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Transport and signalling in Polarized Cells

Final Report Summary - TRANSPOL (Transport and signalling in Polarized Cells)

PUBLISHABLE SUMMARY
The ITN Transpol is an international training network at the intersection of physics and molecular cell biology. The ITNs objectives are a better understanding of ligand/receptor-mediated signaling mechanisms across the membrane, the lipid/protein interface in endocytosis, the signalling properties of endosomes, and deregulated trafficking and signalling pathways in disease. Furthermore, the concept of Transpol comprises the translation of its research program into an interdisciplinary and international training program with trans-national visits for Early stage researchers (ESR) and Experienced Researchers (ER). In total Transpol performed quality training of 14 ESRs and 1 ER.
Our scientific objectives were:
Work Package 1. Signal transduction mechanism of ligand/receptor systems across the plasma membrane. Understanding of the signalling mechanism across the plasma membrane of: a) The receptor for the bacterial toxin ShigaB (Shigatoxin B), the glycolipid Gb3. b) The interferon-receptor. c) The ionotropic glutamate-receptor (AMPA receptor).
Work Package 2. Characterization of protein/lipid interactions and their relation to endocytosis. For this work package the following points were addressed: a.) The shape and force generation by clathrin coats onto lipid membranes. b.) The induction of membrane invagination via Shigatoxin B. c.) Modelling protein-induced membrane curvature using coarse-grained simulations. d.) Impact of lipid mechanics and lateral organization on membrane deformation and trafficking.
Work Package 3. Understanding the molecular relationship of intracellular trafficking and signaling. Scientific objectives within this work package of the ITN are: a.) Analysis of the role of endocytic and trafficking pathways in interferon receptor signaling. b.) Analysis of the role of the protein tyrosine phosphatase in intracellular receptor trafficking. c.) The mechanisms of endosome-to-nucleus trafficking and its impact on transcription. d.) Testing of models to characterize cell adhesion and signalling/trafficking processes using atomic force microscopy
Work Package 4. Impaired intracellular trafficking/signalling in human diseases. The specific scientific objectives are: a.) Characterization of FRMPD2 mediated protein complex b.) Functional characterization of the Parkinson´s disease protein LRRK2. c.) The impact of intracellular trafficking/signalling on the mechanical properties of cells measured with atomic force microscopy and optical stretcher and its relevance to tumor invasion and metastasis.
Summary of the work performed:
WP.1.1. ESR1, Haifei GAO at IC/Institute Curie (36 months). Supervisor: Dr. Ludger Johannes. a) Different molecular species of the Shiga toxin receptor, the glycosphingolipid Gb3 were obtained by chemical synthesis b) Grazing incidence x-ray diffraction (GIXD) was used to investigate lipid reorganization induced by Shiga toxin-binding to Gb3.c) Shiga toxin clustering was studied by fluorescence correlation spectroscopy (FCS) on giant unilamellar vesicles (GUVs) to test on thermal fluctuation-driven aggregation of nanoparticles on lipid membranes, which has been proposed on the basis of theoretical calculations and computer simulation studies.
WP.1.2. ER, Nanaocha Sharma at WEIZ (24 months). Supervisor: Dr. Gideon Schreiber. Systematic insertion of disulfide bonds between the subdomains SD3 and SD4 of IFNAR1 enhances binding affinity toward its ligand. In vitro binding affinity assay correlates with the in vivo gene induction and STAT phosphorylation. Using cross linker and mass spectrometry a disulfide bond between the SD3 and SD4 was identified.
WP.1.3. ESR2, Sandra Lemos at RUB (36 months). Supervisor: Dr. Michael Hollmann. In Xenopus laevis oocytes, the type II-TARPs γ5 and γ7 play a critical role modulating steady-state currents of AMPA receptors. The results prove a strictly AMPAR-selective modulation, as kainate receptor steady-state currents were not altered by co-expression with type II-TARPs.
WP.2.1. ESR3, Valentina Galli at UGEN (36 months). Supervisor: Dr. Aurelien Roux. Dynamin-2 conformational specific antibodies were selected to study the dynamics of membrane scission in vivo: Upon clathrin mediated endocytosis dynamin-2 polymerizes around the neck of the bud in form of a helix and, upon GTP hydrolysis, changes conformation and twists and constricts the neck promoting fission. Conformation specific antibodies were characterized able to recognize the two main states of Dynamin-2 (free or bound to the nucleotide).
