ORCID Profile
0000-0003-3596-2288
Current Organisation
Monash University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Biochemistry and Cell Biology not elsewhere classified | Protein Trafficking | Structural Biology (incl. Macromolecular Modelling) | Receptors and Membrane Biology | Nanoscale Characterisation | Cell Development, Proliferation and Death | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) | Materials Engineering not elsewhere classified | Signal Transduction | Microbiology not elsewhere classified
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences | Reproductive System and Disorders | Expanding Knowledge in the Physical Sciences | Human Pharmaceutical Products not elsewhere classified | Infectious diseases | Cancer and Related Disorders | Manufacturing not elsewhere classified | Cancer and related disorders | Organs, diseases and abnormal conditions not elsewhere classified | Expanding Knowledge in Technology | Infectious Diseases |
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Publications
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy
Publisher: EMBO
Date: 15-08-2022
Abstract: Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co‐ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient‐rich conditions, is unknown. Here, we identify an endosome‐dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα‐derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation‐induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5‐kinase, PIKfyve, to generate PI(3,5)P 2 . The SNX‐BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P 2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve‐dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient‐rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P 2 ‐dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.
High-fat diet increases autophagic flux in pancreatic beta cells in vivo and ex vivo in mice
Publisher: Springer Science and Business Media LLC
Date: 14-06-2015
DOI: 10.1007/S00125-015-3665-X
Abstract: Defective beta cell function during lipid oversupply and type 2 diabetes is associated with dysregulation of lysosomal function and autophagy. Whether this dysregulation represents augmentation or inhibition is unclear because of technical limitations in assaying autophagy. The current aim was to determine the effects of high-fat feeding on true autophagic flux in beta cells in vivo in mice, and to establish the relationship between autophagy, endoplasmic reticulum (ER) stress and apoptosis. Green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) mice were fed chow or high-fat diets for 8-10 weeks and injected with 100 mg kg(-1) day(-1) chloroquine for 5 days, prior to being killed, to block clearance of autophagic markers. Pancreases and livers were fixed and GFP-LC3 aggregates or autophagosomes were detected by fluorescence or electron microscopy, respectively. Independently, islets isolated from chow or high-fat-fed mice were treated for 2 h with chloroquine ex vivo, and immunoblotting was performed for markers of autophagy (LC3 lipidation - LC3II and p62/SQSTM1), ER stress (C/EBP homology protein [CHOP], phosphorylated eukaryotic initiation factor 2α [p-eIFα] and inositol requiring enzyme 1α [p-IRE1α]) and apoptosis (cleaved caspase-3). Numbers of autophagosomes and GFP puncta were increased in beta cells by combined high-fat feeding and chloroquine injection, indicative of enhanced autophagic flux. By contrast, GFP puncta were attenuated in liver under the same conditions. Relative to chow-fed controls, islets isolated from fat-fed mice exhibited higher LC3II levels when treated ex vivo with chloroquine. The combination of high-fat feeding and acute chloroquine treatment induced CHOP, p-eIF2α and caspase-3, but not either treatment alone. We provide the first in vivo demonstrations that high-fat feeding increases autophagic flux in pancreatic beta cells, and that this serves to protect against induction of terminal ER stress. We also highlight an approach for monitoring dietary alterations in autophagic flux using ex vivo manipulation of isolated islets.
INPP5E regulates phosphoinositide-dependent cilia transition zone function.
Publisher: Rockefeller University Press
Date: 20-12-2017
Abstract: Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e−/− embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e−/− ciliopathy phenotypes and “normalizes” Hedgehog signaling. INPP5E’s phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e−/− cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.
Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus
Publisher: Public Library of Science (PLoS)
Date: 05-07-2012
Single-fibre proteomics reveals a greater fibre-type specific mitochondrial remodelling with moderate than high-intensity training
Publisher: Cold Spring Harbor Laboratory
Date: 24-10-2022
DOI: 10.1101/2022.10.23.512956
Abstract: Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle s les, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our MS-based proteomics workflow, we provide new insights into fibre-specific mitochondrial adaptations following two types of exercise training that were very different in terms of metabolic demands and fibre recruitment patterns – moderate-intensity-continuous training (MICT) and sprint interval training (SIT). There was a larger number of differentially expressed proteins in each fibre type following MICT than SIT. Our novel mitochondrial normalisation strategy highlighted that most training-induced changes in mitochondrial protein abundances were stoichiometrically linked to the overall increase in mitochondrial content, except for the decreased abundance of complex IV subunits in both type I and II fibres following SIT, and the increase in proteins associated with fatty acid oxidation in type I fibres.
Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells
Publisher: Rockefeller University Press
Date: 10-2001
Abstract: Immature dendritic cells (DCs) s le their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.
Increased outer membrane vesicle formation in a Helicobacter pylori tolB mutant
Publisher: Wiley
Date: 09-02-2015
DOI: 10.1111/HEL.12196
Abstract: Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes.
Visualising coordination chemistry: fluorescence X-ray absorption near edge structure tomography
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6CC06747F
Abstract: Here we develop a measurement scheme to determine the abundance, distribution, and coordination environment of biological copper complexes in situ , without need for complex s le preparation.
Helicobacter pylori Outer Membrane Vesicle Size Determines Their Mechanisms of Host Cell Entry and Protein Content
Publisher: Frontiers Media SA
Date: 02-07-2018
The rab GTPase-Activating protein TBC1D4/AS160 contains an atypical phosphotyrosine-binding domain that interacts with plasma membrane phospholipids to facilitate GLUT4 trafficking in adipocytes
Publisher: Informa UK Limited
Date: 12-2012
DOI: 10.1128/MCB.00761-12
Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation
Publisher: Rockefeller University Press
Date: 18-11-2016
Abstract: Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members ided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome–lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome–lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.
Methods in renal research: Measurement of autophagic flux in the renal cortex ex vivo
Publisher: Wiley
Date: 22-08-2018
DOI: 10.1111/NEP.13251
Abstract: The role of autophagy in the kidney and many nephrological diseases has gained prominence in recent years. Much of this research has been focused on markers of autophagy that are static and reveal little about the state of this dynamic pathway. Other mechanistic investigations are limited to in vitro studies, that often provide circumstantial evidence of autophagic flux. Here we describe a method for measuring autophagic flux ex vivo that allows more direct observations to be made in situ regarding the state of autophagic flux within the renal cortex of a single animal.
HcRed, a genetically encoded fluorescent binary cross-linking agent for cross-linking of mitochondrial ATP synthase in saccharomyces cerevisiae
Publisher: Public Library of Science (PLoS)
Date: 04-04-2012
Association between Common Variation at the FTO Locus and Changes in Body Mass Index from Infancy to Late Childhood: The Complex Nature of Genetic Association through Growth and Development
Publisher: Public Library of Science (PLoS)
Date: 17-02-2011
Deficiency in apoptosis-inducing factor recapitulates chronic kidney disease via aberrant mitochondrial homeostasis
Publisher: American Diabetes Association
Date: 28-01-2016
DOI: 10.2337/DB15-0864
Abstract: Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.
Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism
Publisher: Cold Spring Harbor Laboratory
Date: 24-04-2020
DOI: 10.1101/2020.04.22.056440
Abstract: Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA) 3 -repeat operator cooperatively and with high affinity. Truncation of an N-terminal extension abolishes cooperative binding. The effector, N -acetylneuraminate, binds NanR and attenuates DNA binding. Crystal structure data show that N -acetylneuraminate binding to NanR causes a domain rearrangement that locks the protein in a conformation that prevents DNA binding. Single-particle cryo-electron microscopy structures of NanR bound to DNA reveal the DNA binding domain is reorganized to engage DNA, while the three dimers assemble in close proximity across the (GGTATA) 3 -repeat operator allowing protein-protein interactions to form via the N-terminal extensions. Our data provides a molecular basis for the regulation of bacterial sialic acid metabolism.
Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway
Publisher: Public Library of Science (PLoS)
Date: 26-01-2017
KBTBD13 is an actin-binding protein that modulates muscle kinetics
Publisher: American Society for Clinical Investigation
Date: 06-01-2020
DOI: 10.1172/JCI124000
Endosomes: multipurpose designs for integrating housekeeping and specialized tasks
Publisher: Springer Science and Business Media LLC
Date: 14-12-2001
DOI: 10.1007/S00418-001-0348-0
Abstract: In most cells the endocytic system is organized following a common concept that allows for the integrative handling of a variety of housekeeping functions. In addition, variations on the general scheme provide for specialized endosome-based pathways that occur only in specific cell types. The ersity of endosomal functions is not only reflected by characteristic molecular compositions, but also mirrored in their morphological organization. In this review we will first describe the general outline of the endocytic system by combining kinetic, morphological, molecular, and functional definitions. In the second part, adaptations of endosomes that allow their functioning in specialized processes, such as antigen presentation, synaptic vesicle formation, and glucose transport, will be addressed.
