No description available.

No description available.

The primary cilium is an immotile, microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Critically, the transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, this work employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. Using SPEED microscopy and single molecule tracking, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins. While the 3D transport pathways in this work are always replicated multiple times with a high degree of consistency, several experimental parameters directly affect the 3D transport routes’ error, such as single molecule localization precision and the number of single molecule localizations. In fact, if these experimental parameters do not meet a minimum threshold, the resultant 3D transport pathways may not have significant resolution to determine any biological details. To estimate the 3D transport routes’ error, this work will explain in detail the component of SPEED microscopy that estimates 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric bio-structures, such as the primary cilium. This component is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions based on prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the sub-structural localization of a particular protein is not. This work will demonstrate how the 2D-to-3D component of SPEED microscopy can be successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as the primary cilium and microtubules. Furthermore, this work will provide comprehensive analyses of this method by using computational simulations which investigate the role of various experimental parameters on the 3D transport pathway error. Lastly, this work will demonstrate that this method can distinguish different types of 3D transport pathway distributions in addition to their locations. / Biology

The nucleus of eukaryotic cells is a vitally important organelle that sequesters the genetic information of the cell, and protects it with the help of two highly evolved structures, the nuclear envelope (NE) and nuclear pore complexes (NPCs). Together, these two structures mediate the bidirectional trafficking of molecules between the nucleus and cytoplasm by forming a barrier. NE transmembrane proteins (NETs) embedded in either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM) play crucial roles in both nuclear structure and functions, including: genome architecture, epigenetics, transcription, splicing, DNA replication, nuclear structure, organization and positioning. Furthermore, numerous human diseases are associated with mutations and mislocalization of NETs on the NE. There are still many fundamental questions that are unresolved with NETs, but we focused on two major questions: First, the localization and transport rate of NETs, and second, the transport route taken by NETs to reach the INM. Since NETs are involved with many of the mechanisms used to maintain cellular homeostasis, it is important to quantitatively determine the spatial locations of NETs along the NE to fully understand their role in these vital processes. However, there are limited available approaches for this task, and moreover, these methods provide no information about the translocation rates of NETs between the two membranes. Furthermore, while the trafficking of soluble proteins between the cytoplasm and the nucleus has been well studied over the years, the path taken by NETs into the nucleus remains in dispute. At least four distinct models have been proposed to suggest how transmembrane proteins destined for the INM cross the NE through NPC-dependent or NPC-independent mechanisms, based on specific features found on the soluble domains of INM proteins. In order to resolve these two major questions, it is necessary to employ techniques with the capabilities to observe these dynamics at the nanoscale. Current experimental techniques are unable to break the temporal and spatial resolution barriers required to study these phenomena. Therefore, we developed and modified single-molecule techniques to answer these questions. First, to study the distribution of NETs on the NE, we developed a new single-molecule microscopy method called single-point single-molecule fluorescence recovery after photobleaching (smFRAP), which is able to provide spatial resolution <10 nm and, furthermore, provide previously unattainable information about NET translocation rates from the ONM to INM. Secondly, to examine the transport route used by NETs destined for the INM, we used a single-molecule microscopy technique previously developed in our lab called single-point edge-excitation sub-diffraction (SPEED) microscopy, which provides spatio-temporal resolution of <10 nm precision and 0.4 ms detection time. The major findings from my doctoral research work can be classified into two categories: (i) Technical developments