WP.2.2. ESR4, Weria Pezeshkian at MEMPH (36 months). Supervisor: Dr. John Ipsen. Aggregation of nano-particles by membrane fluctuation-mediated interactions have been performed: Computer simulations and theoretical calculations have been used to show that the stability of the configuration is affected by the amount the inclusions such as nano-particles perturbing the membrane fluctuations. The results from this study may be of relevance for future strategies of tackling pathogens as a treatment to human health.
WP.2.3. ESR5, Eugenia Cammarota at UCAM (36 months). Supervisor: Dr. Pietro Cicuta. A dynamical phenotype of activated macrophages was shown at the single cell level: The motility, quantified as the mean square displacement (MSD), increase in active cells. The number of cells that have an oscillatory trajectory along the polarization axis increase with the detection of IFN- γ and decrease with LPS. The results represent a fascinating point of contact between fundamental physics (properties of critical points) and biology.
WP.2.4. ESR6, Sergii Shydlovskyi at RUB (36 months). Supervisor: Dr. Christian Herrmann: Here the importance of the farnesyl tail of human Guanylate Binding Protein 1 (hGBP1) for membrane anchorage was demonstrated. Most intriguingly, it was shown that binding of the protein to the membrane is strictly nucleotide dependent. While farnesylated hGBP1 in the presence of GMP or GDP is only found in the soluble fraction it becomes completely attracted to the bilayer surface after addition of GTP.
WP.3.1. ESR7, Daniela Chmiest at IC (36 months). Supervisor: Dr. Christophe Lamaze. A novel role for the endosomal retromer complex was established by elucidating the role of ESCRT proteins in the interferon receptor mediated JAK/STAT signaling pathway. The new downstream mechanism of the JAK/STAT pathway could be used for developing therapeutic targets.
WP.3.2. ESR8, Fangyan Yu at SHEF (12 months RUB, 24 months SHEF). Supervisor: Dr. Kai Erdmann. Interaction of PTP-BL with SDCCAG3 has been identified to be a novel endocytic protein localizing to early, recycling endosomes and cilia. An in-depth study of SDCCAG3 in cilia will help in understanding of cilia formation and mechanism of ciliopathies.
WP3.3: ESR9, Sam Dinesh Stephen at IIMCB (36 months). Supervisor: Dr. Marta Miaczynska. RNAi-based screens to measure transcriptional activity depended on interferon stimulated response (ISRE) activated by type-1 interferons (IFNalpha) has been performed. As a result, components of the ESCRT family of endosomal adaptors may affect the response of cells to interferon. Important for novel therapies employing interferons.
WP.3.4. ESR10, Gilbert Ng at JPK (36 months). Supervisor: Dr. Alex Winkel. Exploration of the mechanisms by which certain cells can sense the stiffness of their surrounding by the use of atomic force microscope (AFM). Establishment of new applications for AFM in basic biological and medical research.
WP3.5 ESR 15, Agnieszka Skowronek at SHEF (21 month) Supervisor: Dr. Kai Erdmann. Identification of new interaction partners of FRMPD2 and characterization of the FRMPD2 mediated protein complex with respect to epithelial cell polarization. This will help us to understand the importance and role of FRMPD2 in the development and functions of living organisms.
WP.4.2. ESR12 Panagiotis Athanasopoulos at RUB (36 months). Supervisor: Dr. R. Heumann Protein Phosphatase 2A (PP2A) was identified as an interacting partner of LRRK2. Additionally, it was demonstrated that only the ROC domain is needed to interact with the three subunits of PP2A in SH-SY5Y cells and in Hela cells. The alpha subunit of PP2A (PP2Aa) might be a novel target for pharmacological intervention.
WP.4.3. ESR13, Chii Jou Chan at UCAM (36 months). Supervisor: Dr. Jochen Guck. A detailed characterization of the effect of heating on the passive and active mechanical response of cells has been performed. A novel active contraction mechanism in cells when heated above 52C was uncovered. The underlying mechanism was the heat-activated influx of Ca-ions through TRPV2 channels, which then led to an increase in the myosin activity and resulting cortex contraction.
WP.4.4. ESR14 at SILAN (36 months). Supervisor: Dr. Hermann Heumann. A Drosophila Parkinson’s Disease model has been established. It was analyzed through SILAC labelling and mass spectrometry. Additionally, the phenotype of the model was tested and described. This will help us to understand better Parkinson’s disease.
Coordinator Contact: Prof. Dr. Rolf Heumann, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Tel:+49-234-3224230 Fax:+49-234-3214105 e-mail: rolf.heumann@rub.de
TRANSPOL-office: e-mail: transpol@rub.de (Logo: see attachment I)