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

Gegenstand der Arbeit sind Zusammenhänge zwischen physikochemischen Eigenschaften und Funktionen der Exine bei Ausbreitung, Bestäubung und Befruchtung. Dabei bewährte sich der Einsatz der 3-kammrigen Sporopolleninkapseln (Zentralkapsel und Sacci) in der Permeationschromatographie. Sowohl kinetisch bedingte chromatographische Dispersion kleiner Moleküle als auch Konzentrationsänderungen von Zuckern und Dextranmolekülen im Medium wurden zur Bestimmung von Permeabilitätskoeffizienten der Nexine genutzt. Die Wasserabsorptionskapazität von Exinefragmenten und die hydraulische Leitfähigkeit der Nexine wurden anhand von Konzentrationsänderungen ausgeschlossener Dextranmoleküle ermittelt. Das Tectum der saccalen Sexine ist eine Mikrofiltermembran mit scharfer Trenngrenze im Submikrometerbereich; daher werden an den Sacci nur Hydrokolloide mit Stokes''schen Radius über 100 nm (z.B. aus nativem Dextran) ausgeschlossen. Die Nexine ist eine nicht-ideale Umkehrosmose-Membran, die in Zucker- und Salzlösungen hohe Reflexionskoeffizienten zeigt; zusätzlich besitzt sie wenige große Poren, die den Austausch von Zuckern und selbst kleinen Polymermolekülen ermöglichen. Die hydraulische Leitfähigkeit der Nexine liegt im Größenbereich derjenigen von Plasmamembranen (0,39-0,48 µm s-1 MPa-1); die Ergebnisse zeigen, dass die Exine weder die Nährstoffaufnahme des Sporoplasten aus der lokulären Flüssigkeit noch dessen rasche Rehydratation in der Mikropyle behindert. Die Einfaltungen der distalen Nexine (oberhalb der Sacci) und die Omega-Faltung der Exine zwischen den Sacci (Leptom) bieten beim Quellvorgang Schutz vor zu schneller Flächenausdehnung der Plasmamembran. Der Corpus kann mit konzentrierten Elektrolytlösungen beladen werden. Beim anschließenden osmotischen Schwellen in Wasser reißt die Exine, und der Sporoplast wird mit anhaftender Intine ausgeschleudert. Wasser und andere polare Flüssigkeiten adhärieren stärker als hydrophobe Flüssigkeiten an Sporopollenin. Die Sporopolleninmatrix weist eine hohe Feststoffdichte auf, ist wenig quellfähig (0,18 mL g-1 TM) und deformationsstabil. Dies ermöglicht die Pulverbildung beim Trocknen. / Subject of this thesis are relationships between physicochemical properties and functions of the exine concerning propagation, pollination and fecundation. Here the application of the 3-chambered sporopollenin-microcapsules (central capsule and sacci) in permeation chromatography proved of value. Both the kinetically dependent dispersion of small molecules and changes in concentration of sugars and dextran molecules in the medium were analysed to determine permeability coefficients of the nexine. The water absorption capacity of exine fragments and the hydraulic conductance of the nexine were calculated by means of changes in concentrations of excluded dextran molecules. The tectum of the saccal sexine is a microfiltration membrane with a sharp cut off in the submicrometer range; thus hydrocolloids with Stokes´radii over 100 nm (e.g. from native dextran) are excluded from the sacci. The nexine is a non-ideal reverse osmosis membrane having high reflexion coefficients in sugar and salt solutions; in addition few large pores allow the exchange of sugars and even of small polymers. The hydraulic conductance of the nexine is in the range typically for plasmamembranes (0.39-0.48 µm s-1 MPa-1); the results indicate that the exine does neither obstruct the uptake of nutrients by the sporoplast from the locular fluid nor hinder the rapid rehydration in the micropyle. When rehydrating, the distal foldings of the nexine (above the sacci) and the omega-like folding of the exine between the sacci (leptom), provide protection for the plasmamembrane when its surface area has to increase too rapidly. The corpus can be loaded with a concentrated electrolyte solution. When subsequently transferred into water the exine rupture and the sporoplast along with the intact intine is ejected. Water and other polar liquids adhere stronger to sporopollenin than hydrophobic ones. The matrix of sporopollenin show a high density in its solid content, water absorption capacity is low (0.18 mL g-1 DM) and it is resistant to deformation. This enable the formation of powder while dehydrating.

