Biological Systems Workbook: Data modelling and simulations at molecular level

Nowadays, there are huge quantities of data surrounding the different fields of biology derived from experiments and theoretical simulations, where results are often stored in biological databases that are growing at a vertiginous rate every year. Therefore, there is an increasing research interest in the application of mathematical and physical models able to produce reliable predictions and explanations to understand and rationalize that information. All these investigations are helping to overcome biological questions pushing forward in the solution of problems faced by our society. In this Biological Systems Workbook, we aim to introduce the basic pieces allowing life to take place, from the 3D structural point of view. We will start learning how to look at the 3D structure of molecules from studying small organic molecules used as drugs. Meanwhile, we will learn some methods that help us to generate models of these structures. Then we will move to more complex natural organic molecules as lipid or carbohydrates, learning how to estimate and reproduce their dynamics. Later, we will revise the structure of more complex macromolecules as proteins or DNA. Along this process, we will refer to different computational tools and databases that will help us to search, analyze and model the different molecular systems studied in this course

Microfluidic Planar Phospholipids Membrane System Advancing Dynamics Studies of Ion Channels and Membrane Physics

The interrogation of lipid membrane and biological ion channels supported within bilayer phospholipid membranes has greatly expanded our understanding of the roles membrane and ion channels play in a host of biological functions. Several key drawbacks of traditional electrophysiology systems used in these studies have long limited our effort to study the ion channels. Firstly, the large volume buffer in this system typically only allows single or multiple additions of reagents, while complete removal either is impossible or requires tedious effort to ensure the stability of membrane. Thus, it has been highly desirable to be able to rapidly and dynamically modulate the (bio)chemical conditions at the membrane site. Second, it is difficult to change temperature effectively with large thermal mass in macro device. Third, traditional PPM device host vertical membranes, therefore incompatible with confocal microscopy techniques. The miniaturization of bilayer phospholipid membrane has shown potential solution to the drawbacks stated above. A simple microfluidic design is developed to enable effective and robust dynamic perfusion of reagents directly to an on-chip planar phospholipid membrane (PPM). It allows ion channel conductance to be readily monitored under different dynamic reagent conditions, with perfusion rates up to 20 µL/min feasible without compromising the membrane integrity. It is estimated that the lower limit of time constant of kinetics that can be resolved by our system is 1 minute. Using this platform, the time-dependent responses of membrane-bound ceramide ion channels to treatments with La3+ and a Bcl-xL mutant were studied and the results were interpreted with a novel elastic biconcave distortion model. Another engineering challenge this dissertation takes on is the integration of fluorescence studies to micro-PPM system. The resulting novel microfluidic system enables high resolution, high magnification and real-time confocal microscope imaging with precise top and bottom (bio)chemical boundary conditions defined by perfusion, by integrating in situ PPM formation method, perfusion capability and microscopy compatibility. To demonstrate such electro-optical chip, lipid micro domains were imaged and quantitatively studied for their movements and responses to different physical parameters. As an extension to this platform, a double PPM system has been developed with the aim to study interactions between two membranes. Potential application in biophysics and biochemistry using those two platforms were discussed. Another important advantage of microfluidics is its lower thermal mass and compatibility with various microfabrication methods which enables potential integration of local temperature controller and sensor. A prototype thermal PPM chip is also discussed together with some preliminary results and their implication on ceramide channel assembly and disassembly mechanism

High-Throughput Automated Patch Clamp Investigations on Ion Channels in Erythrocytes

