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Caveolae sind charakteristische Plasmamembraneinstülpungen, die in vielen Zelltypen vorkommen und deren biologische Funktion umstritten ist. Ihre besondere Form und ihre Häu gkeit in Zellen, die stets mechanischen Belastungen ausgesetzt sind, führten zu der Annahme, dass Caveolae die Plasmamembran vor mechanischen Belastungen schützen und als Membranreservoir dienen. Dies sollte mit dieser Dissertation experimentell geprüft werden. Zunächst wurde der Ein uss der Caveolae auf die Membranspannung von Zellen im Normalzustand untersucht. Dann wurden die Zellen mechanisch belastet. Mit Fluoreszensmikroskopie wurde das Verschwinden von Caveolae nach Strecken der Zellen oder nach einem hypo-osmotischen Schock beobachtet. Messungen der Membranspannung vor und unmittelbar nach dem hypo-osmotischem Schock zeigten, dass Caveolae einen Anstieg der Membranspannung verhindern, unabhängig von ATP und dem Cytoskelett. Die Erzeugung von Membranvesikel mit Caveolae erlaubte es, diesen Effekt der Caveolae in einem vereinfachten Membransystem zu beobachten. Schliesslich wurden Muskelzellen untersucht. Zellen, die genetisch bedingt weniger Caveolae haben und mit Muskelschwundkrankheiten in Verbingung stehen, waren mechanisch weniger belastbar als gesunde Zellen. Zusammenfassend wird mit dieser Dissertation die These bestärkt, dass Caveolae einem Anstieg der Membranspannungen entgegenwirken. Dass dies in Zellen und in Vesikeln unabhängig von Energie und Cytoskelett geschieht, lässt auf einen passiven, mechanisch getriebenen Prozess schliessen. Diese Erkenntnis trägt zum Verständnis der Rolle von Caveolae in Zellen bei und kann dem besseren Verständnis von Krankheiten bedingt durch Caveolin-Mutationen, wie z.B. Muskelschwundkrankheiten, dienen.:I Introduction 9 1 Physical Description of Cellular Membranes 11 1.1 Membrane Physics at Equilibrium . . . . . . . . . . . . . . . . 11 1.1.1 Elastic Membrane Properties . . . . . . . . . . . . . . 13 1.1.2 Mathematical Description of the Membrane . . . . . . 16 1.1.3 Membrane Tension . . . . . . . . . . . . . . . . . . . . 17 1.2 Techniques to Measure Mechanical Properties of Membranes . 20 1.2.1 The Micropipette Aspiration Technique . . . . . . . . . 21 1.2.2 Tether Extraction . . . . . . . . . . . . . . . . . . . . . 24 1.2.3 Force and Radius of a Tether . . . . . . . . . . . . . . 25 2 From Vesicles to Cells 30 2.1 Structure of the Cell . . . . . . . . . . . . . . . . . . . 31 2.2 Cytoskeleton of Cells . . . . . . . . . . . . . . . . . . . 33 2.2.1 Actin Filaments . . . . . . . . . . . . . . . . . . . . . . 35 2.2.2 Actin Cortex Impairing Drugs . . . . . . . . . . . . . . 37 2.3 Cellular Membranes . . . . . . . . . . . . . . . . . . . . 38 2.4 Membrane Area and Membrane Tension Regulation . . . . 39 2.5 Tether Extraction From Cells . . . . . . . . . . . . . . . . . . 41 3 Caveolae 44 3.1 The De nition of Caveolae . . . . . . . . . . . . . . . . . . . . 44 3.2 The Caveolin Protein Family . . . . . . . . . . . . . . . . . . . 46 3.2.1 The Structure of Caveolin . . . . . . . . . . . . . . . . 47 3.3 The Cavin Protein Family . . . . . . . . . . . . . . . . . . . . 50 3.3.1 Cavin1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.2 Cavin2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.3 Cavin3 . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.3.4 Cavin4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 13.4 The Assembly of Caveolae . . . . . . . . . . . . . . . . .54 3.4.1 Caveolin is Synthesized in the Endoplasmic Reticulum, and Assembles in The Golgi Apparatus .54 3.4.2 Cavin Enters the Stage for Caveola Formation . . . . . 56 3.4.3 The Lipid Composition of Caveolae . . . . . . . . . . . 59 3.5 Caveolae Are Stable Structures at the Plasma Membrane . . 60 3.6 Endocytosis of Caveolae . . . . . . . . . . . . . . . . . . 61 3.7 Caveolae/Caveolin Proteins and Signaling Processes . . . . . 62 3.7.1 Ion-pumps in Caveolae . . . . . . . . . . . . . . . . . . 63 3.7.2 Regulation of eNOS . . . . . . . . . . . . . . . . . . . . 63 3.8 Caveolae in Muscle Cells . . . . . . . . . . . . . .. . . . 64 3.8.1 Interaction Partners of Cav3 in Myotubes . . . . . . . 64 3.8.2 Muscular Dystrophies . . . . . . . . . . . . . . . . . . . 69 4 Mechanical Role of Caveolae 74 II Materials and Methods 82 5 Cells and Reagents 84 5.1 Cell Types and Cell Culture . . . . . . . . . . . . . . . . . . 