Membrane proteins are of vital importance for human life. They reside in the plasma membrane, where they provide the adhesive forces needed for cells to spatially organise themselves relative to one another. Additionally, given their unique localisation - being in contact with both sides of the membrane - membrane proteins enable various modes of sensing and transmembrane signalling. For instance, channel proteins regulate the selective membrane permeation to ions and nutrients. Various other membrane proteins transduce information across the membrane by structural changes in response to direct binding interactions on either the intra- or extracellular side.

In the first chapter, we briefly introduce the role of a particular type of membrane proteins, namely synaptic cell adhesion molecules (synCAM) and their importance in organising neuronal networks. We discuss the role of synCAMS in recognizing extracellular cues which constitutes the mechanistic basis for network formation, and focus on the structural basis of synCAM binding specificity.

In the second chapter, teneurin-3 (Ten3) is investigated as a model synCAM for splicing-dependent binding specificity. We resolve the structures of various isoforms to reveal how small splice inserts completely reorganise intramolecular domain configuration. These structural changes are shown to underlie trans-cellular clustering and the guidance of axon outgrowth.

In the third chapter, we investigate the molecular basis of a neurological disease called microphthalmia ("small eye disease"). A unique patient-derived Ten3 missense mutation resides at a intramolecular domain-domain interface specific to two of the splicing isoforms. We showhowthe point mutation disrupts trans-cellular binding which may explain the eye and brain malformations, and thereby the cognitive impairments typical inmicrophthalmia patients.

In the fourth chapter, we look into the structural basis of glycosylation-dependent binding specificity between synCAMs contactin 1 and neurofascin 155. Only specific glycan chains at the interface allow formation of the complex, and subsequently, enable the trans-cellular contactin 1 - neurofascin 155 interactions.

In the fifth chapter, we set up a workflow for the reconstitution of purified neuronal membrane proteins in the lipid bilayers of vesicles over which transmembrane voltages can be induced. This will allow structural studies of neuronal membrane proteins in an environment more representative of the neuronal membrane, which is subjected to the dynamical voltages of the action potential.

In the sixth chapter, we provide an overview of techniques that have enabled the reconstruction of small, low signal-to-noise proteins using electron cryo-microscopy (cryo-EM). These approaches typically involve appending additional protein domains to the target, thereby facilitating particle picking and alignment needed for high-resolution cryo-EM single-particle analysis. This methodological review is aimed to select an approach for reconstructing the membrane-embedded proteins of chapter 5.

The cell is the fundamental unit of life, composed of smaller, non-living components. This raises a key question: what truly gives rise to life? This thesis explores cytoskeletal organization and dynamics, focusing on microtubules and spindle positioning, with the broader aim of reconstituting these processes in synthetic cells.

Encapsulation of biological components is central to synthetic cell research. We evaluated different methods to create cell-like compartments, finding that while droplets are easy to work with, cDICE offers greater flexibility for functional encapsulation. Using high-speed imaging, we studied GUV formation in cDICE and discovered a size-selective crossing of droplets at the interface. We also found that proteins in the inner solution affect GUV formation by increasing viscosity and altering lipid adsorption.

Tubulin, an essential protein in cells, remains difficult to work with in vitro. Using different encapsulation methods, we observed that tubulin influences the stability of lipid bilayers, and using a membrane interaction assay, we found that it can even disrupt the membranes.

To increase biological relevance, we combined major cellular components like microtubule asters with an actin cortex or a nucleus mimic, and explored external tools for spatiotemporal control. We successfully assembled a mitotic spindle-like organization in droplets. We incorporated an optogenetic switch to control dynein in order to achieve asymmetric spindle positioning, inspired by the first cell division of the C. elegans embryo. However, light-induced transport remains limited.

Finally, we adapted the bacterial ParMRC DNA segregation system for synthetic cell, with light-regulated control via iLID. Although individual components react to light activation, further optimization is needed to make the full system responsive.

These reconstitutions provide insight into fundamental mechanisms of spindle positioning and are basic steps toward building a minimal synthetic spindle.