Deregulated Stat3 signaling dissociates pulmonary inflammation from emphysema in gp130 mutant mice
Publisher: American Physiological Society
Date: 04-2012
DOI: 10.1152/AJPLUNG.00285.2011
Abstract: Interleukin (IL)-6 is a potent immunomodulatory cytokine that is associated with emphysema, a major component of chronic obstructive pulmonary disease (COPD). IL-6 signaling via the gp130 coreceptor is coupled to multiple signaling pathways, especially the latent transcription factor signal transducer and activator of transcription (Stat)3. However, the pathological role of endogenous gp130-dependent Stat3 activation in emphysema is ill defined. To elucidate the role of the IL-6/gp130/Stat3 signaling axis in the cellular and molecular pathogenesis of emphysema, we employed a genetic complementation strategy using emphysematous gp130 F/F mice displaying hyperactivation of endogenous Stat3 that were interbred with mice to impede Stat3 activity. Resected human lung tissue from patients with COPD and COPD-free in iduals was also evaluated by immunohistochemistry. Genetic reduction of Stat3 hyperactivity in gp130 F/F : Stat3 −/+ mice prevented lung inflammation and excessive protease activity however, emphysema still developed. In support of these findings, Stat3 activation levels in human lung tissue correlated with the extent of pulmonary inflammation but not airflow obstruction in COPD. Furthermore, COPD lung tissue displayed increased levels of IL-6 and apoptotic alveolar cells, supporting our previous observation that increased endogenous IL-6 expression in the lungs of gp130 F/F mice contributes to emphysema by promoting alveolar cell apoptosis. Collectively, our data suggest that IL-6 promotes emphysema via upregulation of Stat3-independent apoptosis, whereas IL-6 induction of lung inflammation occurs via Stat3. We propose that while discrete targeting of Stat3 may alleviate pulmonary inflammation, global targeting of IL-6 potentially represents a therapeutically advantageous approach to combat COPD phenotypes where emphysema predominates.
Nonplasmacytoid, High IFN-α–Producing, Bone Marrow Dendritic Cells
Publisher: The American Association of Immunologists
Date: 15-04-2012
Abstract: Plasmacytoid dendritic cells (pDC) are the producers of type I IFNs in response to TLR9 ligands. However, we have found that when bone marrow is depleted of pDC, the IFN-α produced in response to TLR9 ligands is not fully removed. We assign the source of this non-pDC IFN-α as a newly described DC type. It displays the high IFN-α producing activity of pDC but to a more limited range of viruses. Unlike pDC, the novel DC display high T cell stimulation capacity. Moreover, unlike mouse pDC, they are matured with GM-CSF and are less prone to apoptosis upon activation stimuli, including viruses. We propose that these DC constitute a novel bone marrow inflammatory DC type, ideally geared to linking innate and adaptive immune responses in bone marrow via their potent IFN-α production and high T cell stimulatory capacity.
Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-golgi network in rat adipocytes
Publisher: American Society for Cell Biology (ASCB)
Date: 12-2000
Abstract: Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-d resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system.
Tetraspanin CD37 Regulates β2 Integrin-Mediated Adhesion and Migration in Neutrophils.
Publisher: The American Association of Immunologists
Date: 15-12-2015
Abstract: Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo β2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the β2 integrin ligand, ICAM-1, despite the normal display of high-affinity β2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates β2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect β2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated β2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.
Basal spot junctions of epithelial tissues respond to morphogenetic forces and regulate Hippo signaling
Publisher: Cold Spring Harbor Laboratory
Date: 17-09-2023
The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1
Publisher: Springer Science and Business Media LLC
Date: 19-09-2019
DOI: 10.1038/S41467-019-12279-2
Abstract: Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.