A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar, com o uso de inibidores do ciclo celular, o período do ciclo em que isto ocorre; b) verificar se o aumento da sensibilidade a pH baixo está relacionado com a expansão celular ou com alterações no potencial osmótico da célula; c) examinar o efeito da aplicação de H2O2 ou ascorbato sobre a resposta de células sensíveis a pH baixo; d) testar a hipótese de que a sensibilidade a pH baixo pode ser revertida por meio de um choque hipo-osmótico prévio; e) avaliar o possível papel da modulação oxidativa da parede celular na reversão de sensibilidade das células a pH baixo expostas ao choque hipo-osmótico. A retomada do ciclo celular é necessária para que ocorra a alteração de sensibilidade a pH baixo, pois a remoção de auxina (2,4-D) ou a adição de bloqueadores de canais de K+ impediu ou atrasou, respectivamente, a alteração na sensibilidade a pH baixo. O uso de inibidores do ciclo celular demonstrou que as células de BY-2 se tornam mais sensíveis a pH baixo durante o final da fase G1 mas antes do ponto de checagem da transição G1/S do ciclo celular. A aplicação de H2O2, diminuiu a suscetibilidade das células a pH baixo, ao contrário da aplicação de ascorbato. Foi demonstrado que a aplicação prévia de tratamento hipo-osmótico por 60 min reverteu a sensibilidade de células a pH baixo. A aplicação de inibidores de NAPDH oxidase da membrana plasmática e de peroxidases revelou a participação destas enzimas na reversão de sensibilidade das células a pH baixo, indicando a possibilidade de geração de ROS e de modulação oxidativa da parede. Embora já tenha sido descrito que ocorre uma explosão oxidativa com choque hipo-osmótico, ainda não havia sido demonstrado a conseqüência disto. Este trabalho fornece indícios de que uma explosão oxidativa poderia modificar a parede tornando-a mais resistente e a célula menos suscetível a pH baixo / Soil acidity is a major factor limiting plant growth worldwide. Although aluminum toxicity, which occurs only at low pH, has been extensively studied, little attention has been given to stress caused by low pH. There are marked differences in the sensitivity of cells to low pH which are contingent on the growth and developmental stage of the cells. These differences should be explored to further the understanding of the factors governing sensitivity and tolerance to low pH. In at least some cases, the susceptibility of cells to low pH is related to derangements in the wall of growing cells, which can cause ruptures or bursting of the cells, as has been clearly demonstrated in expanding root hairs. On the other hand, the oxidative metabolism and generation of reactive oxygen species (ROS) can modulate cell wall extensibility by breaking or making bonds within and between cell wall polymers. In tobacco (Nicotiana tabacum) cv. BY-2 cells, there is a sharp increase in sensitivity to low pH at the end of the lag phase of the cell culture, which occurs between 12 and 24 h of subculture. The objectives of this study were: a) determine if the changes in sensitivity to low pH occurred during the restart of the cell cycle and, by employing cell cycle inhibitors, at which points of the cycle does this occur; b) examine if the changes in sensitivity to low pH are related to cell expansion or changes in osmotic potential of the cell; c) examine how the application of H2O2 or ascorbate affects the response of cells to low pH; d) test the hypothesis that sensitivity of cells to low pH can be reverted by the previous application of a hypo-osmotic shock; e) evaluate the possible role of oxidative modulation of the cell wall in hypo-osmotic-induced reversal of the sensitivity of cells to low pH. The restart of the cell cycle was shown to be necessary for the change in sensitivity to low pH occur, since the absence of auxin (2,4-D) or the addition of K+ channel blockers prevented or delayed this change, respectively. The use of cell cycle inhibitors demonstrated that BY-2 cells become sensitive to low pH at the end of G1 but before the G1/S transition restriction point of the cell cycle. Exogenous H2O2, but not ascorbate, reduced the effect of low pH on sensitive cells. Sensitive cells submitted to 60 min hypo-osmotic treatment became insensitive to low pH. This reversal of sensitivity depended on the activity of plasma membrane NADPH oxidase and peroxidase, as evidenced by the use of DPI and SHAM, inhibitors of these enzymes, respectively. This suggests that ROS is generated and that oxidative modifications of the cell wall occur. Although hypo-osmotic treatments have been shown to generate an oxidative burst, its purpose or implication has not yet been shown. This study provides evidence that an oxidative burst might modify and strengthen the cell wall, making cells less susceptible to low pH

