Coiled-coil motifs are part of 5–10 % of the eukaryotic proteome and are involved in important cellular processes such as membrane trafficking, chromosome segregation or mechanosensing. Their canonical form is well understood and based on a heptad repeat with hydrophobic amino acids at positions 1 and 4. A sequence of these peptides folds into an α-helix and two, or more, of these helices bind together by winding around each other, covering up the hydrophobic residues and giving rise to the coiled-coil structure. Coiled-coil proteins appearing in nature do, however, deviate from this form by introducing discontinuities into the heptad repeat. The effect of these imperfections on the structure is only known for few cases and not generally understood or predictable. The additional impact of these discontinuities on the dynamic function of coiled-coil domains is unknown altogether. Here, in order to tackle these questions, the adhesive forces between the α-helices are studied in single-molecule experiments. To measure these small forces (∼ pN) with a high spatial and temporal resolution, a dual-trap optical tweezers setup was constructed. Special emphasis was put on realizing the required high resolution, a large degree of automation and versatility during the building process. The instrument’s performance was assessed by recording force-extension curves of DNA yielding results for the molecular parameters persistence length and stretch modulus in good agreement with those found in the literature. Additionally, the Allan deviation was computed for different configurations of beads and a high stability and resolution of the instrument was found with optimal performance on the time scale of 1–10 s. Optical tweezers require calibration to accurately measure forces. To this end, generally a scheme is used that leverages the Brownian motion of a trapped object in the harmonic potential, created by the laser focus, to determine the parameters required to convert the analog voltage signal to distances and forces. However, this approach requires prior knowledge of the bead’s drag coefficient. A method was suggested previously that allows to measure this parameter by exciting the trapped bead through an external fluid flow and observing its response. Yet, this scheme was proposed for single-trap devices only. The precision and versatility of the new instrument was increased by extending this technique to work with two traps and implementing it in the apparatus. To this aim, the underlying equations of a trapped bead’s motion were modified to include hydrodynamic interactions between the objects resulting from the external fluid flow. It was found that a single multiplicative factor is sufficient to correct the calibration results for the hydrodynamic effects and ensure precise calibration. The drag coefficient of several beads yielded the same result for a single and two traps within the measurement error thus confirming the validity of the method. The newly built instrument was then used to study the coiled-coil protein early endosome antigen 1 (EEA1). This 200 nm long homodimer was shown to undergo an entropic collapse upon binding a small GTPase at the N-terminus. For further investigations of this effect and the adhesives forces at play, an experiment was designed here to unzip the two α-helices of the protein. To this end, DNA handles were attached to each of the two helices using a sortase A based ligation reaction as force moderators and first optical tweezers experiments were performed with the protein-DNA chimera. Thus, the necessary tools for unzipping assays of EEA1 are now at hand to further research the entropic collapse process. To summarize, a dual-trap optical tweezers setup was built, the calibration routine extended and realized in a more precise way and the instrument was used to investigate binding energies of EEA1 α-helices. / Coiled-Coil Strukturmotive sind in 5–10 % aller Proteine von Eukaryoten vertreten und wichtiger Teil zellulärer Prozesse wie Membrantransport, Segregation von Chromosomen oder Mechanoperzeption. Ihre grundlegende Struktur besteht aus dem sogenannten Heptadenmuster, einer Sequenz aus sieben Aminosäuren mit hydrophoben Molekülen an Position eins und vier. Eine Reihe