reversible activation of class A and B G protein-coupled receptors using tethered pharmacology
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Philipp Leippe, Julia Ast, Nicholas H. F. Fine, Teresa Buenaventura, Ben Jones, Daniela Nasteska, Anja Hoffmann-Röder, Dirk Trauner, Alejandra Tomas, David J. Hodson, Céline M'Kadmi, Jean Louis Banères, Johannes Broichhagen, N Kanda, Tom Podewin, Jacky Marie, Manuel Gailer, and Medical Research Council
- Subjects
0301 basic medicine ,Agonist ,Reducing agent ,medicine.drug_class ,General Chemical Engineering ,media_common.quotation_subject ,Growth hormone secretagogue receptor ,Peptide ,Cleavage (embryo) ,01 natural sciences ,03 medical and health sciences ,medicine ,Receptor ,Internalization ,QD1-999 ,media_common ,G protein-coupled receptor ,chemistry.chemical_classification ,010405 organic chemistry ,General Chemistry ,0104 chemical sciences ,3. Good health ,Cell biology ,Chemistry ,030104 developmental biology ,chemistry ,hormones, hormone substitutes, and hormone antagonists ,Research Article - Abstract
Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of ExONatide, a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. ExONatide covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca2+ rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent beta-mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using GhrelON, a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, ExONatide and GhrelON allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both in vitro and in vivo, with applicability across GPCRs., SNAP-tag-directed activation of class A and B G protein-coupled receptors can be achieved in a conditional and reversible manner using peptidic reductively cleavable agONists (RECONs).
- Published
- 2017
28. Non-human primate and rodent embryonic stem cells are differentially sensitive to embryotoxic compounds
- Author
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Laura Baumgartner, Julia Ast, Kevin C. Keller, Lauren Walker, Nicole I. zur Nieden, Susanne Trettner, and Publica
- Subjects
Health, Toxicology and Mutagenesis ,Clinical Sciences ,Rhesus ,Stem Cell Research - Embryonic - Non-Human ,Old World monkey ,Regenerative Medicine ,Toxicology ,Embryonic stem cell test ,Article ,Medicinal and Biomolecular Chemistry ,Sensitivity ,lcsh:RA1190-1270 ,Osteogenesis ,biology.animal ,Primate ,Viability assay ,lcsh:Toxicology. Poisons ,New World monkey ,biology ,Marmoset ,Anatomy ,Stem Cell Research ,biology.organism_classification ,Embryonic stem cell ,Teratology ,In vitro ,Cell biology ,embryonic structures ,Embryotoxicity ,Other Chemical Sciences - Abstract
Many industrial chemicals and their respective by-products need to be comprehensively evaluated for toxicity using reliable and efficient assays. In terms of teratogenicity evaluations, the murine-based embryonic stem cell test (EST) offers a promising solution to screen for multiple tissue endpoints. However, use of a mouse model in the EST can yield only a limited understanding of human development, anatomy, and physiology. Non-human primate or human in vitro models have been suggested to be a pharmacologically and pathophysiologically desirable alternative to murine in vitro models. Here, we comparatively evaluated the sensitivity of embryonic stem cells (ESCs) of a non-human primate to skeletal teratogens with mouse ESCs hypothesizing that inclusion of non-human primate cells in in vitro tests would increase the reliability of safety predictions for humans.First, osteogenic capacity was compared between ESCs from the mouse and a New World monkey, the common marmoset. Then, cells were treated with compounds that have been previously reported to induce bone teratogenicity. Calcification and MTT assays evaluated effects on osteogenesis and cell viability, respectively. Our data indicated that marmoset ESCs responded differently than mouse ESCs in such embryotoxicity screens with no obvious dependency on chemical or compound classes and thus suggest that embryotoxicity screening results could be affected by species-driven response variation. In addition, ESCs derived from rhesus monkey, an Old World monkey, and phylogenetically closer to humans than the marmoset, were observed to respond differently to test compounds than marmoset ESCs. Together these results indicate that there are significant differences in the responses of non-human primate and mouse ESC to embryotoxic agents.
- Published
- 2014
29. Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi
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Johannes Freitag, Uwe Linne, Thorsten Stehlik, Björn Sandrock, Michael Bölker, Julia Ast, and Domenica Martorana
- Subjects
Mutant ,Wild type ,Fatty acid degradation ,Peroxisome ,Biology ,Microbiology ,carbohydrates (lipids) ,Cytosol ,chemistry.chemical_compound ,Glycolipid ,Biochemistry ,chemistry ,Biosynthesis ,hemic and lymphatic diseases ,neoplasms ,Molecular Biology ,Peroxisomal targeting signal - Abstract
Summary Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.