to study NETs in vivo, and (ii) biological findings from employing these microscopy techniques. In regards to technical contributions, we created and validated of a new single-molecule microscopy method, smFRAP, to accurately determine the localization and distribution ratios of NETs on both the ONM and INM in live cells. Second, we adapted SPEED microscopy to study transmembrane protein translocation in vivo. My work has also contributed four main biological findings to the field: first, we determined the in vivo translocation rates for lamin-B receptor (LBR), a major INM protein found in the nucleus of cells. Second, we verified the existence of peripheral channels in the scaffolding of NPCs and, for the first time, directly observed the transit of INM proteins through these channels in live cells. Third, our research has elucidated the roles that both the nuclear localization signal (NLS) and intrinsically disordered (ID) domains play in INM protein transport. Finally, my work has elucidated which transport routes are used by NETs destined to localize in the INM. / Biology

The DNA origami method was established by Paul Rothemund in 2009. It allows to produce self-assembling 2D nanostructures with precise geometry and tunable mechanical properties that can be equipped with a broad range of functionalizations. It was extended to 3D by the group of William Shih in 2009 which also presented caDNAno, a software that made the design of nanostructures easier and more accessible. Since then, DNA origami nanostructures were utilized in a broad range of applications, which enabled unprecedented insight into mechanisms and processes of biological systems at the nanoscale. In this thesis multiple nanostructures were designed and manufactured to perform studies at the single-molecule level, which yielded a number of scientifically relevant contributions in the fields of biophysics and nanotechnology. Development of DNA origami nanostructures to mimic the properties and function of membrane proteins As a first application, DNA origami nanostructures with defined geometric and mechanical properties were designed, that mimic the behaviour and function of membrane proteins. To this end, rod-shaped nanostructures were equipped with precisely placed, lipid-integrating cholesterol modifications as well as fluorescent dyes. Subsequently their interaction with lipid membranes was studied. It was found that the prepared nanostructures specifically bound to lipid membranes and could diffuse on their surface, for which the rotational and translational diffusion coefficients were determined. The presence of magnesium thereby promoted the nanostructures to migrate into specific lipid domains in a reversible, switchable manner. Furthermore, their high aspect ratio allowed to investigate crowding effects, which are considered important mechanisms for the self-organisation of membrane proteins. In addition, block-shaped DNA origami nanostructures that organized into micrometre-sized super-structures were designed and produced. They were capable of deforming lipid membranes on the scale of micrometres in a similar fashion to biological counterparts. Establishing ultra-fast twist and torque measurements using DNA origami nanorotors In an additional application, DNA origami nanorotors were developed to perform ultra-fast single-molecule twist and torque measurements, allowing to resolve subtle changes in real-time. This also required the development of a new measurement setup that extended magnetic tweezers with the capability to detect the scattered light of gold nanoparticles. Hence, a complex setup was constructed and calibrated that enabled magnetic tweezers measurements with up to 4 kHz and simultaneously track gold nanoparticles at 4 kHz as well. In an alternative configuration the setup allowed simultaneous magnetic tweezers and single-molecule fluorescence and FRET measurements. DNA origami nanorotors which were embedded within DNA constructs and carried the gold nanoparticles were then obtained and used to perform ultra-fast twist and torque measurements. This constituted improvements in the spatio-temporal resolution over previous methods by one to three orders of magnitude, as demonstrated by direct measurements on the torsional response of DNA to external twists and the unwinding of DNA by an enzyme. Direct measurements of the energy landscape and dynamics of the R-loop formation by the CRISPR-Cas surveillance complex Cascade Using the DNA origami nanorotor enhanced ultra-fast twist measurements, the target recognition process of the CRISPR-Cas surveillance complex Cascade was directly observed. Effector complexes of CRISPR-Cas systems have been widely applied in genome editing recently, since they can be programmed to bind practically any genomic target by their intrinsic RNA (crRNA) component. They have, however, considerable