Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c <i>In Vitro</i>

Background: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes. Methodology/Principal Findings Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex. Conclusion/Significance Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

Competition between Allowed and First-Forbidden β Decay: The Case of Hg 208 → Tl 208

6 pags., 4 figs., 1 tab.The β decay of Hg208 into the one-proton hole, one neutron-particle Tl81208127 nucleus was investigated at CERN-ISOLDE. Shell-model calculations describe well the level scheme deduced, validating the proton-neutron interactions used, with implications for the whole of the N>126, Z<82 quadrant of neutron-rich nuclei. While both negative and positive parity states with spin 0 and 1 are expected within the Qβ window, only three negative parity states are populated directly in the β decay. The data provide a unique test of the competition between allowed Gamow-Teller and Fermi, and first-forbidden β decays, essential for the understanding of the nucleosynthesis of heavy nuclei in the rapid neutron capture process. Furthermore, the observation of the parity changing 0+→0-β decay where the daughter state is core excited is unique, and can provide information on mesonic corrections of effective operators.This work was supported by the European Union under Contracts No. 262010 (ENSAR) and No. 654002 (ENSAR2), the Science and Technology Facilities Council (UK), the German BMBF under Contract No. 05P18PKCIA and “Verbundprojekt 05P2018,” the MINECO Projects No. FPA2015-65035-P, No. RTI2018- 098868-B-I00, No. FPA2015-64969-P, and No. FPA2017- 87568-P (Spain), FWO-Vlaanderen (Belgium), GOA/ 2015/010 (BOF KU Leuven), the Excellence of Science programme (EOS-FWO), the Interuniversity Attraction Poles Programme initiated by the Belgian Science Policy Office (BriX network P7/12), the Romanian IFA project CERN-RO/ISOLDE and the Polish National Science Centre under Contracts No. UMO-2015/18/M/ST2/00523 and No. UMO-2019/33/N/ST2/03023. P. H. R. and S. M. J. acknowledge support from the UK Department for Business, Energy and Industrial Strategy via the National Measurement Office. Zs. P. acknowledges support from the ExtreMe Matter Institute EMMI at the GSI Helmholtzzentrum fr Schwerionenforschung, Darmstadt, Germa

The Actin-Binding Protein Capulet Genetically Interacts with the Microtubule Motor Kinesin to Maintain Neuronal Dendrite Homeostasis

BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease

Octupole states in Tl-207 studied through beta decay

The beta decay of Hg-207 into the single-proton-hole nucleus Tl-207 has been studied through gamma-ray spectroscopy at the ISOLDE Decay Station (IDS) with the aim of identifying states resulting from coupling of the pi s(1/2)(-1), pi d(3/2)(-1) and pi h(11/2)(-1) shell model orbitals to the collective octupole vibration. Twenty-two states were observed lying between 2.6 and 4.0 MeV, eleven of which were observed for the first time, and 78 new transitions were placed. Two octupole states (s(3/2)-coupled) are identified and three more states (d(3/2)-coupled) are tentatively assigned using spin-parity inferences, while further h(11/2)-coupled states may also have been observed for the first time. Comparisons are made with state-of-the-art large-scale shell model calculations and previous observations made in this region, and systematic underestimation of the energy of the octupole vibrational states is noted. We suggest that in order to resolve the difference in predicted energies for collective and noncollective t = 1 states (t is the number of nucleons breaking the Pb-208 core), the effect of t = 2 mixing may be reduced for octupole-coupled states. The inclusion of mixing with t = 0, 2, 3 excitations is necessary to replicate all t = 1 state energies accurately.Peer reviewe

Competition between Allowed and First-Forbidden beta Decay : The Case of Hg-208 -> Tl-2(0)8

The beta decay of Hg-208 into the one-proton hole, one neutron-particle Tl-208(81)127 nucleus was investigated at CERN-ISOLDE. Shell-model calculations describe well the level scheme deduced, validating the proton-neutron interactions used, with implications for the whole of the N > 126, Z 0(-)beta decay where the daughter state is core excited is unique, and can provide information on mesonic corrections of effective operators.Peer reviewe

Hearing shapes of drums - mathematical and physical aspects of isospectrality

In a celebrated paper '"Can one hear the shape of a drum?"' M. Kac [Amer. Math. Monthly 73, 1 (1966)] asked his famous question about the existence of nonisometric billiards having the same spectrum of the Laplacian. This question was eventually answered positively in 1992 by the construction of noncongruent planar isospectral pairs. This review highlights mathematical and physical aspects of isospectrality.Comment: 42 pages, 60 figure

Shell evolution of N = 40 isotones towards 60Ca: First spectroscopy of 62Ti

Excited states in the N=40 isotone 62Ti were populated via the 63V(p,2p)62Ti reaction at ∼200 MeV/nucleon at the Radioactive Isotope Beam Factory and studied using γ-ray spectroscopy. The energies of the 21+→0gs+ and 41+→21+ transitions, observed here for the first time, indicate a deformed 62Ti ground state. These energies are increased compared to the neighboring 64Cr and 66Fe isotones, suggesting a small decrease of quadrupole collectivity. The present measurement is well reproduced by large-scale shell-model calculations based on effective interactions, while ab initio and beyond mean-field calculations do not yet reproduce our findings. The shell-model calculations for 62Ti show a dominant configuration with four neutrons excited across the N=40 gap. Likewise, they indicate that the N=40 island of inversion extends down to Z=20, disfavoring a possible doubly magic character of the elusive 60Ca