84 5.1.1 HeLa-PFPIG . . . . . . . . . . . . . . . . . . . . . . . 85 5.1.2 Mouse Lung Endothelial Cells . . . . . . . . . . . . . . 85 5.1.3 Mouse Embryonic Fibroblast . . . . . . . . . . . . . . . 86 5.1.4 Human Muscle Cells . . . . . . . . . . . . . . . . . . . 86 5.2 Treatments Altering the Cell . . . . . . . . . . . . . . . . . 88 5.2.1 Expression of Proteins . . . . . . . . . . . . . . . . . . 88 5.2.2 Altering Actin Dynamics . . . . . . . . . . . . . . . . . 89 5.2.3 ATP depletion . . . . . . . . . . . . . . . . . . . . . . . 89 5.2.4 Cholesterol Depletion . . . . . . . . . . . . . . . . . . . 90 5.3 Vesicles out of Cellular Plasma Membranes . . . . . . . . . . . 91 5.3.1 Giant Plasma Membrane Vesicles (GPMV) . . . . . . . 93 5.3.2 CytochalasinD-Blebs . . . . . . . . . . . . . . . . . . . 94 5.3.3 Plasma Membrane Spheres (PMS) . . . . . . . . . . . . 94 6 Experimental Set-Up 96 6.1 Tether Extraction . . . . . . . . . . . . . . . . . . . . . . . 96 6.1.1 Epi-OT . . . . . . . . . . . . . . . . . . . . . . . . . . 96 6.1.2 Con-OT . . . . . . . . . . . . . . . . . . . . . . . . . . 99 6.1.3 Cell Stage and Pipette Holder . . . . . . . . . . . . . . 102 6.1.4 Hypo-osmotic Shock System . . . . . . . . . . . . . . . 104 6.1.5 Fabrication of Micropipettes . . . . . . . . . . . . . . . 105 6.1.6 Aspiration Control System . . . . . . . . . . . . . . . . 106 6.1.7 Beads and Bead-coatings . . . . . . . . . . . . . . . . . 108 6.1.8 Online Tracking with MatLab . . . . . . . . . . . . . . 108 6.1.9 Calibration . . . . . . . . . . . . . . . . . . . . . . . . 109 6.2 TIRF-microscopy . . . . . . . . . . . . . . . . . . . . . . . 114 6.2.1 TIRF Set-up . . . . . . . . . . . . . . . . . . . . . . . 114 III Results 115 7 Tether Extraction From Adherent Cells 117 7.1 Typical Tether Force Traces . . . . . . . . . . . . . . . . . . . 117 7.2 Preliminary Remarks and Comments on the Relation Between Tether Force and Membrane Tension on Cells . . . . . . . . 120 8 Do Caveolae Contribute to Setting the Resting Cell Tension? 123 8.1 The E ective Tension of MLEC is A ected by the Presence Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 8.2 The E ective Tension in MEFs Does not Depend on the Presence of Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . 126 8.3 Challenging the E ective Cell Tension by Chemical and Biological Treatments . . . . . . . . . . . . . . . . . . . . . . . . 127 8.3.1 Alterations of the Cytoskeleton Decrease the E ective Cell Tension . . . . . . . . . . . . . . . . . . . . . . . . 128 8.3.2 ATP depletion Decreases the Membrane Tension . . . . 130 8.3.3 Interaction of Cav1 with Src-kinase . . . . . . . . . . . 131 8.3.4 Cav3 Re-establishes the Cell Tension of Cav1−/− MLEC 133 8.4 Summary . . . . . . . . . . . . . . . . . . . . . . . 135 9 Caveola-mediated Membrane Tension Bu ering Upon Acute Mechanical Stress: Experiments on Cells 137 9.1 Application of Acute Mechanical Stress and Cell Response Observed by TIRF and EM . . . . . . . . . . . 137 9.1.1 Mechanical Stress Leads to the Partial Disappearance of Caveolae from the Plasma Membrane .138 9.1.2 Partial Disappearance of Caveolae Observed by EM . 144 9.2 Membrane Tension Measurements During Hypo-osmotic Shock 147 9.2.1 Caveolae are Required for Bu ering the Tension Surge Due to Hypo-osmotic Shock . . . . . . . . . . . . . . . 147 9.2.2 Clathrin Coated Pits do not Bu er the Membrane Tension 151 9.2.3 Disassembly of Caveolae During Mechanical Stress . . . 153 9.3 Correlation Between the Observed Loss of Caveolae and the Excess of Membrane Area Required to Bu er Membrane Tension 156 10 Caveola-mediated Membrane Tension Bu ering upon Mechanical Stress: Experiments on Plasma Membrane Spheres 159 10.1 Plasma Membrane Spheres Contain Caveolae and Are Devoid of Actin Filaments . . . . . 161 10.1.1 Production of PMS from HeLa-PGFPIG . . . . . . . . 161 10.1.2 Production of PMS from MLEC . . . . . . . . . . . . . 163 10.2 Micropipette Aspiration of PMS Induces Disassembly of Caveolae 166 10.2.1 Quantitative Analysis of Micropipette Aspiration of PMS 167 11 Experiments on Muscle Cells The Role of Caveolin-3 Mutations in Muscular Dystrophy 174 11.1 Tether Force of Di erentiated Muscle Cells . . . . . . . . . . . 176 11.2 Reaction of Myotubes with Cav3-Mutations upon Acute Mechanical Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 11.3 Contracting Myotubes . . . . . . . . . . . . . . . . . . . . .181 IV Discussion 182 12 Caveolae as a Security Device for the Cell Membrane 183 12.1 Comparison of Experimental Data with the Theoretical Model (Sens and Turner) . . . . . . . . . 