dieser Muster kann sich zu einer α-Helix falten und zwei, oder mehr, solcher Helices sich umeinander winden, um die hydrophoben Moleküle abzuschirmen. Das Ergebnis ist eine Coiled-Coil- oder Doppelwendelstruktur. Natürlich vorkommende Coiled-Coil Proteine weichen jedoch durch Fehlstellen im Heptadenmuster von dieser kanonischen Form ab. Die Auswirkung dieser Störstellen auf die Struktur des gesamten Moleküls ist bisher nur für einige wenige Fälle untersucht und nicht allgemein vorstanden oder vorhersagbar. Der zusätzliche Einfluss dieser Fehlstellen auf die Funktion und dynamischen Prozesse solcher Proteine ist gänzlich unbekannt. Um diesen Fragen nachzugehen werden hier die Bindungskräfte zwischen den α-Helices in Einzelmolekülstudien untersucht. Um diese winzigen Kräfte (∼ pN) mit hoher räumlicher und zeitlicher Auflösung untersuchen zu können, wurde im Rahmen der vorliegenden Arbeit eine optische Doppelfalle konstruiert. Besonderes Augenmerk lag dabei auf dem Erreichen der erforderlichen Auflösung, einem hohen Grad an Automatisierung und der vielfälting Einsatzfähigkeit des Instruments. Die Leistungsfähigkeit dieses Kraftmikroskops wurde besonders durch zwei Experimente überprüft und sichergestellt. Zum einen wurden DNA Moleküle gedehnt und die Polymerparameter Persistenzlänge und Zugmodul gemessen, welche sehr gut mit veröffentlichten Referenzwerten übereinstimmten. Zum anderen wurde die Allan Schwankung für verschiedene experimentelle Konfigurationen von mikroskopischen Kugeln ermittelt, was eine hohe Stabilität und Auflösung des Gerätes, mit optimaler Leistung bei Mittelung auf Zeitskalen von 1–10 s, bestätigte. Optische Fallen müssen kalibriert werden, um Kräfte exakt messen zu können. Im Allgemeinen kommt dafür ein Verfahren zum Einsatz, welches die brownsche Bewegung eines gefangenen Objektes im harmonischen Potential des Laserfokus ausnutzt. Aus diesen Fluktuationen werden die benötigten Parameter ermittelt, um das gemessene analoge Spannungssignal in Abstände und Kräfte umzuwandeln. Dieser Ansatz erfordert jedoch die Kenntnis des Reibungskoeffizienten des gehaltenen Objektes, meist einer mikroskopischen Kugel. Daher wurde eine Methode vorgeschlagen, die durch ein oszillierendes Flussfeld eine zusätzliche Bewegung der Kugel anregt aus welcher der Reibungskoeffizient bestimmt werden kann. Dieses Vorgehen reduziert die im vornherein benötigten Informationen, wurde jedoch nur für eine einzelne optische Falle entwickelt. Der Ansatz wurde in dieser Arbeit erweitert, indem die zu zugrundeliegenden Bewegungsgleichungen einer gefangenen Kugel um hydrodynamische Wechselwirkungen zwischen mehreren Objekten ergänzt und die Kalibrationparameter basierend darauf hergeleitet wurden. Im Ergebnis konnte gezeigt werden, dass ein einzelner multiplikativer Faktor ausreicht, um die Hydrodynamik zu berücksichtigen und die exakte Kalibration des Instruments sicherzustellen. Dieses Vorgehen wurde überprüft, indem der Reibungskoeffizient einer einzelnen oder mehrerer mikroskopischer Kugeln gleichzeitig durch Anlegen eines externen Flussfeldes gemessen wurde. Die Ergebnisse stimmen im Rahmen der Messgenauigkeit überein und bestätigen damit den gewählten Ansatz. Das neu implementierte Kraftmikroskop wurde im Folgenden eingesetzt, um das Coiled-Coil Protein Early Endosome Antigen 1 (EEA1) zu erforschen. Dieser 200 nm lange Homodimer kollabiert aufgrund entropischer Kräfte sobald eine kleine GTPase an seinen N-Terminus bindet. Um diesen Effekt und die wirkenden Bindungskräfte besser zu verstehen, wurde hier ein Experiment entwickelt bei dem die beiden α-Helicen auseinandergezogen werden. Dazu wurde mittels einer Sortase A basierten Ligationsreaktion an jede Helix ein DNA-Stück gebunden, über welches Kräfte auf das Molekül übertragen werden können. Erste Experimente wurden mit der optischen Doppelfalle und dieser Protein-DNA Chimäre durchgeführt. Somit sind alle benötigten Werkzeuge zum weiteren Studium des entropischen Kollapses von EEA1 verfügbar, indem die Bindungskräfte der α-Helicen untersucht werden. Zusammenfassend wurde eine hoch auflösende Doppelfalle konstruiert, die Kalibrationsmethode weiterentwickelt und verfeinert und das Kraftmikroskop zur Erforschung der Bindungskräfte der α-Helicen von EEA1 eingesetzt.