Trotz ihrer morphologischen Einfachheit ist die Membran der roten Blutkörperchen (Erythrozyten) mit einer Reihe von Transportern und Ionenkanälen ausgestattet, die bisher nicht vollständig charakterisiert sind und deren biologische Rolle noch wenig verstanden ist. Die meisten Techniken zur Untersuchung von Ionenkanälen messen summierte Effekte großer Zellpopulationen und verbergen so jede mutmaßliche Variabilität von Zelle zu Zelle. Die Patch-Clamp-Technik hat sich als effektives Werkzeug zur Entdeckung und Charakterisierung von Ionenkanälen auf Einzelzellenebene erwiesen. Dies besonders wichtig für Erythrozyten von Säugetieren, die eine hohe Heterogenität der Leitfähigkeit zwischen verschiedenen Spendern, und auch zwischen Zellen desselben Spenders aufweisen (Kaestner et al., 2004; Minetti et al., 2013). Die Entwicklung des automatisierten Patch-Clamps ermöglichte es, eine hohe Anzahl von Zellen gleichzeitig unter identischen experimentellen Bedingungen zu untersuchen, wodurch Zellheterogenität erstmals umfassend bestimmt wurde. In dieser Arbeit wurden Gárdos- und Piezo1-Kanäle als Hauptuntersuchungsziele ausgewählt, da sie eine prominente Rolle in erythrozytären Erkrankungen, im Einzelnen Gárdos-Kanalopathie (Fermo et al., 2017) und hereditäre Xerozytose (Zarychanski et al., 2012; Bae et al., 2013), spielen. Ziel dieser Arbeit war es, automatisierte Patch-Clamp-Assays zur Charakterisierung dieser Kanäle in Erythrozyten zu entwickeln. Es gibt bisher nur vereinzelte Publikationen zu whole-cell Patch-Clamp-Messungen von Gárdos-Kanälen in Erythrozyten (Grygorczyk et al., 1984; Wolff et al., 1988), wahrscheinlich aufgrund der geringen Expression des Proteins in zirkulierenden Erythrozyten. Der hochparallelisierte Ansatz der automatisierten Patch-Clamp-Technologie ermöglicht zuverlässig die Identifizierung von Gárdos-Strömen in Zelltypen mit einer oft geringen Anzahl von Kanälen und einer großen Heterogenität der Expression, wie bei Erythrozyten. Bisherige Piezo1-Kanaluntersuchungen zeigen, dass die Substanz Yoda1 Piezo1-Ströme bewirken kann, die empfindlich auf GdCl3 (unspezifischer Inhibitor dehnungsaktivierter Kanäle), nicht jedoch auf TRAM-34 (spezifischer Gárdos-Kanalinhibitor) reagieren. Die Anwendung dieses Assays auf Erythrozyten von Patienten mit einer neuartigen PIEZO1 R2110W-Mutation zeigte eine erhöhte Anzahl der Yoda1-empfindlichen Zellen und eine stärkere Antwort auf Yoda1 bei Patienten im Vergleich zu Kontroll-Erythrozyten. In Kombination mit der Untersuchung der Proteinstruktur, die den R2110W-Rests in einem gating-sensitiven Bereich des Kanals lokalisiert, deuten die Patch-Clamp-Ergebnisse darauf hin, dass die neue Piezo1-Mutation eine gain-of-function-Mutation ist (Rotordam et al., 2019). Zusammenfassend zeigt diese Arbeit, dass die automatisierte Patch-Clamp-Methode robuste Assays zur Untersuchung von Ionenkanälen (Gárdos und Piezo1) in Primärzellen liefert. Die Hochdurchsatztechnologie ermöglichte die Entwicklung eines zuverlässigen Assays für gering exprimierte Ionenkanäle bei hoher Heterogenität der Zellen. So war es möglich, eine neuartige Kanalmutation auf funktioneller Ebene direkt in Patientenzellen zu charakterisieren, ohne die Mutation in einem heterologen Expressionssystem