Genome-Wide Association Meta-Analysis of Cortical Bone Mineral Density Unravels Allelic Heterogeneity at the RANKL Locus and Potential Pleiotropic Effects on Bone
Publisher: Public Library of Science (PLoS)
Date: 18-11-2010
The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells
Publisher: Informa UK Limited
Date: 03-11-2013
DOI: 10.4161/AUTO.26557
Abstract: Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.
Dissecting the mechanism of insulin resistance using a novel heterodimerization strategy to activate akt
Publisher: Elsevier BV
Date: 02-2010
Cluster analysis of insulin action in adipocytes reveals a key role for Akt at the plasma membrane
Publisher: Elsevier BV
Date: 2010
The Burkholderia pseudomallei type III secretion system and BopA are required for evasion of LC3-associated phagocytosis
Publisher: Public Library of Science (PLoS)
Date: 11-03-2011
Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.
Publisher: American Association for Cancer Research (AACR)
Date: 03-2022
DOI: 10.1158/2159-8290.CD-21-0522
Abstract: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587
Immuno correlative light and electron microscopy on tokuyasu cryosections
Publisher: Elsevier
Date: 2014
CaMKII-Mediated Phosphorylation of the Myosin Motor Myo1c Is Required for Insulin-Stimulated GLUT4 Translocation in Adipocytes
Publisher: Elsevier BV
Date: 11-2008
DOI: 10.1016/J.CMET.2008.09.011
Abstract: The unconventional myosin Myo1c has been implicated in insulin-regulated GLUT4 translocation to the plasma membrane in adipocytes. We show that Myo1c undergoes insulin-dependent phosphorylation at S701. Phosphorylation was accompanied by enhanced 14-3-3 binding and reduced calmodulin binding. Recombinant CaMKII phosphorylated Myo1c in vitro and siRNA knockdown of CaMKIIdelta abolished insulin-dependent Myo1c phosphorylation in vivo. CaMKII activity was increased upon insulin treatment and the CaMKII inhibitors CN21 and KN-62 or the Ca(2+) chelator BAPTA-AM blocked insulin-dependent Myo1c phosphorylation and insulin-stimulated glucose transport in adipocytes. Myo1c ATPase activity was increased after CaMKII phosphorylation in vitro and after insulin stimulation of CHO/IR/IRS-1 cells. Expression of wild-type Myo1c, but not S701A or ATPase dead mutant K111A, rescued the inhibition of GLUT4 translocation by siRNA-mediated Myo1c knockdown. These data suggest that insulin regulates Myo1c function via CaMKII-dependent phosphorylation, and these events play a role in insulin-regulated GLUT4 trafficking in adipocytes likely involving Myo1c motor activity.
Rapid Activation of Akt2 Is Sufficient to Stimulate GLUT4 Translocation in 3T3-L1 Adipocytes
Publisher: Elsevier BV
Date: 04-2008
DOI: 10.1016/J.CMET.2008.02.008
Abstract: The serine/threonine kinase Akt2 has been implicated in insulin-regulated glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. However, it remains unclear whether activation of Akt2 is sufficient since a role for alternate signaling pathways has been proposed. Here we have engineered 3T3-L1 adipocytes to express a rapidly inducible Akt2 system based on drug-inducible heterodimerization. Addition of the dimerizer rapalog resulted in activation of Akt2 within 5 min, concomitant with phosphorylation of the Akt substrates AS160 and GSK3. Comparison with insulin stimulation revealed that the level of Akt2 activity observed with rapalog was within the physiological range, reducing the likelihood of off-target effects. Transient activation of Akt2 also increased glucose transport and GLUT4 translocation to the plasma membrane. These results show that activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes to an extent similar to insulin.
The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex
Publisher: Wiley
Date: 23-04-2012
Role for the Burkholderia pseudomallei Type Three Secretion System Cluster 1 bpscN gne in vrulence
Publisher: American Society for Microbiology
Date: 09-2011
DOI: 10.1128/IAI.01351-10
Abstract: Burkholderia pseudomallei , the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. The B. pseudomallei genome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved in B. pseudomallei virulence in a hamster infection model. We have characterized a B. pseudomallei mutant lacking expression of the predicted TTSS1 ATPase encoded by bpscN . This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analyses in vitro showed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1 bpscN gene product plays an important role in the intracellular survival of B. pseudomallei and the pathogenesis of murine infection.