- Published
- 2014
30. Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi
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Michael Bölker, Johannes Freitag, and Julia Ast
- Subjects
Molecular Sequence Data ,Mutant ,Protein Sorting Signals ,Aspergillus nidulans ,Peroxisomes ,Ustilago ,Amino Acid Sequence ,Peroxisomal targeting signal ,Glyceraldehyde 3-phosphate dehydrogenase ,Multidisciplinary ,Base Sequence ,Virulence ,biology ,Alternative splicing ,Aldolase A ,Fungi ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Peroxisome ,biology.organism_classification ,Isoenzymes ,Alternative Splicing ,Phosphoglycerate Kinase ,Protein Transport ,Biochemistry ,Cytoplasm ,Codon, Terminator ,biology.protein ,Glycolysis - Abstract
Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids. Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1), which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.
- Published
- 2012
31. Network science: Applications for sustainable agroecosystems and food security
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Fredric M. Windsor, Dolors Armenteras, Ana Paula A. Assis, Julia Astegiano, Pamela C. Santana, Luciano Cagnolo, Luísa G. Carvalheiro, Clive Emary, Hugo Fort, Xavier I. Gonzalez, James J.N. Kitson, Ana C.F. Lacerda, Marcelo Lois, Viviana Márquez-Velásquez, Kirsten E. Miller, Marcos Monasterolo, Marina Omacini, Kate P. Maia, Tania Paula Palacios, Michael J.O. Pocock, Santiago L. Poggio, Isabela G. Varassin, Diego P. Vázquez, Julia Tavella, Débora C. Rother, Mariano Devoto, Paulo R. Guimarães, Jr., and Darren M. Evans
- Subjects
Agriculture ,Biodiversity ,Ecological networks ,Social networks ,Crops ,Food production ,Ecology ,QH540-549.5 ,General. Including nature conservation, geographical distribution ,QH1-199.5 - Abstract
The global challenge of feeding two billion more people by 2050, using more sustainable agricultural practices whilst dealing with uncertainties associated with environmental change, requires a transformation of food systems. We present a new perspective for how advances in network science can provide novel ways to better understand, harness, and restore multiple ecological processes in agricultural environments. We describe: (i) a network-focused framework for managing agro-ecosystems that accounts for the multiple interactions between biodiversity and associated ecosystem services; (ii) guidance for incorporating socio-economic factors into ecological networks; and (iii) the potential to upscale network methods to inform efforts to build resilience, including global food-supply chains. In doing so we aim to facilitate the application of network science as a systems-based way to tackle the challenges of securing an equitable distribution of food.
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- 2022
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32. Ribosomal Readthrough at a Short UGA Stop Codon Context Triggers Dual Localization of Metabolic Enzymes in Fungi and Animals
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Thorsten Stehlik, Michael Bölker, Britta A. M. Tillmann, Julia Ast, Kay Oliver Schink, Johannes Freitag, and Alina C. Stiebler
- Subjects
Cancer Research ,lcsh:QH426-470 ,Peroxisome-Targeting Signal 1 Receptor ,viruses ,Receptors, Cytoplasmic and Nuclear ,Gene Expression ,Context (language use) ,Biology ,Ribosome ,Malate Dehydrogenase ,ddc:570 ,Translational regulation ,Peroxisomes ,Ustilago ,Translation Termination ,Genetics ,Humans ,Fungal Genetics ,RNA, Messenger ,Nucleotide Motifs ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,L-Lactate Dehydrogenase ,Peroxisomal Targeting Signal 1 ,fungi ,Translational readthrough ,Fungi ,Biology and Life Sciences ,Cell Biology ,Malate dehydrogenase 1 ,Stop codon ,Isoenzymes ,lcsh:Genetics ,Open reading frame ,Protein Biosynthesis ,Codon, Terminator ,Protein Translation ,Cellular Structures and Organelles ,Ribosomes ,HeLa Cells ,Research Article - Abstract
Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated., Author Summary Eukaryotic organisms use various strategies to generate protein isoforms with different function or intracellular localization from a single gene. While differential splicing of mRNA is the most common mechanism to expand the number of encoded proteins, translational readthrough of stop codons is an alternative strategy to create protein variants with C-terminally extended proteins. Recently, it has been shown that fungi use both alternative splicing and translational readthrough to specify peroxisomal isoforms of glycolytic enzymes. Here we show that stop codon readthrough is also used in the animal kingdom to target important metabolic enzymes to peroxisomes. Interestingly, several of these enzymes have a function in peroxisomal redox homeostasis and energy metabolism. It has been described that termination fidelity is modulated by oxidation of specific ribosomal proteins. This suggests that dual targeting via translational readthrough allows adaptation of peroxisomal metabolism to the oxidative status of the cell.