A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar, com o uso de inibidores do ciclo celular, o período do ciclo em que isto ocorre; b) verificar se o aumento da sensibilidade a pH baixo está relacionado com a expansão celular ou com alterações no potencial osmótico da célula; c) examinar o efeito da aplicação de H2O2 ou ascorbato sobre a resposta de células sensíveis a pH baixo; d) testar a hipótese de que a sensibilidade a pH baixo pode ser revertida por meio de um choque hipo-osmótico prévio; e) avaliar o possível papel da modulação oxidativa da parede celular na reversão de sensibilidade das células a pH baixo expostas ao choque hipo-osmótico. A retomada do ciclo celular é necessária para que ocorra a alteração de sensibilidade a pH baixo, pois a remoção de auxina (2,4-D) ou a adição de bloqueadores de canais de K+ impediu ou atrasou, respectivamente, a alteração na sensibilidade a pH baixo. O uso de inibidores do ciclo celular demonstrou que as células de BY-2 se tornam mais sensíveis a pH baixo durante o final da fase G1 mas antes do ponto de checagem da transição G1/S do ciclo celular. A aplicação de H2O2, diminuiu a suscetibilidade das células a pH baixo, ao contrário da aplicação de ascorbato. Foi demonstrado que a aplicação prévia de tratamento hipo-osmótico por 60 min reverteu a sensibilidade de células a pH baixo. A aplicação de inibidores de NAPDH oxidase da membrana plasmática e de peroxidases revelou a participação destas enzimas na reversão de sensibilidade das células a pH baixo, indicando a possibilidade de geração de ROS e de modulação oxidativa da parede. Embora já tenha sido descrito que ocorre uma explosão oxidativa com choque hipo-osmótico, ainda não havia sido demonstrado a conseqüência disto. Este trabalho fornece indícios de que uma explosão oxidativa poderia modificar a parede tornando-a mais resistente e a célula menos suscetível a pH baixo / Soil acidity is a major factor limiting plant growth worldwide. Although aluminum toxicity, which occurs only at low pH, has been extensively studied, little attention has been given to stress caused by low pH. There are marked differences in the sensitivity of cells to low pH which are contingent on the growth and developmental stage of the cells. These differences should be explored to further the understanding of the factors governing sensitivity and tolerance to low pH. In at least some cases, the susceptibility of cells to low pH is related to derangements in the wall of growing cells, which can cause ruptures or bursting of the cells, as has been clearly demonstrated in expanding root hairs. On the other hand, the oxidative metabolism and generation of reactive oxygen species (ROS) can modulate cell wall extensibility by breaking or making bonds within and between cell wall polymers. In tobacco (Nicotiana tabacum) cv. BY-2 cells, there is a sharp increase in sensitivity to low pH at the end of the lag phase of the cell culture, which occurs between 12 and 24 h of subculture. The objectives of this study were: a) determine if the changes in sensitivity to low pH occurred during the restart of the cell cycle and, by employing cell cycle inhibitors, at which points of the cycle does this occur; b) examine if the changes in sensitivity to low pH are related to cell expansion or changes in osmotic potential of the cell; c) examine how the application of H2O2 or ascorbate affects the response of cells to low pH; d) test the hypothesis that sensitivity of cells to low pH can be reverted by the previous application of a hypo-osmotic shock; e) evaluate the possible role of oxidative modulation of the cell wall in hypo-osmotic-induced reversal of the sensitivity of cells to low pH. The restart of the cell cycle was shown to be necessary for the change in sensitivity to low pH occur, since the absence of auxin (2,4-D) or the addition of K+ channel blockers prevented or delayed this change, respectively. The use of cell cycle inhibitors demonstrated that BY-2 cells become sensitive to low pH at the end of G1 but before the G1/S transition restriction point of the cell cycle. Exogenous H2O2, but not ascorbate, reduced the effect of low pH on sensitive cells. Sensitive cells submitted to 60 min hypo-osmotic treatment became insensitive to low pH. This reversal of sensitivity depended on the activity of plasma membrane NADPH oxidase and peroxidase, as evidenced by the use of DPI and SHAM, inhibitors of these enzymes, respectively. This suggests that ROS is generated and that oxidative modifications of the cell wall occur. Although hypo-osmotic treatments have been shown to generate an oxidative burst, its purpose or implication has not yet been shown. This study provides evidence that an oxidative burst might modify and strengthen the cell wall, making cells less susceptible to low pH