tolerance for mismatches between their RNA and their intended DNA target. For Cascade, after binding with a protein motif to a DNA target, base-pairing between crRNA and the double-stranded DNA target is initiated, resulting in the formation of an R-loop structure which leads to unwinding of the DNA. This was directly measured using the nanorotor, which provided unprecedented insight in the R-loop formation by Cascade, allowing to determine the underlying energy landscape and the dynamics of the process. It was shown that R-loop progression occurs on 6-bp kinetic intermediate steps with an underlying single base pair stepping on fast time scales. Furthermore the effect of mutations in the target DNA on the R-loop formation process was investigated, indicating that the global shape of the energy landscape allows for a highly specific kinetic discrimination of mismatched targets. Investigations into the locking transition, a conformational change that occurs after the full formation of the R-loop and is a prerequisite for subsequent DNA degradation, completed the study. Overall, the findings provide a better understanding of the target recognition process of Cascade, which will contribute to the construction of more precise gene-editing tools in the future. Furthermore, the nanorotor-assisted measurements are applicable to many twist and torque inducing mechanisms and processes that can be investigated in further studies.:1. Introduction 2. Multifunctional magnetic tweezers 3. Applications of DNA origami 4. Ultra-Fast torque measurements on supercoiled DNA 5. R-loop dynamics of the CRISPR-Cas Cascade complex 6. Summary and Discussion Bibliography List of Figures List of Tables List of Publications A. Appendix / Die DNA Origami Methode wurde im Jahr 2006 durch Paul Rothemund begründet. Sie erlaubt es selbst-assemblierende 2D Nanostrukturen mit präzisen Geometrien und kalibrierbaren mechanischen Eigenschaften zu erstellen, die zudem mit einer Vielzahl an Funktionalisierungen ausgestattet werden können. Die Methode wurde 2009 in der Gruppe von William Shih auf 3D Nanostrukturen erweitert, wobei zudem caDNAno präsentiert wurde, eine Software die die Erstellung solcher Nanostrukturen wesentlich einfacher und zugänglicher machte. Seitdem wurden DNA Origami Nanostrukturen in vielfältigen Anwendungen genutzt, die nie dagewesene Einblicke in Mechanismen und Prozesse von biologischen Systemen auf der Nanoskala erlaubten. In dieser Arbeit wird anhand mehrerer Beispiele gezeigt, wie solche Nanostrukturen genutzt werden können, um Studien auf der Einzelmolekül-Ebene durchzuführen. Entwicklung von DNA Origami Nanostrukturen, welche die Eigenschaften und Funktionen von Membranproteinen imitieren In einer ersten Anwendung wurden DNA Origami Nanostrukturen mit definierten geometrischen und mechanischen Eigenschaften entworfen, welche das Verhalten und die Funktion von Membranproteinen nachahmten. Dazu wurden stabförmige Nanostrukturen mit präzise platzierten, lipidintegrierenden Cholesterinmodifikationen und fluoreszierenden Farbstoffen ausgestattet. Anschließend wurde ihre Interaktion mit Lipidmembranen untersucht. Es zeigte sich, dass die Nanostrukturen spezifisch an Lipidmembranen binden und auf deren Oberfläche diffundieren konnten. Hierbei wurden die Diffusionskoeffizienten der Rotations- und Translationsbewegungen bestimmt. Zudem bewirkte die An- oder Abwesenheit freier Magnesiumionen die steuerbare und reversible Anreicherung in verschiedenen Lipiddomänen. Die längliche Form der Nanostrukturen erlaubte es zudem, Verdrängungseffekte zu untersuchen, die als wichtiger Mechanismus für die Selbstorganisation von Membranproteinen gelten. Des weiteren wurden blockartige, multimerisierende DNA Origami Nanostrukturen entwickelt, die mikrometer-große Superstrukturen bilden konnten. Im ähnlichen Maße wie biologische Vorbilder, waren diese Strukturen in der Lage, Lipidmembranen über mehrere Mikrometer hinweg zu verformen. Etablierung ultraschneller Verdrehungs- und Torsionsmessungen mit DNA Origami Nanorotoren In einer weiteren Anwendung wurden DNA Origami Nanorotoren entwickelt, um ultraschnelle Einzelmolekül-Verdrehungs- und Torsionsmessungen durchzuführen, bei denen kleinste Veränderungen in Echtzeit beobachtet werden konnten. Dazu wurde eine neue Messapparatur entwickelt, bei der eine Magnetische Pinzette um die Fähigkeit Goldnanopartikeln zu detektieren erweitert wurde. Dies erlaubte die Konstruktion und Kalibrierung eines komplexen Messaufbaus, mit dem es möglich war Magnetische-Pinzetten-Messungen mit 4 kHz durchzuführen und gleichzeitig Goldnanopartikel mit ebenfalls 4 kHz zu verfolgen. Zudem konnten in einer alternativen Konfiguration Magnetische-Pinzetten-Messungen mit Einzelmolekül-Fluoreszenz- und FRET-Messungen kombiniert werden. Mit Goldnanopartikeln funktionalisierte DNA-Origami-Nanorotoren wurden anschließend