- Published
- 2014
33. Unravelling the gender productivity gap in science: a meta-analytical review
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Julia Astegiano, Esther Sebastián-González, and Camila de Toledo Castanho
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gender bias ,h-index ,science impact ,success rate ,women in science ,women underrepresentation ,Science - Abstract
Women underrepresentation in science has frequently been associated with women being less productive than men (i.e. the gender productivity gap), which may be explained by women having lower success rates, producing science of lower impact and/or suffering gender bias. By performing global meta-analyses, we show that there is a gender productivity gap mostly supported by a larger scientific production ascribed to men. However, women and men show similar success rates when the researchers' work is directly evaluated (i.e. publishing articles). Men's success rate is higher only in productivity proxies involving peer recognition (e.g. evaluation committees, academic positions). Men's articles showed a tendency to have higher global impact but only if studies include self-citations. We detected gender bias against women in research fields where women are underrepresented (i.e. those different from Psychology). Historical numerical unbalance, socio-psychological aspects and cultural factors may influence differences in success rate, science impact and gender bias. Thus, the maintenance of a women-unfriendly academic and non-academic environment may perpetuate the gender productivity gap. New policies to build a more egalitarian and heterogeneous scientific community and society are needed to close the gender gap in science.
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- 2019
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34. Optical control of a receptor-linked guanylyl cyclase using a photoswitchable peptidic hormone
- Author
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Christina V. Frost, Dirk Trauner, Nicholas H. F. Fine, David J. Hodson, Anja Hoffmann-Röder, Dieter Groneberg, Johannes Broichhagen, Tom Podewin, Andreas Friebe, Julia Ast, Helena Meyer-Berg, and Martin Zacharias
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Photoswitch ,Peptidomimetic ,Chemistry ,General Chemistry ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Amino acid ,03 medical and health sciences ,030104 developmental biology ,Atrial natriuretic peptide ,Biochemistry ,Cell surface receptor ,Biophysics ,Receptor ,Intracellular ,Homeostasis - Abstract
The photoswitchable peptidomimetic hormone TOP271 allows the precise optical control of cGMP generation via the receptor-linked enzyme NPR-A in explanted aortic rings and islets of Langerhans., The optical control over biological function with small photoswitchable molecules has gathered significant attention in the last decade. Herein, we describe the design and synthesis of a small library of photoswitchable peptidomimetics based upon human atrial natriuretic peptide (ANP), in which the photochromic amino acid [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP) is incorporated into the peptide backbone. The endogeneous hormone ANP signals via the natriuretic peptide receptor A (NPR-A) through raising intracellular cGMP concentrations, and is involved in blood pressure regulation and sodium homeostasis, as well as lipid metabolism and pancreatic function. The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro. Despite this seemingly small difference, TOP271 enables large, optically-induced conformational changes ex vivo and transforms the NPR-A into an endogenous photoswitch. Thus, application of TOP271 allows the reversible generation of cGMP using light and remote control can be afforded over vasoactivity in explanted murine aortic rings, as well as pancreatic beta cell function in islets of Langerhans. This study demonstrates the broad applicability of TOP271 to enzyme-dependent signalling processes, extends the toolbox of photoswitchable molecules to all classes of transmembrane receptors and utilizes photopharmacology to deduce receptor activation on a molecular level.
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35. The robustness of plant-pollinator assemblages: linking plant interaction patterns and sensitivity to pollinator loss.