exprimieren zu müssen. Dieser Ansatz kann zum Nachweis und zur Charakterisierung weiterer Kanalopathien verwendet werden, die nicht auf Erythrozyten beschränkt sind, und kann generell als zur Gensequenzierung komplementärer Routine-Screening-Assay für Krankheiten dienen, die mit Ionenkanalstörungen zusammenhängen.Despite the morphological simplicity, the Red Blood Cell (RBC) membrane is endowed with a number of transporters and ion channels, yet not fully characterized and whose biological role is still poorly understood. Most of the techniques used to investigate ion channels are addressed to large populations of cells, thus concealing any putative cell-to-cell variability. The patch clamp technique has proven to be a valid tool for the discovery and characterization of ion channels at a single-cell level. This is of particular relevance for mammalian RBCs, which present a high heterogeneity of conductance not only between different donors but also among cells of the same donor (Kaestner et al., 2004; Minetti et al., 2013). The advent of automated patch clamp allowed to probe an increased number of cells at the same time under identical experimental conditions, thus tackling cell heterogeneity issues. In this thesis, Gárdos and Piezo1 channels were selected as main targets of investigation due to their relevance in RBC-related diseases, i.e. Gárdos channelopathy (Fermo et al., 2017) and hereditary xerocytosis (Zarychanski et al., 2012; Bae et al., 2013). The aim of this work was to develop automated patch clamp assays for characterizing those channels in RBCs. As for Gárdos channels, whole cell recordings reported so far are fragmentary probably due to the low expression of the protein in circulating RBCs (Grygorczyk et al., 1984; Wolff et al., 1988). By increasing the number of cells recorded at the same time, the automated patch clamp technology allowed to identify Gárdos-mediated currents in primary cells with a low-copy number of channels and a large heterogeneity of conductance as RBCs. Piezo1 channels investigations confirmed that application of Yoda1 alone is able to elicit currents sensitive to GdCl3 (non-specific stretch-activated channels inhibitor) but not TRAM-34 (specific Gárdos channel blocker). When transferred to patients carrying a novel PIEZO1 R2110W mutation, the assay revealed that the number of responders and the magnitude of the response to Yoda1 increased in patient compared to control RBCs. This result, combined with structural studies identifying the R2110W residue in a gating sensitive area of the channel, suggested that the novel Piezo1 mutation is gain-of-function (Rotordam et al., 2019). Altogether, this work demonstrates that automated patch clamping provides robust assays to investigate ion channels (Gárdos and Piezo1) in primary cells. The high-throughput technology allowed to tackle issues as response heterogeneity and low expression of the channels, and to characterize a novel channel mutation at a functional level directly from patient cells, without having to express the mutation in a heterologous expression system. This approach may be used to detect other channelopathies not limited to RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing

Hydrogel encapsulated droplet interface bilayer networks as a chassis for artificial cells and a platform for membrane studies

There has been increasing interest in droplet interface bilayers (DIBs) as novel devices for the study of lipid membranes and the development of artificial cell systems. Although DIBs have demonstrated to be useful in a number of laboratory applications, their wider use is hampered by a limited ability to exist untethered and remain mechanically stable beyond controlled laboratory environments. In this thesis, a microfluidic system is developed which enables the facile generation of hydrogel-encapsulated DIB networks which are freestanding and can exist in air, water and oil environments, without compromise to their ability to interface with the surrounding environment. Electrophysiology is employed in order to demonstrate the formation of bilayers between the encapsulated DIBs (eDIBs) and their external environment, achieved via the incorporation of the transmembrane pore α-Hemolysin. The eDIBs produced here are able to form higher-order structures akin to tissues via their assembly and adherence to one another, further demonstrating their potential to act as a chassis for artificial cells. Furthermore, the potential of eDIBs to be used as a platform for membrane studies is demonstrated via their use as a high-throughput array for membrane disruption fluorescence measurements using a plate reader, which makes use of the ability of eDIBs to be generated in large numbers as well as to be mechanically handled and placed in the wells of a 96-well plate. Fluorescence measurements were taken on up to 47 eDIBs simultaneously, and were able to detect bilayer leakage through pores as well as bilayer failure. The above experiments comprise the design, manufacture and use of a novel kind of DIB construct as a chassis for artificial cells and a platform for high-throughput membrane studies. It is proposed that eDIBs may help in realising the unfulfilled potential of DIB networks in applications in healthcare and beyond

Microfluidic construction and operation of artificial cell chassis encapsulating living cells and pharmaceutical compounds towards their controlled interaction

