A Python based automated tracking routine for myosin II filaments

The study of motor protein dynamics within cytoskeletal networks is of high interest to physicists and biologists to understand how the dynamics and properties of individual motors lead to cooperative effects and control of overall network behaviour. Here, we report a method to detect and track muscular myosin II filaments within an actin network tethered to supported lipid bilayers. Based on the characteristic shape of myosin II filaments, this automated tracking routine allowed us to follow the position and orientation of myosin II filaments over time, and to reliably classify their dynamics into segments of diffusive and processive motion based on the analysis of displacements and angular changes between time steps. This automated, high throughput method will allow scientists to efficiently analyse motor dynamics in different conditions, and will grant access to more detailed information than provided by common tracking methods, without any need for time consuming manual tracking or generation of kymographs

Role of Caveolae in Membrane Tension

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 . . . . . . . . . . . . . . . . . . . . . . . 212Caveolae, 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

Myosin II filament dynamics in actin networks revealed with interferometric scattering microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network

Calponin-homology domain mediated bending of membrane-associated actin filaments

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that ‘curly’, a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Although F-actin is semi-flexible with a persistence length of ~10 μm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 μm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures

Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression ofcaveolin-3. Our study reveals that under mechanical stress the regulation of mechan-oprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling inmuscle cells and that this regulation is absent in Cav3-associated dystrophic patients

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress

Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement

Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. Whereas, in muscle cells, tropomyosin is subject to calcium regulation, its regulation in non-muscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of non-muscle tropomyosins, which in fission yeast controls actin filament stability and division site placement

Role of Caveolae in Membrane Tension

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 . . . . . . . . . . . . . . . . . . . . . . . 212Caveolae, 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

Role of Caveolae in Membrane Tension

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 . . . . . . . . . . . . . . . . . . . . . . . 212Caveolae, 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

Cortical actin and the plasma membrane : inextricably intertwined

The plasma membrane serves as a barrier, separating the cell from its external environment. Simultaneously it acts as a site for information transduction, entry of nutrients, receptor signaling, and adapts to the shape of the cell. This requires local control of organization at multiple scales in this heterogeneous fluid lipid bilayer with a plethora of proteins and a closely juxtaposed dynamic cortical cytoskeleton. New membrane models highlight the influence of the underlying cortical actin on the diffusion of membrane components. Myosin motors as well as proteins that remodel actin filaments have additionally been implicated in defining the organization of many membrane constituents. Here we provide a perspective of the intimate relationship of the membrane lipid matrix and the underlying cytoskeleton