186 13 Mechanical Stress and the Role of Caveolae in Signaling 189 14 Towards a Better Understanding of Muscular Dystrophies 191 15 Other Caveolin Related Diseases 194 V Appendices 196 A Cell Speci c Protocols 197 A.1 General Cell Handling . . . . . . . . . . . . . . . . . . . . 197 A.1.1 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 197 A.2 Mouse Lung Endothelial Cells . . . . . . . . . . . . . . . . . . 198 A.2.1 Cell Type Description . . . . . . . . . . . . . . . . . . 198 A.2.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 198 A.2.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 198 A.2.4 Transfection . . . . . . . . . . . . . . . . . . . . . . . . 199 A.3 HeLa and Mouse Embryonic Fibroblast Cells . . . . . . . . . . 199 A.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 199 A.3.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4 Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4.1 Cell Type Description . . . . . . . . . . . . . . . . . . 200 A.4.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 201 A.4.4 Transfection . . . . . . . . . . . . . . . . . . . . . . . . 202 B Cav1-Reconstitution in Lipid Vesicles 203 B.1 Puri cation of Cav1-GST . . . . . . . . . . . . . . . . . . . . 203 B.1.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 203 B.1.2 Puri cation . . . . . . . . . . . . . . . . . . . . . . . . 205 B.2 puri cation of Cav1-His . . . . . . . . . . . . . . . . . . . . . 206 B.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 206 B.2.2 Puri cation . . . . . . . . . . . . . . . . . . . . . . . . 207 B.3 Incorporation of Cav1 in Lipid Vesicles . . . . . . . . . . . . . 208 B.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 208 B.3.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 209 B.4 GUV Electro formation . . . . . . . . . . . . . . . . . . . . . . 209 B.4.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 209 B.4.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 210 5 B.5 Check of Cav1 Association with Lipids . . . . . . . . . . . . . 210 B.5.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 210 B.5.2 Cav1-SUVs . . . . . . . . . . . . . . . . . . . . . . . . 211 B.5.3 Run Sucrose Gradient . . . . . . . . . . . . . . . . . . 211 B.5.4 TCA precipitation and Western Blot . . . . . . . . . . 212 B.5.5 SDS Page . . . . . . . . . . . . . . . . . . . . . . . . . 212 B.5.6 Western Blot . . . . . . . . . . . . . . . . . . . . . . . 212 / Caveolae, the characteristic plasma membrane invaginations present in many cells, have been associated with numerous functions that still remain debated. Taking into account the particular abundance of caveolae in cells experiencing mechanical stress, it was proposed that caveolae constitute a membrane reservoir and bu er the membrane tension upon mechanical stress. The present work aimed to check this proposition experimentally. First, the in uence of caveolae on the membrane tension was studied on mouse lung endothelial cells in resting conditions using tether extraction with optically trapped beads. Second, experiments on cells upon acute mechanical stress showed that caveolae serve as a membrane reservoir bu ering surges in membrane tension in their immediate, ATP- and cytoskeleton-independent attening and disassembly. Third, caveolae incorporated in membrane vesicles also showed the tension bu ering. Finally, in a physiologically more relevant case, human muscle cells were studied, and it was shown that mutations with impaired caveolae which are described in muscular dystrophies render muscle cells less resistant to mechanical stress. In Summary the present work provides experimental evidence for the hypothesis that caveolae bu er the membrane tension upon mechanical stress. The fact that this was observed in cells and membrane vesicles in an ATP and cytoskeleton independent manner reveals a passive, mechanically driven process. This could be a leap forward in the comprehension of the role of caveolae in the cell, and in the understanding of genetic diseases like muscular dystrophies.:I Introduction 9 1 Physical Description of Cellular Membranes 11 1.1 Membrane Physics at Equilibrium . . . . . . . . . . . . . . . . 11 1.1.1 Elastic Membrane Properties . . . . . . . . . . . . . . 13 1.1.2 Mathematical Description of the Membrane . . . . . . 