Regulation of glucose transporter 4 translocation by the rab guanosine triphosphatase-activating protein AS160/TBC1D4: Role of phosphorylation and membrane association
Publisher: The Endocrine Society
Date: 12-2008
DOI: 10.1210/ME.2008-0111
Live-Cell CLEM of Subcellular Targets
Publisher: Elsevier
Date: 2014
Zebrafish models of BAG3 myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency
Publisher: Springer Science and Business Media LLC
Date: 02-10-2014
DOI: 10.1007/S00401-014-1344-5
Abstract: Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) can cause myofibrillar myopathy (MFM), a childhood-onset progressive muscle disease, characterized by the formation of protein aggregates and myofibrillar disintegration. In contrast to other MFM-causing proteins, BAG3 has no direct structural role, but regulates autophagy and the degradation of misfolded proteins. To investigate the mechanism of disease in BAG3-related MFM, we expressed wild-type BAG3 or the dominant MFM-causing BAG3 (BAG3(P209L)) in zebrafish. Expression of the mutant protein results in the formation of aggregates that contain wild-type BAG3. Through the stimulation and inhibition of autophagy, we tested the prevailing hypothesis that impaired autophagic function is responsible for the formation of protein aggregates. Contrary to the existing theory, our studies reveal that inhibition of autophagy is not sufficient to induce protein aggregation. Expression of the mutant protein, however, did not induce myofibrillar disintegration and we therefore examined the effect of knocking down Bag3 function. Loss of Bag3 resulted in myofibrillar disintegration, but not in the formation of protein aggregates. Remarkably, BAG3(P209L) is able to rescue the myofibrillar disintegration phenotype, further demonstrating that its function is not impaired. Together, our knockdown and overexpression experiments identify a mechanism whereby BAG3(P209L) aggregates form, gradually reducing the pool of available BAG3, which eventually results in BAG3 insufficiency and myofibrillar disintegration. This mechanism is consistent with the childhood onset and progressive nature of MFM and suggests that reducing aggregation through enhanced degradation or inhibition of nucleation would be an effective therapy for this disease.
FLNC myofibrillar myopathy results from impaired autophagy and protein insufficiency
Publisher: Oxford University Press (OUP)
Date: 11-03-2016
DOI: 10.1093/HMG/DDW080
Abstract: Myofibrillar myopathy is a progressive muscle disease characterized by the disintegration of muscle fibers and formation of protein aggregates. Causative mutations have been identified in nine genes encoding Z-disk proteins, including the actin binding protein filamin C (FLNC). To investigate the mechanism of disease in FLNC
BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-02-2018
Abstract: Mitochondrial DNA (mtDNA) is a potent damage-associated molecular pattern that, if it reaches the cytoplasm or extracellular milieu, triggers innate immune pathways. mtDNA signaling has been implicated in a wide range of diseases however, the mechanisms of mtDNA release are unclear, and the process has not been observed in real time thus far. McArthur et al. used live-cell lattice light-sheet microscopy to look at mtDNA release during intrinsic apoptosis. Activation of the pro-death proteins BAK and BAX resulted in the formation of large macro-pores in the mitochondrial outer membrane. These massive holes caused the inner mitochondrial membrane to balloon out into the cytoplasm, resulting in mitochondrial herniation. This process allowed the contents of the mitochondrial matrix, including mtDNA, to escape into the cytoplasm. Science , this issue p. eaao6047
The first transmembrane region of complement component-9 acts as a brake on its self-assembly
Publisher: Springer Science and Business Media LLC
Date: 15-08-2018
DOI: 10.1038/S41467-018-05717-0
Abstract: Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.
The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.BIOCEL.2006.03.009
Abstract: Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, in idually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.