in DNA-Konstrukte eingebettet und mit Hilfe des Messaufbaus für ultraschnelle Verdrehungs- und Torsionsmessungen genutzt. Gegenüber vorheriger Methoden wurde dadurch die räumlich-zeitliche Auflösung um eine bis drei Größenordnungen verbessert. Dies wurde anhand der Bestimmung der Torsionsreaktion von DNA auf Verdrehungen sowie deren Entwindung durch ein Enzym demonstriert. Direkte Bestimmung der Energielandschaft und Dynamiken der R-loop Entstehung des CRISPR-Cas Überwachungskomplexes Cascade Die entwickelten DNA Origami Nanorotoren ermöglichten zudem ultraschnelle Verdrehungsmessungen durchzuführen, um den Zielerkennungsprozess des CRISPR-Cas Überwachungskomplexes Cascade direkt zu beobachten. Effektorkomplexe von CRISPR-Cas Systemen werden zunehmend als Geneditierwerkzeuge eingesetzt, da sie aufgrund ihrer intrinsischen RNA-Komponente (crRNA) darauf programmiert werden können an praktisch jede DNA-Sequenz zu binden. Allerdings zeigen Sie eine beträchtliche Toleranz gegenüber Abweichungen zwischen der Sequenz ihrer RNA und der bestimmungsgemäßen DNA-Zielsequenz. Grundsätzlich bindet Cascade zunächst mit einem Protein-Motiv an eine kurze DNA-Sequenz, woraufhin es zur Basenpaarung zwischen der crRNA und der doppelsträngigen DNA-Zielsequenz kommt. Dabei entsteht ein R-loop, was zur Entwindung der DNA führt. Dies wurde direkt mit Hilfe der Nanorotoren gemessen, was nie dagewesene Einblicke in diesen Prozess erlaubte und die Bestimmung der zugrundeliegenden Energielandschaft und Dynamiken ermöglichte. Es wurde gezeigt, dass Längenänderungen des R-loops in kinetischen Zwischenschritten von 6 Basenpaaren erfolgen, denen Einzel-Basenpaar-Schritte auf schnelleren Zeitskalen zugrunde liegen. Des weiteren wurde der Effekt von Mutationen der DNA-Zielsequenz auf die R-loop Entstehung untersucht. Hierbei zeigte sich, dass die globale Form der Energielandschaft eine hochspezifische kinetische Differenzierung von inkongruenten Zielsequenzen erlaubt. Untersuchungen des 'locking' Mechanismus, ein struktureller Übergang der nach der vollständigen Ausbildung des R-loops erfolgt und eine Voraussetzung für die nachfolgende Zersetzung von DNA darstellt, rundeten die Untersuchungen ab. Insgesamt wurde gezeigt, dass mit Hilfe der ultraschnellen Verdrehungsmessungen, neue, detaillierte Einblicke in den Zielerkennungsprozess von Cascade gewonnen wurden, die zur Erstellung präziserer Genmanipulationswerkzeuge in der Zukunft beitragen können. Darüber hinaus eignen sich die Nanorotoren zur Untersuchung weiterer verdrehungs- und torsionserzeugender Mechanismen und Prozesse, die in weiteren Studien erforscht werden können.:1. Introduction 2. Multifunctional magnetic tweezers 3. Applications of DNA origami 4. Ultra-Fast torque measurements on supercoiled DNA 5. R-loop dynamics of the CRISPR-Cas Cascade complex 6. Summary and Discussion Bibliography List of Figures List of Tables List of Publications A. Appendix

The DNA origami method was established by Paul Rothemund in 2009. It allows to produce self-assembling 2D nanostructures with precise geometry and tunable mechanical properties that can be equipped with a broad range of functionalizations. It was extended to 3D by the group of William Shih in 2009 which also presented caDNAno, a software that made the design of nanostructures easier and more accessible. Since then, DNA origami nanostructures were utilized in a broad range of applications, which enabled unprecedented insight into mechanisms and processes of biological systems at the nanoscale. In this thesis multiple nanostructures were designed and manufactured to perform studies at the single-molecule level, which yielded a number of scientifically relevant contributions in the fields of biophysics and nanotechnology. Development of DNA origami nanostructures to mimic the properties and function of membrane proteins As a first application, DNA origami nanostructures with defined geometric and mechanical properties were designed, that mimic the behaviour and function of membrane proteins. To this end, rod-shaped nanostructures were equipped with precisely placed, lipid-integrating cholesterol modifications as well as fluorescent dyes. Subsequently their interaction with lipid membranes was studied. It was found that the prepared nanostructures specifically bound to lipid membranes and could diffuse on their surface, for which the rotational and translational diffusion coefficients were determined. The presence of magnesium thereby promoted the nanostructures to migrate into specific lipid domains in a reversible, switchable manner. Furthermore, their high aspect ratio allowed to investigate crowding effects, which are considered important mechanisms for the self-organisation of membrane proteins. In addition, block-shaped DNA origami nanostructures that organized into micrometre-sized super-structures were designed and produced. They