Droplet-based microfluidic devices can generate complex, soft-matter emulsion systems towards drug screening applications and artificial cell membrane studies. This thesis investigates a methodology for the eventual ‘programmed’ release of pharmaceuticals to treat breast cancer cells that are encapsulated and cultured within small diameter (<2 mm), artificial cell chassis hydrogel capsules. A pharmaceutical analogue was compartmentalised within smaller, membrane-bound, inner cores, that are arranged inside the overall hydrogel capsule. The membrane was based upon droplet interface bilayers (DIBs), which are widely employed for the study of artificial cell membrane transport properties. The whole capsule and contents were produced using enclosed 3D-printed multi-material, microfluidic devices. Methods to control the (programmed) release of compounds from the inner cores to the hydrogel shell, were investigated. The application-specific study was used as an exemplar for a more generally applicable model system. Monolithic microfluidic devices were fabricated using 3D printing and filaments of cyclic olefin copolymer (COC) and nylon for the production of single, double and triple emulsions. With these devices, monodispersed single-emulsion microgels suitable for cell encapsulation were produced, whilst dual-junction devices generated double-emulsion capsules with a controlled number of oil cores. Multi-junction devices also produced triple emulsion, encapsulated droplet interface bilayers (eDIBs), which were subsequently monitored and characterised. Additionally, to demonstrate the ability of eDIBs to act as programmed pharmaceutical delivery systems, assays were performed to induce core release, using membrane modulation by lysolipids (LPC). Computational simulations and DIB electrophysiology experiments were performed to investigate the effect of LPC on the system. MCF-7 model breast cancer cells were encapsulated in alginate-collagen emulsion capsules and their viability was assessed. Moreover, multicellular tumour spheroids (MCTSs) in oil core microgels showed no response to tested doxorubicin concentrations, while proliferated at certain LPC concentrations. Encapsulated cells in eDIBs formed tumour spheroids, however, the DIB survival was low. The integration of living cells and artificial cell membranes within a single entity presents a hybrid model for studying their interaction, towards applications in synthetic biology and drug delivery/screening

Improving Droplet Interface Bilayers as Models for Cell Membranes

This work describes research aimed at improving the droplet interface bilayer (DIB) platform for creating and characterizing biologically relevant model cell membranes. Improvements are made possible in part through the development of a portable, compact platform for controlling temperature with DIBs. Feedback-controlled heating allows studies to be conducted across a range of temperatures, from ambient up to at least 80°C, and also provides new understanding of methods to form DIBs using mixtures of total lipids extracted from bacterial and eukaryotic cells. The membranes formed from total lipid extracts (TLE) are introduced along with evidence that model membranes formed using lipid compositions mimicking natural biological membranes (here using total lipids extracted from Escherichia coli and from porcine brain) behave significantly different than simple single-component membranes. Results provided herein indicate TLE DIBs display high sensitivity to antimicrobial peptide (alamethicin) insertion at room temperature which is explained by evidence of thermotropic phase behavior not encountered with single-lipid DIBs. These results highlight the importance of considering lipid composition when using lipid bilayers as models of cell membranes, and new techniques are described that facilitate the study of composition-dependent phase transitions in model membranes. The proposed research is also aimed at developing new non-ionic methods for characterizing membranes and the interactions of membrane-active molecules that do not necessarily affect membrane conductance and are thus difficult to study. A new tool is developed that utilizes the Young-Lippmann and Young-Dupre relations to enable measurement of membrane specific capacitance and surface tension in a single DIB experiment. The method is introduced, applied, and validated through studies of DPhPC DIBs incorporating various amounts of cholesterol and solvent molecules. The new method is also applied to provide insight toward the interactions of cell-penetrating mixed-monolayer protected nanoparticles in lipid bilayers. Lastly, the method is proven useful for tracking transitions in monolayer and bilayer surface tension that result from thermotropic phase transitions in total lipid extract DIBs

Bottom-up reconstitution using giant unilamellar vesicles as membrane compartments