16 1.1.3 Membrane Tension . . . . . . . . . . . . . . . . . . . . 17 1.2 Techniques to Measure Mechanical Properties of Membranes . 20 1.2.1 The Micropipette Aspiration Technique . . . . . . . . . 21 1.2.2 Tether Extraction . . . . . . . . . . . . . . . . . . . . . 24 1.2.3 Force and Radius of a Tether . . . . . . . . . . . . . . 25 2 From Vesicles to Cells 30 2.1 Structure of the Cell . . . . . . . . . . . . . . . . . . . 31 2.2 Cytoskeleton of Cells . . . . . . . . . . . . . . . . . . . 33 2.2.1 Actin Filaments . . . . . . . . . . . . . . . . . . . . . . 35 2.2.2 Actin Cortex Impairing Drugs . . . . . . . . . . . . . . 37 2.3 Cellular Membranes . . . . . . . . . . . . . . . . . . . . 38 2.4 Membrane Area and Membrane Tension Regulation . . . . 39 2.5 Tether Extraction From Cells . . . . . . . . . . . . . . . . . . 41 3 Caveolae 44 3.1 The De nition of Caveolae . . . . . . . . . . . . . . . . . . . . 44 3.2 The Caveolin Protein Family . . . . . . . . . . . . . . . . . . . 46 3.2.1 The Structure of Caveolin . . . . . . . . . . . . . . . . 47 3.3 The Cavin Protein Family . . . . . . . . . . . . . . . . . . . . 50 3.3.1 Cavin1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.2 Cavin2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.3 Cavin3 . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.3.4 Cavin4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 13.4 The Assembly of Caveolae . . . . . . . . . . . . . . . . .54 3.4.1 Caveolin is Synthesized in the Endoplasmic Reticulum, and Assembles in The Golgi Apparatus .54 3.4.2 Cavin Enters the Stage for Caveola Formation . . . . . 56 3.4.3 The Lipid Composition of Caveolae . . . . . . . . . . . 59 3.5 Caveolae Are Stable Structures at the Plasma Membrane . . 60 3.6 Endocytosis of Caveolae . . . . . . . . . . . . . . . . . . 61 3.7 Caveolae/Caveolin Proteins and Signaling Processes . . . . . 62 3.7.1 Ion-pumps in Caveolae . . . . . . . . . . . . . . . . . . 63 3.7.2 Regulation of eNOS . . . . . . . . . . . . . . . . . . . . 63 3.8 Caveolae in Muscle Cells . . . . . . . . . . . . . .. . . . 64 3.8.1 Interaction Partners of Cav3 in Myotubes . . . . . . . 64 3.8.2 Muscular Dystrophies . . . . . . . . . . . . . . . . . . . 69 4 Mechanical Role of Caveolae 74 II Materials and Methods 82 5 Cells and Reagents 84 5.1 Cell Types and Cell Culture . . . . . . . . . . . . . . . . . . 84 5.1.1 HeLa-PFPIG . . . . . . . . . . . . . . . . . . . . . . . 85 5.1.2 Mouse Lung Endothelial Cells . . . . . . . . . . . . . . 85 5.1.3 Mouse Embryonic Fibroblast . . . . . . . . . . . . . . . 86 5.1.4 Human Muscle Cells . . . . . . . . . . . . . . . . . . . 86 5.2 Treatments Altering the Cell . . . . . . . . . . . . . . . . . 88 5.2.1 Expression of Proteins . . . . . . . . . . . . . . . . . . 88 5.2.2 Altering Actin Dynamics . . . . . . . . . . . . . . . . . 89 5.2.3 ATP depletion . . . . . . . . . . . . . . . . . . . . . . . 89 5.2.4 Cholesterol Depletion . . . . . . . . . . . . . . . . . . . 90 5.3 Vesicles out of Cellular Plasma Membranes . . . . . . . . . . . 91 5.3.1 Giant Plasma Membrane Vesicles (GPMV) . . . . . . . 93 5.3.2 CytochalasinD-Blebs . . . . . . . . . . . . . . . . . . . 94 5.3.3 Plasma Membrane Spheres (PMS) . . . . . . . . . . . . 94 6 Experimental Set-Up 96 6.1 Tether Extraction . . . . . . . . . . . . . . . . . . . . . . . 96 6.1.1 Epi-OT . . . . . . . . . . . . . . . . . . . . . . . . . . 96 6.1.2 Con-OT . . . . . . . . . . . . . . . . . . . . . . . . . . 99 6.1.3 Cell Stage and Pipette Holder . . . . . . . . . . . . . . 102 6.1.4 Hypo-osmotic Shock System . . . . . . . . . . . . . . . 104 6.1.5 Fabrication of Micropipettes . . . . . . . . . . . . . . . 105 6.1.6 Aspiration Control System . . . . . . . . . . . . . . . . 106 6.1.7 Beads and Bead-coatings . . . . . . . . . . . . . . . . . 108 6.1.8 Online Tracking with MatLab . . . . . . . . . . . . . . 108 6.1.9 Calibration . . . . . . . . . . . . . . . . . . . . . . . . 109 6.2 TIRF-microscopy . . . . . . . . . . . . . . . . . . . . . . . 114 6.2.1 TIRF Set-up . . . . . . . . . . . . . . . . . . . . . . . 114 III Results 115 7 Tether Extraction From Adherent Cells 117 7.1 Typical Tether Force Traces . . . . . . . . . . . . . . . . . . . 117 7.2 Preliminary Remarks and Comments on the Relation Between Tether Force and Membrane Tension on Cells . . . . . . . . 120 8 Do Caveolae Contribute to Setting the Resting Cell Tension? 