were capable of deforming lipid membranes on the scale of micrometres in a similar fashion to biological counterparts. Establishing ultra-fast twist and torque measurements using DNA origami nanorotors In an additional application, DNA origami nanorotors were developed to perform ultra-fast single-molecule twist and torque measurements, allowing to resolve subtle changes in real-time. This also required the development of a new measurement setup that extended magnetic tweezers with the capability to detect the scattered light of gold nanoparticles. Hence, a complex setup was constructed and calibrated that enabled magnetic tweezers measurements with up to 4 kHz and simultaneously track gold nanoparticles at 4 kHz as well. In an alternative configuration the setup allowed simultaneous magnetic tweezers and single-molecule fluorescence and FRET measurements. DNA origami nanorotors which were embedded within DNA constructs and carried the gold nanoparticles were then obtained and used to perform ultra-fast twist and torque measurements. This constituted improvements in the spatio-temporal resolution over previous methods by one to three orders of magnitude, as demonstrated by direct measurements on the torsional response of DNA to external twists and the unwinding of DNA by an enzyme. Direct measurements of the energy landscape and dynamics of the R-loop formation by the CRISPR-Cas surveillance complex Cascade Using the DNA origami nanorotor enhanced ultra-fast twist measurements, the target recognition process of the CRISPR-Cas surveillance complex Cascade was directly observed. Effector complexes of CRISPR-Cas systems have been widely applied in genome editing recently, since they can be programmed to bind practically any genomic target by their intrinsic RNA (crRNA) component. They have, however, considerable tolerance for mismatches between their RNA and their intended DNA target. For Cascade, after binding with a protein motif to a DNA target, base-pairing between crRNA and the double-stranded DNA target is initiated, resulting in the formation of an R-loop structure which leads to unwinding of the DNA. This was directly measured using the nanorotor, which provided unprecedented insight in the R-loop formation by Cascade, allowing to determine the underlying energy landscape and the dynamics of the process. It was shown that R-loop progression occurs on 6-bp kinetic intermediate steps with an underlying single base pair stepping on fast time scales. Furthermore the effect of mutations in the target DNA on the R-loop formation process was investigated, indicating that the global shape of the energy landscape allows for a highly specific kinetic discrimination of mismatched targets. Investigations into the locking transition, a conformational change that occurs after the full formation of the R-loop and is a prerequisite for subsequent DNA degradation, completed the study. Overall, the findings provide a better understanding of the target recognition process of Cascade, which will contribute to the construction of more precise gene-editing tools in the future. Furthermore, the nanorotor-assisted measurements are applicable to many twist and torque inducing mechanisms and processes that can be investigated in further studies.:1. Introduction 2. Multifunctional magnetic tweezers 3. Applications of DNA origami 4. Ultra-Fast torque measurements on supercoiled DNA 5. R-loop dynamics of the CRISPR-Cas Cascade complex 6. Summary and Discussion Bibliography List of Figures List of Tables List of Publications A. Appendix / Die DNA Origami Methode wurde im Jahr 2006 durch Paul Rothemund begründet. Sie erlaubt es selbst-assemblierende 2D Nanostrukturen mit präzisen Geometrien und kalibrierbaren mechanischen Eigenschaften zu erstellen, die zudem mit einer Vielzahl an Funktionalisierungen ausgestattet werden können. Die Methode wurde 2009 in der Gruppe von William Shih auf 3D Nanostrukturen erweitert, wobei zudem caDNAno präsentiert wurde, eine Software die die Erstellung solcher Nanostrukturen wesentlich einfacher und zugänglicher machte. Seitdem wurden DNA Origami Nanostrukturen in vielfältigen Anwendungen genutzt, die nie dagewesene Einblicke in Mechanismen und Prozesse von biologischen Systemen auf der Nanoskala erlaubten. In dieser Arbeit wird anhand mehrerer Beispiele gezeigt, wie solche Nanostrukturen genutzt werden können, um Studien auf der Einzelmolekül-Ebene durchzuführen. Entwicklung von DNA Origami Nanostrukturen, welche die Eigenschaften und Funktionen von Membranproteinen imitieren In einer ersten Anwendung wurden DNA Origami Nanostrukturen mit definierten geometrischen und mechanischen Eigenschaften entworfen, welche das Verhalten und die Funktion von Membranproteinen nachahmten. Dazu wurden stabförmige Nanostrukturen mit präzise platzierten, lipidintegrierenden Cholesterinmodifikationen und