One of the most basic defining features of a living biological cell is its ability to confine its molecular components within an isolating boundary. In all living cells known to date, phospholipid membranes play this central role by separating cellular machinery from the outside environment. Compartmentalization by membranes is a key principle of life, not only on the scale of the cell as a whole, but, in many cases, within cells as well. This unification and isolation of essential processes within a single, definable unit has made life as we know it possible. As such, compartmentalization represents an essential task that had to be achieved in the emergence of life, as only a confining barrier to the environment allows for distinction of individual units of life and therefore for Darwinian evolution. Another core task of life, is the ability to reproduce. Modern cells are able to split into two daughter cells by deforming their compartmentalizing membrane barrier, all with a division machinery which is situated within this barrier itself – a quite remarkable feat. In this thesis, I present my doctoral work, which aimed to investigate these key features of life through bottom-up reconstitution approaches. Bottom-up cell biology uses isolated well-characterized components, like purified proteins, and tries to recreate cellular processes from these simple building blocks. One of the core challenges of classical cell biology is how to tackle the intrinsic complexity of biological systems with an analytical approach. Complexity is one of the most intrinsic features of life on earth, owing to billions of years of evolution. Biological cells are not designed to be understood and in fact, complexity itself contributes to the adaptability and robustness of biological function. However, by reconstitution of biological structure and function from the bottom-up with minimal components, scientists can observe and try to understand the emergence of biological complexity. Membranes, of course, play a great role in many cellular processes, and contribute significantly to this complexity. However, they can be difficult to incorporate into bottom-up reconstitution approaches, limiting our ability to get a complete view of such processes from in vitro experiments. In this thesis, I present a number of projects, which use giant unilamellar vesicles (GUVs) as mimicries of the compartmentalizing membranes of cells to investigate basic biological functions. I’ve encapsulated a diverse array of biological components in GUVs to achieve biomimetic behavior and function. In each case, I made use of the unique properties of the GUVs to achieve behaviors or make observations that would have not been possible with other approaches, including alternative model membrane systems. Overall, my work presents a step forward toward the reconstitution of complex and dynamic cellular processes under cell-like conditions.Eines der grundlegendsten Definitionsmerkmale einer lebenden biologischen Zelle ist ihre Fähigkeit, ihre molekularen Komponenten innerhalb einer isolierenden Barriere einzuschließen. In allen bis dato bekannten lebenden Zellen spielen Phospholipidmembranen diese zentrale Rolle, indem sie die zelluläre Maschinerie von der äußeren Umgebung abtrennen. Die Kompartmentalisierung durch Membranen ist ein Schlüsselprinzip des Lebens, nicht nur auf der Ebene der kompletten Zelle, sondern oft auch innerhalb von Zellen. Diese Einschließung und Isolierung wesentlicher Prozesse innerhalb einer einzigen, definierbaren Einheit hat Leben, wie wir es kennen, möglich gemacht. Damit stellt die Kompartimentierung eine wesentliche Aufgabe dar, die bei der Entstehung des Lebens realisiert werden musste, denn nur eine abgrenzende Barriere zur Umwelt ermöglicht die Unterscheidung einzelner Lebenseinheiten und damit die darwinistische Evolution. Eine weitere Kernaufgabe des Lebens, ist die Fähigkeit zur Vermehrung. Moderne Zellen sind in der Lage, sich durch Verformung ihrer kompartimentierenden Membranbarriere in zwei Tochterzellen zu teilen, und zwar mit einer Teilungsmaschinerie, die sich innerhalb dieser Barriere befindet - eine bemerkenswerte Leistung. In dieser Dissertation stelle ich meine Forschungsergebnisse vor, deren Ziel es war, diese Schlüsselmerkmale des Lebens durch Bottom-up-Rekonstruktionsansätze zu untersuchen. Die Bottom-up-Zellbiologie verwendet isolierte, gut charakterisierte Komponenten, wie gereinigte Proteine, und versucht, zelluläre Prozesse aus diesen einfachen Bausteinen zu rekonstruieren. Eine der Kernherausforderungen der klassischen Zellbiologie ist, wie man die intrinsische Komplexität biologischer Systeme mit einem analytischen Ansatz angehen kann. Komplexität ist eines der intrinsischen Merkmale des Lebens auf der Erde, das auf Milliarden von Jahren der Evolution zurückzuführen ist. Biologische Zellen sind nicht darauf ausgelegt, verstanden zu werden, und vielmehr trägt Komplexität zur Anpassungsfähigkeit und Robustheit biologischer Funktionsweisen bei. Durch die Rekonstruktion von biologischen Strukturen und Funktionen mit minimalen Komponenten können Wissenschaftler jedoch die Entstehung biologischer Komplexität beobachten und zu verstehen versuchen. Membranen spielen eine große Rolle in vielen zellulären Prozessen und tragen wesentlich zu dieser Komplexität bei. Sie können allerdings schwer in Bottom-up- Rekonstitutionsansätze zu integrieren sein, was die Möglichkeiten einschränken kann, einen vollständigen Blick auf solche Prozesse durch in vitro-Experimenten zu erhalten. In dieser Arbeit stelle ich eine Reihe von Projekten vor, die riesige unilamellare Vesikel (GUVs) als Nachahmung der kompartimentierenden Membranen von Zellen verwenden, um dabei grundlegende biologische Funktionen zu untersuchen. Dafür habe ich eine Vielzahl von biologischen Komponenten in GUVs eingekapselt, um biomimetisches Verhalten und Funktion zu erzielen. Dabei habe ich jeweils die einzigartigen Eigenschaften der GUVs genutzt, um Verhaltensweisen zu erreichen oder Beobachtungen zu machen, die mit anderen Ansätzen, einschließlich alternativer Modellmembransystemen, nicht möglich gewesen wären. Insgesamt stellt meine Arbeit einen Schritt vorwärts in Richtung der Rekonstruktion komplexer und dynamischer zellulärer Prozesse unter zellähnlichen Bedingungen dar