The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression
Publisher: Public Library of Science (PLoS)
Date: 05-04-2012
Publisher Correction: Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality
Publisher: Springer Science and Business Media LLC
Date: 09-07-2020
DOI: 10.1038/S41467-020-17441-9
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM
Publisher: Public Library of Science (PLoS)
Date: 22-04-2014
Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase
Publisher: American Diabetes Association
Date: 10-2006
DOI: 10.2337/DB05-1404
Abstract: Although interleukin-6 (IL-6) has been associated with insulin resistance, little is known regarding the effects of IL-6 on insulin sensitivity in humans in vivo. Here, we show that IL-6 infusion increases glucose disposal without affecting the complete suppression of endogenous glucose production during a hyperinsulinemic-euglycemic cl in healthy humans. Because skeletal muscle accounts for most of the insulin-stimulated glucose disposal in vivo, we examined the mechanism(s) by which IL-6 may affect muscle metabolism using L6 myotubes. IL-6 treatment increased fatty acid oxidation, basal and insulin-stimulated glucose uptake, and translocation of GLUT4 to the plasma membrane. Furthermore, IL-6 rapidly and markedly increased AMP-activated protein kinase (AMPK). To determine whether the activation of AMPK mediated cellular metabolic events, we conducted experiments using L6 myotubes infected with dominant-negative AMPK α-subunit. The effects described above were abrogated in AMPK dominant-negative–infected cells. Our results demonstrate that acute IL-6 treatment enhances insulin-stimulated glucose disposal in humans in vivo, while the effects of IL-6 on glucose and fatty acid metabolism in vitro appear to be mediated by AMPK.
Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality
Publisher: Springer Science and Business Media LLC
Date: 05-06-2020
DOI: 10.1038/S41467-020-16633-7
Abstract: Virtual memory T (T VM ) cells are antigen-naïve CD8 + T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (T MEM ) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of T VM cells and their altered functionality with age, here we investigate T VM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of T VM , but not T MEM , cells and it increases with age in both subsets. The elevated SRC observed in aged mouse T VM cells and human CD8 + T cells from older in iduals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of T VM , but not T MEM , cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8 + T cells.
Characterization of the Role of the Rab GTPase-activating Protein AS160 in Insulin-regulated GLUT4 Trafficking
Publisher: Elsevier BV
Date: 11-2005
INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer
Publisher: Springer Science and Business Media LLC
Date: 25-05-2021
DOI: 10.1038/S41467-021-23241-6
Abstract: INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P 2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA -mutant ER + breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA -mutant ER + breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P 2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER + breast cancer via PI(3,4)P 2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies.
Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination
Publisher: Society for Neuroscience
Date: 06-05-2019
The serine/threonine kinase ULK1 is a target of multiple phosphorylation events
Publisher: Portland Press Ltd.
Date: 14-11-2011
DOI: 10.1042/BJ20101894
Abstract: Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser555in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser774 and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr180 that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.
TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages.
Publisher: Springer Science and Business Media LLC
Date: 13-01-2021
DOI: 10.1038/S41598-020-79637-9
Abstract: Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno -correlative light and electron microscopy ( i CLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of i CLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The i CLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.
Periaxonal and nodal plasticities modulate action potential conduction in the adult mouse brain
Publisher: Elsevier BV
Date: 2021
DOI: 10.1016/J.CELREP.2020.108641
Abstract: Central nervous system myelination increases action potential conduction velocity. However, it is unclear how myelination is coordinated to ensure the temporally precise arrival of action potentials and facilitate information processing within cortical and associative circuits. Here, we show that myelin sheaths, supported by mature oligodendrocytes, remain plastic in the adult mouse brain and undergo subtle structural modifications to influence action potential conduction velocity. Repetitive transcranial magnetic stimulation and spatial learning, two stimuli that modify neuronal activity, alter the length of the nodes of Ranvier and the size of the periaxonal space within active brain regions. This change in the axon-glial configuration is independent of oligodendrogenesis and robustly alters action potential conduction velocity. Because aptitude in the spatial learning task was found to correlate with action potential conduction velocity in the fimbria-fornix pathway, modifying the axon-glial configuration may be a mechanism that facilitates learning in the adult mouse brain.
Assembly of the secretion pores GspD , W za and CsgG into bacterial outer membranes does not require the O mp85 proteins BamA or TamA
Publisher: Wiley
Date: 06-06-2015
DOI: 10.1111/MMI.13055
Abstract: In Gram-negative bacteria, β-barrel proteins are integrated into the outer membrane by the β-barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo-electron microscopy show a erse set of secretion pores in Gram-negative bacteria, with α-helix (Wza and GspD) or β-strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG. Like the well characterised pilotins for GspD and other secretins, small periplasmic proteins enhance the assembly of the CsgG β-barrel. We discuss a model for integral protein assembly into the bacterial outer membrane, focusing on the commonalities and differences in the assembly of Wza, GspD and CsgG.