fluoreszierenden Farbstoffen ausgestattet. Anschließend wurde ihre Interaktion mit Lipidmembranen untersucht. Es zeigte sich, dass die Nanostrukturen spezifisch an Lipidmembranen binden und auf deren Oberfläche diffundieren konnten. Hierbei wurden die Diffusionskoeffizienten der Rotations- und Translationsbewegungen bestimmt. Zudem bewirkte die An- oder Abwesenheit freier Magnesiumionen die steuerbare und reversible Anreicherung in verschiedenen Lipiddomänen. Die längliche Form der Nanostrukturen erlaubte es zudem, Verdrängungseffekte zu untersuchen, die als wichtiger Mechanismus für die Selbstorganisation von Membranproteinen gelten. Des weiteren wurden blockartige, multimerisierende DNA Origami Nanostrukturen entwickelt, die mikrometer-große Superstrukturen bilden konnten. Im ähnlichen Maße wie biologische Vorbilder, waren diese Strukturen in der Lage, Lipidmembranen über mehrere Mikrometer hinweg zu verformen. Etablierung ultraschneller Verdrehungs- und Torsionsmessungen mit DNA Origami Nanorotoren In einer weiteren Anwendung wurden DNA Origami Nanorotoren entwickelt, um ultraschnelle Einzelmolekül-Verdrehungs- und Torsionsmessungen durchzuführen, bei denen kleinste Veränderungen in Echtzeit beobachtet werden konnten. Dazu wurde eine neue Messapparatur entwickelt, bei der eine Magnetische Pinzette um die Fähigkeit Goldnanopartikeln zu detektieren erweitert wurde. Dies erlaubte die Konstruktion und Kalibrierung eines komplexen Messaufbaus, mit dem es möglich war Magnetische-Pinzetten-Messungen mit 4 kHz durchzuführen und gleichzeitig Goldnanopartikel mit ebenfalls 4 kHz zu verfolgen. Zudem konnten in einer alternativen Konfiguration Magnetische-Pinzetten-Messungen mit Einzelmolekül-Fluoreszenz- und FRET-Messungen kombiniert werden. Mit Goldnanopartikeln funktionalisierte DNA-Origami-Nanorotoren wurden anschließend in DNA-Konstrukte eingebettet und mit Hilfe des Messaufbaus für ultraschnelle Verdrehungs- und Torsionsmessungen genutzt. Gegenüber vorheriger Methoden wurde dadurch die räumlich-zeitliche Auflösung um eine bis drei Größenordnungen verbessert. Dies wurde anhand der Bestimmung der Torsionsreaktion von DNA auf Verdrehungen sowie deren Entwindung durch ein Enzym demonstriert. Direkte Bestimmung der Energielandschaft und Dynamiken der R-loop Entstehung des CRISPR-Cas Überwachungskomplexes Cascade Die entwickelten DNA Origami Nanorotoren ermöglichten zudem ultraschnelle Verdrehungsmessungen durchzuführen, um den Zielerkennungsprozess des CRISPR-Cas Überwachungskomplexes Cascade direkt zu beobachten. Effektorkomplexe von CRISPR-Cas Systemen werden zunehmend als Geneditierwerkzeuge eingesetzt, da sie aufgrund ihrer intrinsischen RNA-Komponente (crRNA) darauf programmiert werden können an praktisch jede DNA-Sequenz zu binden. Allerdings zeigen Sie eine beträchtliche Toleranz gegenüber Abweichungen zwischen der Sequenz ihrer RNA und der bestimmungsgemäßen DNA-Zielsequenz. Grundsätzlich bindet Cascade zunächst mit einem Protein-Motiv an eine kurze DNA-Sequenz, woraufhin es zur Basenpaarung zwischen der crRNA und der doppelsträngigen DNA-Zielsequenz kommt. Dabei entsteht ein R-loop, was zur Entwindung der DNA führt. Dies wurde direkt mit Hilfe der Nanorotoren gemessen, was nie dagewesene Einblicke in diesen Prozess erlaubte und die Bestimmung der zugrundeliegenden Energielandschaft und Dynamiken ermöglichte. Es wurde gezeigt, dass Längenänderungen des R-loops in kinetischen Zwischenschritten von 6 Basenpaaren erfolgen, denen Einzel-Basenpaar-Schritte auf schnelleren Zeitskalen zugrunde liegen. Des weiteren wurde der Effekt von Mutationen der DNA-Zielsequenz auf die R-loop Entstehung untersucht. Hierbei zeigte sich, dass die globale Form der Energielandschaft eine hochspezifische kinetische Differenzierung von inkongruenten Zielsequenzen erlaubt. Untersuchungen des 'locking' Mechanismus, ein struktureller Übergang der nach der vollständigen Ausbildung des R-loops erfolgt und eine Voraussetzung für die nachfolgende Zersetzung von DNA darstellt, rundeten die Untersuchungen ab. Insgesamt wurde gezeigt, dass mit Hilfe der ultraschnellen Verdrehungsmessungen, neue, detaillierte Einblicke in den Zielerkennungsprozess von Cascade gewonnen wurden, die zur Erstellung präziserer Genmanipulationswerkzeuge in der Zukunft beitragen können. Darüber hinaus eignen sich die Nanorotoren zur Untersuchung weiterer verdrehungs- und torsionserzeugender Mechanismen und Prozesse, die in weiteren Studien erforscht werden können.:1. Introduction 2. Multifunctional magnetic tweezers 3. Applications of DNA origami 4. Ultra-Fast torque measurements on supercoiled DNA 5. R-loop dynamics of the CRISPR-Cas Cascade complex 6. Summary and Discussion Bibliography List of Figures List of Tables List of Publications A. Appendix

Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity. This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics.