Optical tweezers are a sensitive position and force transducer widely employed in physics and biology. In a focussed laser, forces due to radiation pressure enable to trap and manipulate small dielectric particles used as probes for various experiments. For sensitive biophysical measurements, microspheres are often used as a handle for the molecule of interest. The force range of optical traps well covers the piconewton forces generated by individual biomolecules such as kinesin molecular motors. However, cellular processes are often driven by ensembles of molecular machines generating forces exceeding a nanonewton and thus the capabilities of optical tweezers. In this thesis I focused, fifirst, on extending the force range of optical tweezers by improving the trapping e fficiency of the probes and, second, on applying the optical tweezers technology to understand the mechanics of molecular motors. I designed and fabricated photonically-structured probes: Anti-reflection-coated, high-refractive-index, core-shell particles composed of titania. With these probes, I significantly increased the maximum optical force beyond a nanonewton. These particles open up new research possibilities in both biology and physics, for example, to measure hydrodynamic resonances associated with the colored nature of the noise of Brownian motion. With respect to biophysical applications, I used the optical tweezers to study the mechanics of single kinesin-8. Kinesin-8 has been shown to be a very processive, plus-end directed microtubule depolymerase. The underlying mechanism for the high processivity and how stepping is affected by force is unclear. Therefore, I tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads. We found that kinesin-8 is a low-force motor protein, which stalled at loads of only 1 pN. In addition, we discovered a force-induced stick-slip motion, which may be an adaptation for the high processivity. Further improvement in optical tweezers probes and the instrument will broaden the scope of feasible optical trapping experiments in the future.

Die Proteinfaltung ist ein Prozess der molekularen Selbstorganisation, bei dem sich eine lineare Kette von Aminosäuren zu einer definierten, funktionellen dreidimensionalen Struktur zusammensetzt. Der Prozess der Faltung ist ein thermisch getriebener diffusiver Prozess durch eine Gibbs-Energie-Landschaft im Konformationsraum für die Struktur der minimalen Energie. Während dieses Prozesses zeigt die freie Enthalpie des Systems nicht immer eine monotone Abnahme; stattdessen führt eine suboptimale Kompensation der Enthalpie- und der Entropieänderung während jedes Faltungsschrittes zur Bildung von Freien-Enthalpie-Faltungsbarrieren. Diese Barrieren und damit verbundenen hochenergetischen Übergangszustände, die wichtige Informationen über Mechanismen der Proteinfaltung enthalten, sind jedoch kinetisch unzugänglich. Um den Prozess der Barrierebildung und die strukturellen Merkmale von Übergangszuständen aufzudecken, werden Proteine genutzt, die über barrierefreie Pfade falten – so genannte “downhill folder“. Aufgrund der geringen Faltungsbarrieren werden wichtige Interaktionen der Faltung zugänglich und erlauben Einblicke in die ratenbegrenzenden Faltungsvorgänge. In dieser Arbeit vergleichen wir die Faltungsdynamiken von drei verschiedenen Varianten eines Lambda-Repressor-Fragments, bestehend aus den Aminosäuren 6 bis 85: ein Zwei-Zustands-Falter λWT (Y22W) und zwei downhill-folder-artige Varianten, λYA (Y22W/Q33Y/ G46,48A) und λHA (Y22W/Q33H/G46,48A). Um auf die Kinetik und die strukturelle Dynamik zu greifen zu können, werden Einzelmolekülkraftspektroskopische Experimente mit optische Pinzetten mit Submillisekunden- und Nanometer-Auflösung verwendet. Ich fand, dass die niedrige denaturierende Kraft die Mikrosekunden Faltungskinetik von downhill foldern auf eine Millisekunden-Zeitskala verlangsamt, sodass das System für Einzelmolekülstudien gut zugänglich ist. Interessanterweise zeigten sich unter Krafteinwirkung die downhill-folder-artigen Varianten des Lambda-Repressors als kooperative Zwei-Zustands-Falter mit deutlich unterschiedlicher Faltungskinetik und Kraftabhängigkeit. Drei Varianten des Proteins zeigten ein hoch konformes Verhalten unter Last. Die modellfreie Rekonstruktion von Freien-Enthalpie-Landschaften ermöglichte es uns, die feinen Details der Transformation