Low-cost solid state nanopore biosensing technology towards early disease detection

Solid-state nanopore based biosensors are cost effective, high-throughput engines for single molecule detection of biomolecules, which is useful for detecting epigenetic modifications on DNA; one of these being the potentially cancerous hypo, or hypermethylation of CpG islands. Despite its immense potential in the realm of disease diagnostics, nanopore detection as it stands faces various limitations that inhibit it from widespread commercial use. These include the complex method of solid-state nanopore fabrication, fast DNA translocations through the pore causing poor resolution, and poor signal to noise ratio. The following work aims to improve the efficacy of the solid-state nanopore biosensing platform as a disease diagnostic tool by improving ease of fabrication with automated MATLAB instrument control and controlled dielectric breakdown fabrication technique and increase signal resolution by using lithium chloride salt concentration gradients. In addition, methylated DNA labeled with certain methyl-binding proteins were tested in an attempt to localize areas of methylation on the DNA strand. These experiments yielded transport events that showed multilevel electrical signals that, in some instances, were able to distinguish between regions of bound protein and unbound DNA on the same strand. Increasing the accuracy of these multilevel event readings will aid in pinpointing localized regions of methylation on DNA and thereby increase the efficacy the solid-state nanopore platform for biosensing

The Challenges of Measuring Membrane Protein Function in Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are a desired membrane‐mimetic system for the study of many membrane‐related phenomena, the function of MPs and the creation of synthetic cells. These micrometer sized vesicles are similar in size to bacteria and eukaryotic cells, and thus mimic these organisms more closely in terms of surface and volume. The size allows the integration of complex systems and entire metabolic processes such as transcription and translation, as well as the investigation of individual vesicles using light microscopy techniques, potentially cutting the costs of purified MPs needed to perform experiments by a factor of 100 compared to bulk methods. This makes them a very attractive system to investigate MPs, for example to test and develop novel drugs, and to create a bottom‐up synthetic cell. However, lipids do not spontaneously assemble into cell‐sized vesicles which has prompted the development of several different techniques for GUV formation. For the same reason, these vesicles are more fragile towards the use of detergents, which complicates MP reconstitution. To harness the power of light microscopy measurements, GUVs have to be immobilized to enable real‐time observation over several minutes to hours. Lastly, the success of measuring MP function in a GUV also depends on the choice of detection system. In previous work in our lab, GUV electroformation on indium‐tin‐oxide (ITO) coated glass slides and reconstitution of MPs using charge‐mediated fusion of oppositely charged vesicles was established. GUVs were immobilized using a streptavidin‐biotin system to enable measurement of MP function. One of the disadvantages of GUV electroformation on ITO coated glass slides is the poor compatibility with high ionic solution, which could result in low protein activities due to formation at non‐physiological conditions. One of the aims of this project was to establish GUV formation under physiologically relevant conditions to allow formation in buffer compositions optimal for MP function. We thus compared previously established electroformation on ITO coated glass slides with electroformation on platinum (Pt) wires and the more recently developed polymer assisted swelling using PVA. We observed that both Pt wire and PVA formation produced GUVs using various buffer compositions and that polymer assisted swelling produced a high yield of GUVs without much optimization, showing the potential and versatility of this method. Interestingly, we discovered that the immobilization was affected by the buffer composition, and that strong adhesion can lead to leakage and loss of encapsulated cargo, especially in PVA GUVs. This is an important finding as MPs are frequently followed using encapsulated fluorescent dyes, showing that immobilization conditions have to be tuned according to the buffer composition to provide sufficient immobilization while preventing too much cargo loss. Protons play an important role in many cellular processes, they are involved in many transmembrane transport reactions as well as in the production of ATP by the ATP synthase. Thus, GUVs should be able to maintain a proton gradient. Our measurements suggest that this is indeed the case also in immobilized vesicles that have not leaked encapsulated fluorophores. We further presented a simple strategy that could be used to estimate the protein concentration in GUVs after charge‐mediated reconstitution by fusion of GUVs with small vesicles containing labeled lipids and labeled MP. We could show that GUVs with more lipid‐coupled dye signal also showed more MP‐coupled dye signal, which could simplify the quantification of MPs in GUVs after fusion by following lipid‐coupled dye incorporation without the need for MP labeling. However, this strategy is not able to distinguish between simple adhesion or hemifusion of small vesicles and full fusion, which would be required for functional reconstitution of a MP. However, the same is true for simply following labeled MP signal, meaning that potentially other methods such as content mixing assays would be needed to get a better idea on the amount of functionally reconstituted MP. Finally, knowledge gained from the characterization of GUVs was applied for the reconstitution and measurement of cytochrome c oxidase from Rhodobacter sphaeroides using carboxyfluorescein and pyranine (HPTS). The former produced only weak and unclear signals and was prone to fast bleaching. Using ratiometric dyes such as HPTS, pH calibration can be performed, where the observed ratios should be independent of dye concentration and bleaching. This proved to be challenging in GUVs, as the vesicles size seemed to have an effect on the observed HPTS ratio. Despite that, cytochrome c oxidase measurements using HPTS yielded better results and by characterization of vesicle shape and measurement of lipid‐coupled dye signal introduced via fusion, a correlation between the proton translocation and the relative amount of MP per vesicle could be observed, showing that thorough characterization of the GUVs can help to relate vesicle activities. Nonetheless, the different sizes of the vesicles were a considerable challenge for data analysis. We thus plan to use monodisperse GUVs produced by microfluidic techniques in collaboration with members of the deMello group form the ETH Zürich and we could show that these GUVs are fusogenic and can be potentially used to measure proton translocation. In a second project, we used bifunctional DNA duplexes to establish a new tool for the measurement of MP function. By linking pH‐sensitive dyes via DNA oligomers to cholesterol moieties, fluorescent probes can be anchored to the lipid membrane, allowing more efficient encapsulation compared to soluble dyes which could safe costs using precious probes. Addition of DNase I allows fast and simple removal of probes facing the liposome exterior, which are exposed to the buffered solution and thus do not contribute to the measurement of proton translocation in or out of the vesicle, a common problem with lipid‐coupled probes. Incorporation of bifunctional DNA into GUVs was slightly more challenging. Depending on the GUV formation method, as well as lipid and buffer composition, differences in the degree of incorporation into the membrane were observed, ranging from no membrane localization to complete incorporation. Formation further seemed to be negatively affected by the probe. Thus, further optimization of the probe might be needed to enable measurement of MP function in GUVs