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Julia Astegiano, François Massol, Mariana Morais Vidal, Pierre-Olivier Cheptou, and Paulo R Guimarães
- Subjects
Medicine ,Science - Abstract
Most flowering plants depend on pollinators to reproduce. Thus, evaluating the robustness of plant-pollinator assemblages to species loss is a major concern. How species interaction patterns are related to species sensitivity to partner loss may influence the robustness of plant-pollinator assemblages. In plants, both reproductive dependence on pollinators (breeding system) and dispersal ability may modulate plant sensitivity to pollinator loss. For instance, species with strong dependence (e.g. dioecious species) and low dispersal (e.g. seeds dispersed by gravity) may be the most sensitive to pollinator loss. We compared the interaction patterns of plants differing in dependence on pollinators and dispersal ability in a meta-dataset comprising 192 plant species from 13 plant-pollinator networks. In addition, network robustness was compared under different scenarios representing sequences of plant extinctions associated with plant sensitivity to pollinator loss. Species with different dependence on pollinators and dispersal ability showed similar levels of generalization. Although plants with low dispersal ability interacted with more generalized pollinators, low-dispersal plants with strong dependence on pollinators (i.e. the most sensitive to pollinator loss) interacted with more particular sets of pollinators (i.e. shared a low proportion of pollinators with other plants). Only two assemblages showed lower robustness under the scenario considering plant generalization, dependence on pollinators and dispersal ability than under the scenario where extinction sequences only depended on plant generalization (i.e. where higher generalization level was associated with lower probability of extinction). Overall, our results support the idea that species generalization and network topology may be good predictors of assemblage robustness to species loss, independently of plant dispersal ability and breeding system. In contrast, since ecological specialization among partners may increase the probability of disruption of interactions, the fact that the plants most sensitive to pollinator loss interacted with more particular pollinator assemblages suggest that the persistence of these plants and their pollinators might be highly compromised.
- Published
- 2015
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- View/download PDF
36. Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates
- Author
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Joshua Levitz, David J. Hodson, Vanessa A. Gutzeit, Bettina Mathes, Elisa D’Este, Johannes Broichhagen, Ben Jones, Julia Ast, Joon Lee, and Pascal Poc
- Subjects
0303 health sciences ,Bioconjugation ,Membrane permeability ,Chemistry ,Protein tag ,010402 general chemistry ,01 natural sciences ,Transmembrane protein ,0104 chemical sciences ,SNAP-tag ,03 medical and health sciences ,Extracellular ,Biophysics ,Intracellular ,030304 developmental biology ,G protein-coupled receptor - Abstract
Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.
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37. Fragmentación de hábitat, riqueza de polinizadores, polinización y reproducción de plantas nativas en el Bosque Chaqueño de Córdoba, Argentina
- Author
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Leonardo Galetto, Ramiro Aguilar, Mariana Musicante, Julia Astegiano, Ana Ferreras, Mariana Jausoro, Carolina Torres, Lorena Ashworth, and Cecilia Eynard
- Subjects
comunidades ,fragmentación de hábitat ,meta-análisis ,polinización ,región Chaqueña ,Environmental sciences ,GE1-350 ,Ecology ,QH540-549.5 - Abstract
En este trabajo se analiza la riqueza de polinizadores y la polinización y reproducción de plantas en un gradiente de fragmentación en el bosque Chaqueño de Córdoba, Argentina. Se espera encontrar una relación directa entre el área de los fragmentos, la riqueza de polinizadores y estos dos procesos. A partir de los datos obtenidos por varios investigadores, se plantearon los siguientes objetivos: i) evaluar, en la escala de sitio, la relación entre el área de los fragmentos, la riqueza de polinizadores y la polinización (medida como frecuencia de visitas a las flores), ii) analizar, en la escala de un conjunto de especies de plantas, los patrones de respuesta para variables relacionadas con la polinización (riqueza de polinizadores, frecuencia promedio de visitas) y reproducción (producción de frutos) considerando un gradiente de fragmentación, iii) comparar las tendencias obtenidas a partir de estas dos aproximaciones metodológicas y iv) discutir los resultados en el contexto de la conservación de la biodiversidad. No se encontró una menor riqueza o frecuencia de visitas de los polinizadores a medida que disminuye el área de los fragmentos. Los resultados obtenidos a través de meta-análisis muestran que las magnitudes del efecto global fueron significativamente diferentes de cero para la riqueza de polinizadores y la producción de frutos. Por el contrario, no se detectó un efecto global significativo en relación a la frecuencia de visitas de los polinizadores. Las tendencias encontradas considerando las dos aproximaciones no fueron uniformes. Las respuestas de los polinizadores y las plantas resultaron más complejas que lo planteado a priori, ya que si se analiza la heterogeneidad en las tendencias encontradas en el conjunto de especies de plantas estudiado dentro del gradiente de fragmentación se sugiere una susceptibilidad especie-específica. Asimismo, varias de las especies que fueron estudiadas en distintas temporadas mostraron variaciones en las tendencias de las variables consideradas en relación a la disminución del área de los fragmentos. Para políticas de conservación del bosque Chaqueño, sería interesante considerar aquellas especies de plantas y sus polinizadores asociados que estarían siendo afectadas negativamente por la fragmentación de hábitat.
- Published
- 2007
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