Autophagic lysosome reformation in health and disease
Publisher: Informa UK Limited
Date: 21-11-2022
DOI: 10.1080/15548627.2022.2128019
Abstract: Lysosomes are the primary degradative compartment within cells and there have been significant advances over the past decade toward understanding how lysosome homeostasis is maintained. Lysosome repopulation ensures sustained autophagy function, a fundamental process that protects against disease. During macroautophagy/autophagy, cellular debris is sequestered into phagophores that mature into autophagosomes, which then fuse with lysosomes to generate autolysosomes in which contents are degraded. Autophagy cannot proceed without the sufficient generation of lysosomes, and this can be achieved via their de novo biogenesis. Alternatively, during autophagic lysosome reformation (ALR), lysosomes are generated via the recycling of autolysosome membranes. During this process, autolysosomes undergo significant membrane remodeling and scission to generate membrane fragments, that mature into functional lysosomes. By utilizing membranes already formed during autophagy, this facilitates an efficient pathway for re-deriving lysosomes, particularly under conditions of prolonged autophagic flux. ALR dysfunction is emerging as an important disease mechanism including for neurodegenerative disorders such as hereditary spastic paraplegia and Parkinson disease, neuropathies including Charcot-Marie-Tooth disease, lysosome storage disorders, muscular dystrophy, metabolic syndrome, and inflammatory and liver disorders. Here, we provide a comprehensive review of ALR, including an overview of its dynamic spatiotemporal regulation by MTOR and phosphoinositides, and the role ALR dysfunction plays in many diseases.
Immuno-correlative light and electron microscopy (iCLEM) using SEM v1
Publisher: ZappyLab, Inc.
Date: 19-05-2021
DOI: 10.17504/PROTOCOLS.IO.BU54NY8W
Abstract: Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy with the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu s le preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for ex le, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-SEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has erse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-SEM provides a large quantifiable overview of 200 nm tissue sections without the presence of grid bars, and thicker sections enhance the immunofluorescent labelling.
GLUT4 trafficking in a test tube
Publisher: Elsevier BV
Date: 09-2005
DOI: 10.1016/J.CMET.2005.08.008
Abstract: Insulin regulates glucose transport in muscle and fat cells by stimulating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In this issue of Cell Metabolism, Holman and colleagues reconstitute this process in vitro, providing a system that promises new breakthroughs in our understanding of this important metabolic process.
Immuno-correlative light and electron microscopy (iCLEM) using STEM v1
Publisher: ZappyLab, Inc.
Date: 30-06-2021
DOI: 10.17504/PROTOCOLS.IO.BV89N9Z6
Abstract: Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy and the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu s le preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for ex le, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-STEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has erse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-STEM involves the examination of ultrathin tissue sections (62-70 nm) using immunofluorescence labelling and subsequent SEM imaging to obtain a high resolution overview of these sections with greater morphological detail compared to iCLEM-SEM. This protocol should be of particular interest to EM facilities with SEM, but not TEM access.
Podocyte-specific Nox4 deletion affords renoprotection in a mouse model of diabetic nephropathy
Publisher: Springer Science and Business Media LLC
Date: 28-10-2015
Guidelines for the use and interpretation of assays for monitoring autophagy
Publisher: Informa UK Limited
Date: 04-2012
DOI: 10.4161/AUTO.19496
Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism
Publisher: Springer Science and Business Media LLC
Date: 31-03-2021
DOI: 10.1038/S41467-021-22253-6
Abstract: Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA) 3 -repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA) 3 -repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N -acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N -acetylneuraminate reveals a domain rearrangement upon N -acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.
Structure of the poly-C9 component of the complement membrane attack complex
Publisher: Springer Science and Business Media LLC
Date: 04-02-2121
DOI: 10.1038/NCOMMS10588
Abstract: The membrane attack complex (MAC) erforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.
Deep multi-omic profiling reveals extensive mitochondrial remodeling driven by glycemia in early diabetic kidney disease
Publisher: Cold Spring Harbor Laboratory
Date: 26-10-2023
Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function
Publisher: Springer Science and Business Media LLC
Date: 05-2015
RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells
Publisher: eLife Sciences Publications, Ltd
Date: 02-12-2020
DOI: 10.7554/ELIFE.63452
Abstract: The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8 + T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.
Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization
Publisher: Elsevier BV
Date: 11-2016
Biogenesis of insulin-responsive GLUT4 vesicles is independent of brefeldin A-sensitive trafficking
Publisher: Wiley
Date: 08-2000
DOI: 10.1034/J.1600-0854.2000.010809.X
Abstract: Insulin stimulates translocation of GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. As a significant amount of GLUT4 is localised to the TGN, independently of the biosynthetic pathway, one possibility is that trafficking via the TGN is important in either intracellular sequestration or insulin-dependent movement to the cell surface. In this study we have used immuno-electron microscopy to show that GLUT4 is localised to AP-1 vesicles in the TGN region in 3T3-L1 adipocytes. To dissect the role of this trafficking pathway we used brefeldin A (BFA) to disrupt AP-1 association with membranes. Despite a reorganisation of GLUT4 compartments following BFA treatment, the intracellular sequestration of GLUT4, and its insulin-dependent movement to the cell surface, was unaffected. BFA increased the half time of reversal of insulin-stimulated glucose transport from 17 to 30 min but did not prevent complete reversal. Furthermore, following reversal restimulation of glucose transport activity by insulin was not compromised. We conclude that under basal conditions GLUT4 cycles between the TGN and endosomes via the AP-1 pathway. However, neither this pathway, nor any other BFA-sensitive pathway, appears to play a major role in insulin-dependent recruitment of GLUT4 to the cell surface.
Immuno-correlative light and electron microscopy (iCLEM) using TEM v1
Publisher: ZappyLab, Inc.
Date: 24-03-2021
DOI: 10.17504/PROTOCOLS.IO.BTMRNK56
Abstract: Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy with the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu s le preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for ex le, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-TEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has erse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-TEM provides high resolution ultrastructural detail of cells, and consecutive ultrathin (62-70 nm) tissue sections can be examined using different labelling techniques involving the use of immunofluorescent and immunogold markers.
A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160
Publisher: Elsevier BV
Date: 09-2006
Metformin rescues muscle function in BAG3 myofibrillar myopathy models
Publisher: Informa UK Limited
Date: 19-10-2020
Hydrogen Production from Methane Cracking in Dielectric Barrier Discharge Catalytic Plasma Reactor using a Nanocatalyst
Publisher: MDPI AG
Date: 10-11-2020
DOI: 10.20944/PREPRINTS202011.0316.V1
Abstract: The study experimentally investigated a novel approach for producing hydrogen from methane cracking in dielectric barrier discharge catalytic plasma reactor using a nanocatalyst. Plasma-catalytic methane (CH4) cracking was undertaken in a dielectric barrier discharge (DBD) catalytic plasma reactor using Ni/MgAl2O4. The Ni/MgAl2O4 was synthesised through co-precipitation followed customised hydrothermal method. The physicochemical properties of the catalyst were examined using X-ray diffraction (XRD), scanning electron microscopy - energy dispersive X-ray spectrometry (SEM-EDX) and thermogravimetric analysis (TGA). The Ni/MgAl2O4 shows a porous structure spinel MgAl2O4 and thermal stability. In the catalytic-plasma methane cracking, the Ni/MgAl2O4 shows 80% of the maximum conversion of CH4 with H2 selectivity 75%. Furthermore, the stability of the catalyst was encouraging 16 hours with CH4 conversion above 75%, and the selectivity of H2 was above 70%. This is attributed to the synergistic effect of the catalyst and plasma. The plasma-catalytic CH4 cracking is a promising technology for the simultaneous H2 and carbon nanotubes (CNTs) production for energy storage applications.
Bacterial cancer therapy in autochthonous colorectal cancer affects tumor growth and metabolic landscape
Publisher: American Society for Clinical Investigation
Date: 28-10-2021
Testing of therapies in a novel nebulin nemaline myopathy model demonstrate a lack of efficacy
Publisher: Springer Science and Business Media LLC
Date: 30-05-2018
Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis
Publisher: Public Library of Science (PLoS)
Date: 30-03-2018
An ultrastructural investigation of tumors undergoing regression mediated by immunotherapy
Publisher: Impact Journals, LLC
Date: 14-12-2017
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