123 8.1 The E ective Tension of MLEC is A ected by the Presence Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 8.2 The E ective Tension in MEFs Does not Depend on the Presence of Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . 126 8.3 Challenging the E ective Cell Tension by Chemical and Biological Treatments . . . . . . . . . . . . . . . . . . . . . . . . 127 8.3.1 Alterations of the Cytoskeleton Decrease the E ective Cell Tension . . . . . . . . . . . . . . . . . . . . . . . . 128 8.3.2 ATP depletion Decreases the Membrane Tension . . . . 130 8.3.3 Interaction of Cav1 with Src-kinase . . . . . . . . . . . 131 8.3.4 Cav3 Re-establishes the Cell Tension of Cav1−/− MLEC 133 8.4 Summary . . . . . . . . . . . . . . . . . . . . . . . 135 9 Caveola-mediated Membrane Tension Bu ering Upon Acute Mechanical Stress: Experiments on Cells 137 9.1 Application of Acute Mechanical Stress and Cell Response Observed by TIRF and EM . . . . . . . . . . . 137 9.1.1 Mechanical Stress Leads to the Partial Disappearance of Caveolae from the Plasma Membrane .138 9.1.2 Partial Disappearance of Caveolae Observed by EM . 144 9.2 Membrane Tension Measurements During Hypo-osmotic Shock 147 9.2.1 Caveolae are Required for Bu ering the Tension Surge Due to Hypo-osmotic Shock . . . . . . . . . . . . . . . 147 9.2.2 Clathrin Coated Pits do not Bu er the Membrane Tension 151 9.2.3 Disassembly of Caveolae During Mechanical Stress . . . 153 9.3 Correlation Between the Observed Loss of Caveolae and the Excess of Membrane Area Required to Bu er Membrane Tension 156 10 Caveola-mediated Membrane Tension Bu ering upon Mechanical Stress: Experiments on Plasma Membrane Spheres 159 10.1 Plasma Membrane Spheres Contain Caveolae and Are Devoid of Actin Filaments . . . . . 161 10.1.1 Production of PMS from HeLa-PGFPIG . . . . . . . . 161 10.1.2 Production of PMS from MLEC . . . . . . . . . . . . . 163 10.2 Micropipette Aspiration of PMS Induces Disassembly of Caveolae 166 10.2.1 Quantitative Analysis of Micropipette Aspiration of PMS 167 11 Experiments on Muscle Cells The Role of Caveolin-3 Mutations in Muscular Dystrophy 174 11.1 Tether Force of Di erentiated Muscle Cells . . . . . . . . . . . 176 11.2 Reaction of Myotubes with Cav3-Mutations upon Acute Mechanical Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 11.3 Contracting Myotubes . . . . . . . . . . . . . . . . . . . . .181 IV Discussion 182 12 Caveolae as a Security Device for the Cell Membrane 183 12.1 Comparison of Experimental Data with the Theoretical Model (Sens and Turner) . . . . . . . . . 186 13 Mechanical Stress and the Role of Caveolae in Signaling 189 14 Towards a Better Understanding of Muscular Dystrophies 191 15 Other Caveolin Related Diseases 194 V Appendices 196 A Cell Speci c Protocols 197 A.1 General Cell Handling . . . . . . . . . . . . . . . . . . . . 197 A.1.1 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 197 A.2 Mouse Lung Endothelial Cells . . . . . . . . . . . . . . . . . . 198 A.2.1 Cell Type Description . . . . . . . . . . . . . . . . . . 198 A.2.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 198 A.2.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 198 A.2.4 Transfection . . . . . . . . . . . . . . . . . . . . . . . . 199 A.3 HeLa and Mouse Embryonic Fibroblast Cells . . . . . . . . . . 199 A.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 199 A.3.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4 Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4.1 Cell Type Description . . . . . . . . . . . . . . . . . . 200 A.4.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 201 A.4.4 Transfection . . . . . . . . . . . . . . . . . . . . . . . . 202 B Cav1-Reconstitution in Lipid Vesicles 203 B.1 Puri cation of Cav1-GST . . . . . . . . . . . . . . . . . . . . 203 B.1.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 203 B.1.2 Puri cation . . . . . . . . . . . . . . . . . . . . . . . . 205 B.2 puri cation of Cav1-His . . . . . . . . . . . . . . . . . . . . . 206 B.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 206 B.2.2 Puri cation . . . . . . . . . . . . . . . . . . . . . . . . 207 B.3 Incorporation of Cav1 in Lipid Vesicles . . . . . . . . . . . . . 208 B.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 208 B.3.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 209 B.4 GUV Electro formation . . . . . . . . . . . . . . . . . . . . . . 209 B.4.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 209 B.4.