des Zwei-Zustands-Faltungspfad direkt in einen downhill-artigen Pfad aufzulösen. Die Auswirkungen von einzelnen Mutationen auf die Proteinstabilität, Bildung der Übergangszustände und die konformationelle Heterogenität der Faltungs- und Entfaltungszustände konnten beobachtet werden. Interessanterweise zeigen unsere Ergebnisse, dass sich die untersuchten Varianten trotz der ultraschnellen Faltungszeit im Bereich von 2 μs in einem kooperativen Prozess über verbleibende Energiebarrieren falten und entfalten, was darauf hindeutet, dass wesentlich schnellere Faltungsraten notwendig sind um ein downhill Limit vollständig zu erreichen.:I Theoretical background 1 1 Introduction 3 2 Protein folding: the downhill scenario 5 2.1 Protein folding as a diffusion on a multidimensional energy landscape 5 2.2 Downhill folding proteins 7 2.2.1 Thermodynamic description of downhill folders 7 2.2.2 Identification criteria for downhill folders 8 2.3 Lambda repressor as a model system for studying downhill folding 9 2.3.1 Wild-type lambda repressor fragment λ{6-85} 10 2.3.2 Acceleration of λ{6-85} folding by specifific point mutations 11 2.3.3 The incipient-downhill λYA and downhill λHA variants 14 2.4 Single-molecule techniques as a promising tool for probing downhill folding dynamics 17 3 Single-molecule protein folding with optical tweezers 19 3.1 Optical tweezers 19 3.1.1 Working principle of optical tweezers 19 3.1.2 The optical tweezers setup 21 3.2 The dumbbell assay 22 3.3 Measurement protocols 23 3.3.1 Constant-velocity experiments 23 3.3.2 Constant-trap-distance experiments (equilibrium experiments) 24 4 Theory and analysis of single-molecule trajectories 27 4.1 Polymer elasticity models 27 4.2 Equilibrium free energies of protein folding in optical tweezers 28 4.3 Signal-pair correlation analysis 29 4.4 Force dependence of transition rate constants 29 4.4.1 Zero-load extrapolation of rates: the Berkemeier-Schlierf model 30 4.4.2 Detailed balance for unfolding and refolding data 31 4.5 Direct measurement of the energy landscape via deconvolution 32 II Results 33 5 Efficient strategy for protein-DNA hybrid formation 35 5.1 Currently available strategies for protein-DNA hybrid formation 35 5.2 Novel assembly of protein-DNA hybrids based on copper-free click chemistry 37 5.3 Click-chemistry based assembly preserves the native protein structure 40 5.4 Summary 42 6 Non-equilibrium mechanical unfolding and refolding of lambda repressor variants 45 6.1 Non-equilibrium unfolding and refolding of lambda repressor λWT 45 6.2 Non-equilibrium unfolding and refolding of incipient-downhill λYA and downhill λHA variants of lambda repressor 48 6.3 Summary 52 7 Equilibrium unfolding and refolding of lambda repressor variants 53 7.1 Importance of the trap stiffness to resolve low-force nanometer transitions 54 7.2 Signal pair-correlation analysis to achieve millisecond transitions 56 7.3 Force-dependent equilibrium kinetics of λWT 59 7.4 Equilibrium folding of incipient-downhill λYA and downhill λHA variants of lambda repressor 61 7.5 Summary 65 8 Model-free energy landscape reconstruction for λWT, incipient-downhill λYA and downhill λHA variants 69 8.1 Direct observation of the effect of a single mutation on the conformational heterogeneity and protein stability 71 8.2 Artifacts of barrier-height determination during deconvolution 75 8.3 Summary 76 9 Conclusions and Outlook 79 / Protein folding is a process of molecular self-assembly in which a linear chain of amino acids assembles into a defined, functional three-dimensional structure. The process of folding is a thermally driven diffusive search on a free-energy landscape in the conformational space for the minimal-energy structure. During that process, the free energy of the system does not always show a monotonic decrease; instead, sub-optimal compensation of enthalpy and entropy change during each folding step leads to formation of folding free-energy barriers. However, these barriers, and associated high-energy transition states, that contain key information about mechanisms of protein folding, are kinetically inaccessible. To reveal the barrier-formation process and structural characteristics of transition states, proteins are employed that fold via barrierless paths – so-called downhill folders. Due to the low folding barriers, the key folding interactions become