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Dynamics of the Plasma Membrane Transporter GLUT4

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Dynamics of the Plasma Membrane Transporter GLUT4 Dynamics of the Plasma Membrane Transporter GLUT4 Silke Morris (née Machauer) BSc, MSc Thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy April 2020 Institute of Molecular, Cell and Systems Biology College of Medical, Veterinary and Life Sciences University of Glasgow 2 Abstract Glucose homeostasis in the human body is maintained by hormones of the pancreas, mostly glucagon and insulin. Insulin is secreted when blood glucose levels are high and triggers a signalling cascade that results in glucose uptake via the glucose transporter GLUT4 in peripheral tissues. GLUT4 is the only glucose transporter that responds to insulin stimulation and it slowly recycles between intracellular storage compartments and the plasma membrane. In the basal state, the majority of GLUT4 is intracellularly localised. Insulin stimulation results in movement (“translocation”) of GLUT4 from these intracellular stores to the plasma membrane. The signalling cascade from insulin binding to its receptor to translocation of GLUT4 is comparatively well understood. Less is known about the dynamics of GLUT4 within the plasma membrane itself. Advances in light microscopy techniques, such as Total Internal Reflection Fluorescence and super-resolution microscopy, have allowed new insights into the events in the membrane. It has recently been proposed that GLUT4 is located in plasma membrane clusters and that another effect of insulin is the dispersal of these GLUT4 clusters. The main objective of this work was to develop a microscopy-based assay to visualise and quantify these clusters and to investigate the molecular mechanisms behind clustering and dispersal of the glucose transporter in response to insulin. The majority of this work has been carried out in 3T3 L1 adipocytes, a widely used cell model for the study of GLUT4. However, this cell line is difficult to maintain, and its genetic manipulation is very challenging. For this reason, we investigated HeLa cells as a suitable substitute cell model for preliminary screenings. Using Total Internal Reflection Fluorescence Microscopy and Spatial Intensity Distribution Analysis, we gained new insight into the dynamics of plasma membrane GLUT4 in both 3T3 L1 adipocytes and HeLa cells. We found that the transporter forms an oligomer of high order in the plasma membrane in both cell types. Further, we compared the dynamics of GLUT4 mobilisation in response to insulin and found similar results. Based on these findings, we carried out an siRNA knock-down screening to determine proteins involved in intracellular 3 GLUT4 trafficking and found that GOSR1 and Ykt6 are promising targets for further examination. Single molecule localisation microscopy allowed us to accomplish our aim to assay GLUT4 clustering and dispersal. Using dSTORM and Ripley’s K-function, as well as Bayesian cluster analysis methods, we showed that GLUT4 is indeed located in clusters in the plasma membrane and that insulin stimulation leads to its dispersal. We found that treatment with Galectin-3, a drug that inhibits glucose uptake, impedes the dispersal. Building upon previous research in our group that identified EFR3a as a membrane-localised protein involved in glucose uptake, we knocked-down EFR3a in 3T3 L1 adipocytes and found that this also disrupts GLUT4 dispersal, which we hypothesise could be a potential drug target for type 2 diabetes. Taken together, the findings presented in this thesis suggest HeLa cells as a suitable cell model for initial assessments of research questions related to GLUT4 trafficking. Furthermore, a robust assay to measure GLUT4 dispersal was established. 4 Table of Contents Abstract ...................................................................................... 2 List of Tables ................................................................................ 8 List of Figures ............................................................................... 9 Acknowledgements ........................................................................ 11 Author’s Declaration ...................................................................... 13 Abbreviations .............................................................................. 14 Chapter 1 Introduction ................................................................. 18 1.1 Glucose Homeostasis ............................................................ 18 1.2 Diabetes Mellitus ................................................................ 20 1.3 Glucose Transporters ........................................................... 21 1.3.1 Glucose Transporter 4 ..................................................... 