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 210 5 B.5 Check of Cav1 Association with Lipids . . . . . . . . . . . . . 210 B.5.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 210 B.5.2 Cav1-SUVs . . . . . . . . . . . . . . . . . . . . . . . . 211 B.5.3 Run Sucrose Gradient . . . . . . . . . . . . . . . . . . 211 B.5.4 TCA precipitation and Western Blot . . . . . . . . . . 212 B.5.5 SDS Page . . . . . . . . . . . . . . . . . . . . . . . . . 212 B.5.6 Western Blot . . . . . . . . . . . . . . . . . . . . . . . 212 / Cavéoles sont des invaginations caractéristiques de la membrane plas- mique présents dans beaucoup de types cellulaires. Ils sont liées à plusieurs fonctions cellulaires, ce qui sont encore débattues. Prenant compte de l importance des cavéoles dans les cellules soumises au stress mécanique, les cavéoles sont proposées de constituer un réservoir membranaire et de tamponner la tension membranaire pendant des stresses mécaniques. Cette étude a eu le but de tester cette hypothèse expérimentalement. En premier, l in uence des cavéoles sur la tension membranaire au repos a été étudiée sur des cellules endothéliales du poumon de la souris. Puis, on a montré que les cavéoles tamponnent l augmentation de la tension membranaire après l application d un stress mécanique. En suite, la réalisation des vésicules membranaires contenant des cavéoles a permit de montrer leur rôle comme réservoir membranaire dans un système simpli é. Finalement, dans un contexte physiologiquement plus relevant, l étude des cellules musculaires a montrée que les mutations du cavéolin associées aux dystrophies musculaires rendent les cellules moins résistante aux stresses mécaniques. En conclusion, cette étude supporte l\''hypothèse que les cavéoles tamponnent la tension membranaire pendant des stresses mécaniques. Le fait que cela se passe dans les cellules et les vésicules indépendamment d ATP et du cytosquelette révèlent un processus passif et mécanique. Cela pourrait servir à une meilleure compréhension du rôle des cavéoles dans la cellule et les maladies génétiques comme les dystrophies musculaires.:I Introduction 9 1 Physical Description of Cellular Membranes 11 1.1 Membrane Physics at Equilibrium . . . . . . . . . . . . . . . . 11 1.1.1 Elastic Membrane Properties . . . . . . . . . . . . . . 13 1.1.2 Mathematical Description of the Membrane . . . . . . 16 1.1.3 Membrane Tension . . . . . . . . . . . . . . . . . . . . 17 1.2 Techniques to Measure Mechanical Properties of Membranes . 20 1.2.1 The Micropipette Aspiration Technique . . . . . . . . . 21 1.2.2 Tether Extraction . . . . . . . . . . . . . . . . . . . . . 24 1.2.3 Force and Radius of a Tether . . . . . . . . . . . . . . 25 2 From Vesicles to Cells 30 2.1 Structure of the Cell . . . . . . . . . . . . . . . . . . . 31 2.2 Cytoskeleton of Cells . . . . . . . . . . . . . . . . . . . 33 2.2.1 Actin Filaments . . . . . . . . . . . . . . . . . . . . . . 35 2.2.2 Actin Cortex Impairing Drugs . . . . . . . . . . . . . . 37 2.3 Cellular Membranes . . . . . . . . . . . . . . . . . . . . 38 2.4 Membrane Area and Membrane Tension Regulation . . . . 39 2.5 Tether Extraction From Cells . . . . . . . . . . . . . . . . . . 41 3 Caveolae 44 3.1 The De nition of Caveolae . . . . . . . . . . . . . . . . . . . . 44 3.2 The Caveolin Protein Family . . . . . . . . . . . . . . . . . . . 46 3.2.1 The Structure of Caveolin . . . . . . . . . . . . . . . . 47 3.3 The Cavin Protein Family . . . . . . . . . . . . . . . . . . . . 50 3.3.1 Cavin1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.2 Cavin2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.3 Cavin3 . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.3.4 Cavin4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 13.4 The Assembly of Caveolae . . . . . . . . . . . . . . . . .54 3.4.1 Caveolin is Synthesized in the Endoplasmic Reticulum, and Assembles in The Golgi Apparatus .54 3.4.2 Cavin Enters the Stage for Caveola Formation . . . . . 56 3.4.3 The Lipid Composition of Caveolae . . . . . . . . . . . 59 3.5 Caveolae Are Stable Structures at the Plasma Membrane . . 60 3.6 Endocytosis of Caveolae . . . . . . . . . . . . . . . . . . 61 3.7 Caveolae/Caveolin Proteins and Signaling Processes . . . . . 62 3.7.1 Ion-pumps in Caveolae . . . . . . . . . . . . . . . . . . 63 3.7.2 Regulation of eNOS . . . . . . . . . . . . . . . . . . . . 63 3.8 Caveolae in Muscle Cells . . . . . . . . . . . . . .. . . . 