Inwardly rectifying potassium (Kir) channels are essential for controlling the excitability of eukaryotic cells, forming a key part of the inter-cellular signalling system in multi-cellular organisms. However, as prokaryotic (KirBac) channels are less technically challenging to study in vitro and have been shown to be directly homologous to eukaryotic channels, they are often studied in lieu of their mammalian counterparts. A vital feature of Kir and KirBac channels is their mechanism for opening and closing, or their gating: this study predominantly features observations of open and/or closed channel populations. A well-characterised member of the KirBac family, KirBac1.1, has been successfully expressed, purified into detergent micelles, and doubly labelled with fluorescent maleimide dyes in order to enable observation of confocal-in-solution Förster Resonance Energy Transfer (FRET) at the single molecule level. Results demonstrate single-molecule FRET signals from KirBac1.1 and therefore represent the first single-molecule FRET observations from a KirBac channel. Perturbation of the open-closed dynamic equilibrium was performed via activatory point mutations, changes in pH, and ligand binding. A protocol for reconstitution into nanodiscs was optimised in order to more closely approximate native conditions, and the single-molecule FRET observations repeated. This thesis presents a comparison between measurements made using the detergent solubilisation system and those made using nanodiscs.

Information about protein structures is important in many areas of life sciences, including structure-based drug design. Gas phase methods, like electrospray ionization and mass spectrometry are powerful tools for the analysis of molecular interactions and conformational changes which complement existing solution phase methods. Novel techniques such as single particle imaging with X-ray free electron lasers are emerging as well. A requirement for using gas phase methods is that we understand what happens to proteins when injected into vacuum, and what is the relationship between the vacuum structure and the solution structure. Molecular dynamics simulations in combination with experiments show that protein structures in the gas phase can be similar to solution structures, and that hydrogen bonding networks and secondary structure elements can be retained. Structural changes near the surface of the protein happen quickly (ns-µs) during transition from solution into vacuum. The native solution structure results in a reasonably well defined gas phase structure, which has high structural similarity to the solution structure. Native charge locations are in some cases also preserved, and structural changes, due to point mutations in solution, can also be observed in vacuo. Proteins do not refold in vacuo: when a denatured protein is injected into vacuum, the resulting gas phase structure is different from the native structure. Native structures can be protected in the gas phase by adjusting electrospray conditions to avoid complete evaporation of water. A water layer with a thickness of less than two water molecules seems enough to preserve native conditions. The results presented in this thesis give confidence in the continued use of gas phase methods for analysis of charge locations, conformational changes and non-covalent interactions, and provide a means to relate gas phase structures and solution structures.

La fonction des canaux ioniques est finement régulée par des changements structuraux de sites clés contrôlant l’ouverture du pore. Ces modulations structurales découlent de l’interaction du canal avec l’environnement local, puisque certains domaines peuvent être suffisamment sensibles à des propriétés physico-chimiques spécifiques. Les mouvements engendrés dans la structure sont notamment perceptibles fonctionnellement lorsque le canal ouvre un passage à certains ions, générant ainsi un courant ionique mesurable selon le potentiel électrochimique. Une description détaillée de ces relations structure-fonction est cependant difficile à obtenir à partir de mesures sur des ensembles de canaux identiques, puisque les fluctuations et les distributions de différentes propriétés individuelles demeurent cachées dans une moyenne. Pour distinguer ces propriétés, des mesures à l’échelle de la molécule unique sont nécessaires. Le but principal de la présente thèse est d’étudier la structure et les mécanismes moléculaires de canaux ioniques par mesures de spectroscopie de fluorescence à l’échelle de la molécule unique. Les études sont particulièrement dirigées vers le développement de nouvelles méthodes ou leur amélioration. Une classe de toxine formeuse de pores a servi de premier modèle d’étude. La fluorescence à l’échelle de la molécule unique a aussi été utilisée pour l’étude d’un récepteur glutamate, d’un récepteur à la glycine et d’un canal potassique procaryote. Le premier volet porte sur l’étude de la stœchiométrie par mesures de photoblanchiment en temps résolu. Cette méthode permet de déterminer directement le nombre de monomères fluorescents dans un complexe isolé par le décompte des sauts discrets de fluorescence suivant les événements de photoblanchiment. Nous présentons ici la première description, à notre connaissance, de l’assemblage dynamique d’une protéine membranaire dans un environnement lipidique. La toxine monomérique purifiée Cry1Aa s’assemble à d’autres monomères selon la concentration et sature en conformation tétramérique. Un programme automatique est ensuite développé pour déterminer la stœchiométrie de protéines membranaires fusionnées à GFP et exprimées à la