23 1.4 The GLUT4 Storage Compartment ............................................ 25 1.4.1 Endocytosis of GLUT4 ...................................................... 30 1.4.2 Other Components and Biogenesis of GSVs ............................. 30 1.5 Insulin Signalling ................................................................. 33 1.5.1 The APS Pathway ........................................................... 34 1.5.2 The PI3K Pathway .......................................................... 34 1.6 GLUT4 Trafficking Proteins .................................................... 36 1.6.1 Rab Proteins ................................................................. 36 1.6.2 SNARE and SM Proteins..................................................... 38 1.6.2.1 The SNARE Complex ................................................... 38 1.6.2.2 VAMPs ................................................................... 39 1.6.2.3 SM Proteins ............................................................. 40 1.6.3 Translocation of GSVs Along Filaments and Microtubules ............ 41 1.7 GLUT4 in the Plasma Membrane ............................................... 42 1.7.1 GSVs in the TIRF Zone ...................................................... 45 1.7.2 GLUT4 Clustering in Response to Insulin ................................ 46 1.7.3 Tethering and Fusion of GSVs with the Plasma Membrane ........... 46 1.7.4 GLUT4 Clustering and Dispersal .......................................... 49 1.7.5 Single Molecule Imaging of GLUT4 in the Plasma Membrane ......... 50 1.7.6 Possible Mechanisms of GLUT4 Clustering .............................. 52 1.7.7 EFR3 and its Potential Role in GLUT4 Dispersal ........................ 53 1.8 Working Hypothesis and Aims of this Study .................................. 54 Chapter 2 General Materials and Methods ........................................... 57 2.1 Materials .......................................................................... 57 2.1.1 Reagents and Enzymes ..................................................... 57 2.1.2 Buffers and Solutions ....................................................... 60 2.1.3 Antibodies ................................................................... 62 5 2.1.3.1 Primary Antibodies .................................................... 62 2.1.3.2 Secondary Antibodies for Western Blotting ........................ 63 2.1.3.3 Secondary Antibodies for Immunofluorescence ................... 63 2.1.4 Plasmids and siRNA ......................................................... 64 2.1.5 Mammalian Cell Lines ...................................................... 65 2.1.6 Materials ..................................................................... 65 2.2 Methods ........................................................................... 65 2.2.1 Cell Culture Methods ....................................................... 65 2.2.1.1 Growth and Maintenance of HeLa and HEK Cells ................. 66 2.2.1.2 Growth and Maintenance of 3T3 L1 Cells .......................... 66 2.2.1.3 Differentiation of 3T3 L1 Cells ....................................... 66 2.2.1.4 Freezing and Resurrecting Cells ..................................... 67 2.2.1.5 Cleaning and Seeding on Cover Glasses ............................ 67 2.2.1.6 Transfection ............................................................ 67 2.2.1.7 siRNA Electroporation of 3T3 L1 Adipocytes ....................... 68 2.2.2 IF Staining ................................................................... 68 2.2.2.1 IF Staining for Permeabilised Cells .................................. 68 2.2.2.2 IF Surface Staining for Confocal Microscopy ....................... 69 2.2.2.3 IF Surface Staining for dSTORM ...................................... 69 2.2.3 Lentivirus .................................................................... 70 2.2.3.1 Lentivirus Production ................................................. 70 2.2.3.2 Lentiviral Infection .................................................... 70 2.2.3.3 Generation of a Stable Cell Line Using Lentivirus. ............... 70 2.2.4 Molecular Biology Methods ................................................ 71 2.2.4.1 Transformation ......................................................... 71 2.2.4.2 Plasmid DNA Purification ............................................. 71 2.2.4.3 Agarose Gel Electrophoresis ......................................... 72 2.2.4.4 Restriction Endonuclease Digest ..................................... 72 2.2.4.5 Cloning .................................................................. 72 2.2.5 Gel Electrophoresis and Western Blotting .............................. 73 2.2.5.1 Cell Lysates ............................................................. 73 2.2.5.2 SDS-PAGE ...............................................................
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