64 3.8.1 Interaction Partners of Cav3 in Myotubes . . . . . . . 64 3.8.2 Muscular Dystrophies . . . . . . . . . . . . . . . . . . . 69 4 Mechanical Role of Caveolae 74 II Materials and Methods 82 5 Cells and Reagents 84 5.1 Cell Types and Cell Culture . . . . . . . . . . . . . . . . . . 84 5.1.1 HeLa-PFPIG . . . . . . . . . . . . . . . . . . . . . . . 85 5.1.2 Mouse Lung Endothelial Cells . . . . . . . . . . . . . . 85 5.1.3 Mouse Embryonic Fibroblast . . . . . . . . . . . . . . . 86 5.1.4 Human Muscle Cells . . . . . . . . . . . . . . . . . . . 86 5.2 Treatments Altering the Cell . . . . . . . . . . . . . . . . . 88 5.2.1 Expression of Proteins . . . . . . . . . . . . . . . . . . 88 5.2.2 Altering Actin Dynamics . . . . . . . . . . . . . . . . . 89 5.2.3 ATP depletion . . . . . . . . . . . . . . . . . . . . . . . 89 5.2.4 Cholesterol Depletion . . . . . . . . . . . . . . . . . . . 90 5.3 Vesicles out of Cellular Plasma Membranes . . . . . . . . . . . 91 5.3.1 Giant Plasma Membrane Vesicles (GPMV) . . . . . . . 93 5.3.2 CytochalasinD-Blebs . . . . . . . . . . . . . . . . . . . 94 5.3.3 Plasma Membrane Spheres (PMS) . . . . . . . . . . . . 94 6 Experimental Set-Up 96 6.1 Tether Extraction . . . . . . . . . . . . . . . . . . . . . . . 96 6.1.1 Epi-OT . . . . . . . . . . . . . . . . . . . . . . . . . . 96 6.1.2 Con-OT . . . . . . . . . . . . . . . . . . . . . . . . . . 99 6.1.3 Cell Stage and Pipette Holder . . . . . . . . . . . . . . 102 6.1.4 Hypo-osmotic Shock System . . . . . . . . . . . . . . . 104 6.1.5 Fabrication of Micropipettes . . . . . . . . . . . . . . . 105 6.1.6 Aspiration Control System . . . . . . . . . . . . . . . . 106 6.1.7 Beads and Bead-coatings . . . . . . . . . . . . . . . . . 108 6.1.8 Online Tracking with MatLab . . . . . . . . . . . . . . 108 6.1.9 Calibration . . . . . . . . . . . . . . . . . . . . . . . . 109 6.2 TIRF-microscopy . . . . . . . . . . . . . . . . . . . . . . . 114 6.2.1 TIRF Set-up . . . . . . . . . . . . . . . . . . . . . . . 114 III Results 115 7 Tether Extraction From Adherent Cells 117 7.1 Typical Tether Force Traces . . . . . . . . . . . . . . . . . . . 117 7.2 Preliminary Remarks and Comments on the Relation Between Tether Force and Membrane Tension on Cells . . . . . . . . 120 8 Do Caveolae Contribute to Setting the Resting Cell Tension? 123 8.1 The E ective Tension of MLEC is A ected by the Presence Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 8.2 The E ective Tension in MEFs Does not Depend on the Presence of Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . 126 8.3 Challenging the E ective Cell Tension by Chemical and Biological Treatments . . . . . . . . . . . . . . . . . . . . . . . . 127 8.3.1 Alterations of the Cytoskeleton Decrease the E ective Cell Tension . . . . . . . . . . . . . . . . . . . . . . . . 128 8.3.2 ATP depletion Decreases the Membrane Tension . . . . 130 8.3.3 Interaction of Cav1 with Src-kinase . . . . . . . . . . . 131 8.3.4 Cav3 Re-establishes the Cell Tension of Cav1−/− MLEC 133 8.4 Summary . . . . . . . . . . . . . . . . . . . . . . . 135 9 Caveola-mediated Membrane Tension Bu ering Upon Acute Mechanical Stress: Experiments on Cells 137 9.1 Application of Acute Mechanical Stress and Cell Response Observed by TIRF and EM . . . . . . . . . . . 137 9.1.1 Mechanical Stress Leads to the Partial Disappearance of Caveolae from the Plasma Membrane .138 9.1.2 Partial Disappearance of Caveolae Observed by EM . 144 9.2 Membrane Tension Measurements During Hypo-osmotic Shock 147 9.2.1 Caveolae are Required for Bu ering the Tension Surge Due to Hypo-osmotic Shock . . . . . . . . . . . . . . . 147 9.2.2 Clathrin Coated Pits do not Bu er the Membrane Tension 151 9.2.3 Disassembly of Caveolae During Mechanical Stress . . . 153 9.3 Correlation Between the Observed Loss of Caveolae and the Excess of Membrane Area Required to Bu er Membrane Tension 156 10 Caveola-mediated Membrane Tension Bu ering upon Mechanical Stress: Experiments on Plasma Membrane Spheres 159 10.1 Plasma Membrane Spheres Contain Caveolae and Are Devoid of Actin Filaments . . . . . 161 10.1.1 Production of PMS from HeLa-PGFPIG . . . . . . . . 161 10.1.2 Production of PMS from MLEC . . . . . . . . . . . . . 163 10.2 Micropipette Aspiration of PMS Induces Disassembly of Caveolae 166 10.2.1 Quantitative Analysis of Micropipette Aspiration of PMS 167 11 Experiments on Muscle Cells The Role of Caveolin-3 Mutations in Muscular Dystrophy 174 11.1 Tether Force of Di erentiated Muscle Cells . . . . . . . . . . . 