surface de cellules mammifères. Bien que ce système d’expression soit approprié pour l’étude de protéines d’origine mammifère, le bruit de fluorescence y est particulièrement important et augmente significativement le risque d’erreur dans le décompte manuel des monomères fluorescents. La méthode présentée permet une analyse rapide et automatique basée sur des critères fixes. L’algorithme chargé d’effectuer le décompte des monomères fluorescents a été optimisé à partir de simulations et ajuste ses paramètres de détection automatiquement selon la trace de fluorescence. La composition de deux canaux ioniques a été vérifiée avec succès par ce programme. Finalement, la fluorescence à l’échelle de la molécule unique est mesurée conjointement au courant ionique de canaux potassiques KcsA avec un système de fluorométrie en voltage imposé. Ces enregistrements combinés permettent de décrire la fonction de canaux ioniques simultanément à leur position et densité alors qu’ils diffusent dans une membrane lipidique dont la composition est choisie. Nous avons observé le regroupement de canaux KcsA pour différentes compositions lipidiques. Ce regroupement ne paraît pas être causé par des interactions protéine-protéine, mais plutôt par des microdomaines induits par la forme des canaux reconstitués dans la membrane. Il semble que des canaux regroupés puissent ensuite devenir couplés, se traduisant en ouvertures et fermetures simultanées où les niveaux de conductance sont un multiple de la conductance « normale » d’un canal isolé. De plus, contrairement à ce qui est actuellement suggéré, KcsA ne requiert pas de phospholipide chargé négativement pour sa fonction. Plusieurs mesures indiquent plutôt que des lipides de forme conique dans la phase cristalline liquide sont suffisants pour permettre l’ouverture de canaux KcsA isolés. Des canaux regroupés peuvent quant à eux surmonter la barrière d’énergie pour s’ouvrir de manière coopérative dans des lipides non chargés de forme cylindrique. / The function of ion channels is finely regulated by structural changes of key domains controlling the pore opening. These structural modulations arise from interactions with the local environment, since several domains can be sensitive to specific physico-chemical properties. Movements generated in the structure become notably perceptible when channels open a passage for some ions, thus generating a measurable ionic current according to the electrochemical potential. A detailed description of these structure-function relationships is however difficult to obtain from measurements involving a set of identical channels, since the fluctuations and distributions of different individual properties remain hidden in an average. To differentiate these properties, single-molecule recordings are required. The main purpose of this thesis is to study the structural aspects and molecular mechanisms of ion channels using fluorescence spectroscopy at the single-molecule level. Studies are oriented towards the development or improvement of new methods. A class of pore-forming toxin served as a first study model. Single-molecule fluorescence was also used to study an ionotropic glutamate receptor, a glycine receptor and a prokaryotic potassium channel. The first part focuses on the study of stoichiometry using fluorescent subunit counting. This method allows a direct measure of the number of fluorescent monomers within a single complex by counting the number of step-wise fluorescence intensity decrease following photobleaching events. Here we present the first description, to our knowledge, of the dynamic assembly of a membrane protein in a lipid environment. The purified monomeric Cry1Aa toxin clusters with other monomers depending on the concentration and saturates in a tetrameric conformation. An automated method has been developed to determine the stoichiometry of GFP-tagged membrane proteins expressed on mammalian cell surface. Although this expression system is suitable for the study of proteins of mammalian origin, background fluorescence is particularly important and significantly increases the risk of error in the manual counting process. The presented method allows a fast and automated analysis based on fixed criteria. The algorithm responsible for counting fluorescent monomers was optimized from simulations and adjusts its detection parameters automatically according to the fluorescence trace recording. The composition of two ion channels was successfully verified using this program. Finally, single-molecule fluorescence is measured together with ionic current of KcsA channels using a voltage-clamp fluorometry setup. These combined recordings allowed us to describe the function of ion channels simultaneously to their position and density as they diffuse in a lipid membrane of defined composition. We observed clustering of KcsA channels for various lipid compositions. Clustering does not appear to be caused by protein-protein interaction, but rather by microdomains induced by the shape of reconstructed channels in the lipid bilayer. It seems that clustered KcsA channels could then become coupled, resulting in cooperative gating events with conductance levels multiple to the “normal” unitary channel conductance. Moreover, as opposed to what is currently suggested, KcsA does not require a negatively charged phospholipid for its function. Several of our recordings rather suggest that conically shaped lipids in the lamellar liquid crystalline phase are sufficient to allow single channel opening. Clustered channels can on the other hand overcome the energy barrier to open cooperatively in uncharged cylindrical lipids.