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.

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Alzheimers sjukdom är den vanligaste orsaken till demens och i samband med att befolkningen blir större och allt äldre ökar även antalet patienter. Vid sjukdomen sker en hjärnatrofi och de mikroskopiska fynd man ser är extracellulära plack av β-amyloid, intracellulära neurofibriller av fosforylerat tau och förlust av nervcellsutskott, axoner, synapser och dendriter. Några av de tidiga patologiska förändringarna man kan se är störningar i nervcellernas lysosomala system som fyller en viktig roll vid nedbrytning av makromolekyler. I en tidigare studie har man påvisat förhöjda nivåer av proteiner från det lysosomala systemet i cerebrospinalvätska. Syftet med den här studien var att mäta nivåer av det lysosomala systemets proteiner i human hjärnvävnad från patienter med Alzheimer och jämföra dessa med kontrollprover. De sex proteiner som analyserades med Western blot var EEA1, PICALM, LAMP-1, LAMP-2, LC3 och TFEB. Resultaten visar på signifikant ökning i temporala cortex av LAMP-1 och LAMP-2 och en signifikant minskning av LC3 och EEA1 hos patienter med Alzheimers sjukdom. För att kunna dra riktiga slutsatser kring hur de ökade nivåerna i cerebrospinalvätska speglar de olika sjukdomsmekanismerna i hjärnan krävs vidare analyser av fler patientprover samt prover från andra områden i hjärnan. / Alzheimer's disease is the most common cause of dementia, and when the population becomes larger and older also the number of patients increase. A cerebral atrophy and microscopic findings of extracellular plaques of β-amyloid, intracellular neurofibrillary of phosphorylated tau and loss of nerve cell protrusions, axons, synapses and dendrites are seen during the disease. One of the early pathological changes is the disruption of the neuronal lysosomal network that plays an important role in the degradation of macromolecules. In a previous study elevated levels of proteins of the lysosomal network in cerebrospinal fluid from Alzheimer’s disease patients was demonstrated. The purpose of this study was to measure levels of the lysosomal network system in the brain. The six proteins EEA1, PICALM, LAMP-1, LAMP -2, LC3 and TFEB were analyzed in human brain tissue from five Alzheimer's disease cases and five control cases by Western blot. The results show a significant increase in the temporal cortex of LAMP-1 and LAMP -2 and a significant decrease of LC3 and EEA1 in patients with Alzheimer's disease. In order to draw proper conclusions about how the increased levels in cerebrospinal fluid reflect the different disease mechanisms in the brain it requires further analysis of more patient samples and from other areas of the brain.

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

The purpose of the thesis is to explore the role of BMP signaling in developing vertebrate midbrain. BMP signaling plays important roles in various tissues and stages of neural development to regulate cell fate, proliferation, differentiation, morphogenesis and more. We observed that several major BMPs are expressed not only at the roof plate but also the floor plate of the midbrain. This has led us to ask the role of BMP signaling in dorsal and ventral midbrain patterning. Despite ventral experiments, we found that BMP signaling does not regulate ventral cell fate specification in the midbrain. Instead BMPs profoundly influence the shape and early morphogenesis of the midbrain neural plate as it closes into a neural tube. During neural tube closure, one of the early events occurring at the ventral midline is median hinge point (MHP) formation. Failure to form MHP leads to neural tube closure defects, the 2nd most common birth defects in humans. However, the molecular mechanisms underlying MHP formation are not well known. We found that the lowest BMP signaling occurs at the MHP during early neurulation and BMP blockade is necessary and sufficient for MHP formation. Interestingly, we also demonstrated that BMP blockade directs MHP formation by regulating the apicobasal polarity pathway and this regulation may be mediated by biochemical interactions between pSMAD5 and the apical protein, PAR3. Additionally, our time-lapse data suggest that BMP blockade slows cell cycle progression by increasing duration of G1 to S transition and S phase which leads cell nuclei stay at the basal location longer. This mimics basal nuclear migration seen at the MHP where low BMP signaling occurs. Thus, we conclude that BMP signaling regulates neural tube closure via the apicobasal polarity pathway and in a cell cycle dependent manner at the ventral midline. We observed that BMP signaling is necessary and sufficient for the dorsal cell fate specification in a context-dependent manner and ventral BMP signaling affects dorsal cell fates. Taken together, we propose the idea that BMP signaling has distinct roles in different contexts. BMPs regulate tissue morphogenesis in the ventral midbrain and dorsally cell fate specification. / text