176 11.2 Reaction of Myotubes with Cav3-Mutations upon Acute Mechanical Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 11.3 Contracting Myotubes . . . . . . . . . . . . . . . . . . . . .181 IV Discussion 182 12 Caveolae as a Security Device for the Cell Membrane 183 12.1 Comparison of Experimental Data with the Theoretical Model (Sens and Turner) . . . . . . . . . 186 13 Mechanical Stress and the Role of Caveolae in Signaling 189 14 Towards a Better Understanding of Muscular Dystrophies 191 15 Other Caveolin Related Diseases 194 V Appendices 196 A Cell Speci c Protocols 197 A.1 General Cell Handling . . . . . . . . . . . . . . . . . . . . 197 A.1.1 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 197 A.2 Mouse Lung Endothelial Cells . . . . . . . . . . . . . . . . . . 198 A.2.1 Cell Type Description . . . . . . . . . . . . . . . . . . 198 A.2.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 198 A.2.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 198 A.2.4 Transfection . . . . . . . . . . . . . . . . . . . . . . . . 199 A.3 HeLa and Mouse Embryonic Fibroblast Cells . . . . . . . . . . 199 A.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 199 A.3.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4 Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4.1 Cell Type Description . . . . . . . . . . . . . . . . . . 200 A.4.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 200 A.4.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 201 A.4.4 Transfection . . . . . . . . . . . . . . . . . . . . . . . . 202 B Cav1-Reconstitution in Lipid Vesicles 203 B.1 Puri cation of Cav1-GST . . . . . . . . . . . . . . . . . . . . 203 B.1.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 203 B.1.2 Puri cation . . . . . . . . . . . . . . . . . . . . . . . . 205 B.2 puri cation of Cav1-His . . . . . . . . . . . . . . . . . . . . . 206 B.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 206 B.2.2 Puri cation . . . . . . . . . . . . . . . . . . . . . . . . 207 B.3 Incorporation of Cav1 in Lipid Vesicles . . . . . . . . . . . . . 208 B.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 208 B.3.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 209 B.4 GUV Electro formation . . . . . . . . . . . . . . . . . . . . . . 209 B.4.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 209 B.4.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 210 5 B.5 Check of Cav1 Association with Lipids . . . . . . . . . . . . . 210 B.5.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 210 B.5.2 Cav1-SUVs . . . . . . . . . . . . . . . . . . . . . . . . 211 B.5.3 Run Sucrose Gradient . . . . . . . . . . . . . . . . . . 211 B.5.4 TCA precipitation and Western Blot . . . . . . . . . . 212 B.5.5 SDS Page . . . . . . . . . . . . . . . . . . . . . . . . . 212 B.5.6 Western Blot . . . . . . . . . . . . . . . . . . . . . . . 212

<p>Peptides, specifically cell-penetrating peptides (CPP), have become wonderful research tools due to their enhanced stability, solubility, and ease of synthesis. They have been used for a wide range of biomedical applications, from insecticides to biosensors and drug-delivery scaffolds. The work presented in this dissertation characterizes the biophysical properties of two different CPPs. The first is the cationic amphiphilic polyproline helix (CAPH) peptide, P14LRR. In addition to cell penetration, this CPP has demonstrated broad spectrum antibacterial properties. Fluorescence polarization (FP) and SEC-MALS were conducted to understand the dissociation constant (KD) and oligomerization effects of P14LRR with respect to its putative molecular target in Staphylococcus aureus (S. aureus). A biotinylated derivative of this peptide was also used as a drug-delivery scaffold to transport fluorescently conjugated streptavidin into mammalian cells. A second CPP, DAN13, was also developed as a biosensor for phosphoinositide lipids, specifically PI(4,5)P2. This was effected through careful calibration using stacked supported lipid bilayers (SSLB) in combination with total internal reflection fluorescence (TIRF) microscopy. This was then used to determine the absolute densities and spatial distribution of PIP2 in live KRas mutant cells.</p>

No description available.

No description available.

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.