accessible, yielding insights about the rate-limiting folding events. Here, I compared the folding dynamics of three different variants of a lambda repressor fragment, containing amino acids 6 to 85: a two-state folder λWT (Y22W) and two downhill-like folding variants, λYA (Y22W/Q33Y/G46,48A) and λHA (Y22W/Q33H/G46,48A). To access the kinetics and structural dynamics, single-molecule optical tweezers with submillisecond and nanometer resolution are used. I found that force perturbation slowed down the microsecond kinetics of downhill folders to a millisecond time-scale, making it accessible to single-molecule studies. Interestingly, under load, the downhill-like variants of lambda repressor appeared as cooperative two-state folders with significantly different folding kinetics and force dependence. The three protein variants displayed a highly compliant behaviour under load. Model-free reconstruction of free-energy landscapes allowed us to directly resolve the fine details of the transformation of the two-state folding path into a downhill-like path. The effect of single mutations on protein stability, transition state formation and conformational heterogeneity of folding and unfolding states was observed. Noteworthy, our results demonstrate, that despite the ultrafast folding time in a range of 2 µs, the studied variants fold and unfold in a cooperative process via residual barriers, suggesting that much faster folding rate constants are required to reach the full-downhill limit.:I Theoretical background 1 1 Introduction 3 2 Protein folding: the downhill scenario 5 2.1 Protein folding as a diffusion on a multidimensional energy landscape 5 2.2 Downhill folding proteins 7 2.2.1 Thermodynamic description of downhill folders 7 2.2.2 Identification criteria for downhill folders 8 2.3 Lambda repressor as a model system for studying downhill folding 9 2.3.1 Wild-type lambda repressor fragment λ{6-85} 10 2.3.2 Acceleration of λ{6-85} folding by specifific point mutations 11 2.3.3 The incipient-downhill λYA and downhill λHA variants 14 2.4 Single-molecule techniques as a promising tool for probing downhill folding dynamics 17 3 Single-molecule protein folding with optical tweezers 19 3.1 Optical tweezers 19 3.1.1 Working principle of optical tweezers 19 3.1.2 The optical tweezers setup 21 3.2 The dumbbell assay 22 3.3 Measurement protocols 23 3.3.1 Constant-velocity experiments 23 3.3.2 Constant-trap-distance experiments (equilibrium experiments) 24 4 Theory and analysis of single-molecule trajectories 27 4.1 Polymer elasticity models 27 4.2 Equilibrium free energies of protein folding in optical tweezers 28 4.3 Signal-pair correlation analysis 29 4.4 Force dependence of transition rate constants 29 4.4.1 Zero-load extrapolation of rates: the Berkemeier-Schlierf model 30 4.4.2 Detailed balance for unfolding and refolding data 31 4.5 Direct measurement of the energy landscape via deconvolution 32 II Results 33 5 Efficient strategy for protein-DNA hybrid formation 35 5.1 Currently available strategies for protein-DNA hybrid formation 35 5.2 Novel assembly of protein-DNA hybrids based on copper-free click chemistry 37 5.3 Click-chemistry based assembly preserves the native protein structure 40 5.4 Summary 42 6 Non-equilibrium mechanical unfolding and refolding of lambda repressor variants 45 6.1 Non-equilibrium unfolding and refolding of lambda repressor λWT 45 6.2 Non-equilibrium unfolding and refolding of incipient-downhill λYA and downhill λHA variants of lambda repressor 48 6.3 Summary 52 7 Equilibrium unfolding and refolding of lambda repressor variants 53 7.1 Importance of the trap stiffness to resolve low-force nanometer transitions 54 7.2 Signal pair-correlation analysis to achieve millisecond transitions 56 7.3 Force-dependent equilibrium kinetics of λWT 59 7.4 Equilibrium folding of incipient-downhill λYA and downhill λHA variants of lambda repressor 61 7.5 Summary 65 8 Model-free energy landscape reconstruction for λWT, incipient-downhill λYA and downhill λHA variants 69 8.1 Direct observation of the effect of a single mutation on the conformational heterogeneity and protein stability 71 8.2 Artifacts of barrier-height determination during deconvolution 75 8.3 Summary 76 9 Conclusions and Outlook 79

The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

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