Biological systems are dynamic and multi-layered, characterized by internal structures with varying levels of complexity that are in constant interplay. For instance, the regulated interaction between gene expression machinery and the biochemical reactions among proteins is crucial for orchestrating cellular functions. This interplay is essential for maintaining life, even in seemingly "simpler" single-cell organisms, amidst an ever-changing external environment. These interactions create a complex web of connections between different biological organization levels, making studying such systems enormously challenging. However, the story does not end there; biological systems have the remarkable ability to evolve. Evolution is fundamental to all living beings on Earth, enabling the vast diversity of forms, shapes, lifestyles, and colors observed in nature. Anticipating evolutionary outcomes has long perplexed scientists. In some cases, evolution appears to follow reproducible trajectories, providing opportunities to investigate factors that may constrain evolutionary paths while controlling for external environmental in_uences. One such factor, discovered in the past century, is epistasis, which refers to the variable effect of a gene mutation depending on the presence or absence of mutations in other genes. This concept has played a pivotal role in shaping our understanding of evolutionary processes. In this thesis, we examine the effects of epistatic mutations on a genome-wide scale within a speci_c evolutionary trajectory...

Cells are the fundamental unit of life. All living matter is made of cells: from the small systems imperceptible to our eye, like bacteria or archaea; to bigger systems, like plants or magnificent trees, fungi, and animals - including ourselves: the humans. All these systems vary in size yet they are all alive. And the common element of these systems is that they are all composed of cells. Cells are therefore fundamental, but also very complex systems. One may say, broadly, that cells are a cocktail of subsystems that, in combination and in the right balance, can become this basic unit of life. Understanding cells, and their diverse mechanisms, would therefore imply that, eventually, curious scientists (like the author herself ) may eventually be able to (better) understand life.
From a physics perspective, cells are fascinating because they constantly endure mechanical stresses and strains that challenge their survival, yet they also actively deform themselves. Our own cells exhibit remarkable deformability in response to external forces such as blood flow or muscle contraction. They also actively alter their own shape. During wound healing, cells in the skin for instance move as coherent cell sheets to heal wounds and renew tissue. And upon cellular division and differentiation, cells experience considerable shape transformations and therefore endure big deformations. Cell deformability is also an important factor in many diseases. During cancer metastasis, tumor cells for instance squeeze themselves through tissues and vessel and lymph node walls. All in all: cells are often pushed to deform, yet they somehow manage to endure those changes. So we may ask ourselves: How do they do this? To address this question, we can take a closer look at the units that forma cell....

Genetic information encoded in the DNA sequences is maintained, replicated, and transcribed across the tree of life. Organisms evolved multiple hierarchical layers of chromosome organization which ensure that DNA can be contained but also processed within individual cells. This thesis focuses on SMC (Structural Maintenance of Chromosomes) protein complexes, an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. In this thesis, we utilized single-molecule fluorescence and magnetic tweezers to study the intricacies of DNA loop extrusion by SMC complexes in a controlled in vitro environment. We observed, described, and analyzed the behaviour of SMCs which yielded detailed yet important contributions to the resolution of the DNA loop extrusion mechanism as well as to the physiological impact on genome structure. In particular, we observed that large DNA-tethered roadblocks cannot stall loop extrusion efficiently and concluded that DNA loop extrusion can occur in a non-topological manner. In contrast, we found that a small DNA-binding protein, CCCTC-binding factor (CTCF) stalls cohesin through direct protein-protein interactions with cohesin’s N-terminal region. Furthermore, we could reconcile previous results that suggested different loop extrusion mechanisms for different members of the SMC protein family. We demonstrated that all eukaryotic SMCs extrude DNA asymmetrically, i.e. only from one side at a time, and that frequent direction switches yield the previously observed ‘symmetric’ loop extrusion. Furthermore, we showed that DNA supercoiling is intimately linked to loop extrusion for all eukaryotic SMC complexes, postulating a common DNA loop extrusion mechanism.

Cryogenic electron microscopy (cryo-EM) has become a powerful technique to understand the structure and function of proteins. Thanks to substantial technical advancements in the 2010s, the frequent determination of atomic structures is now possible. This thesis encompasses chapters that contribute to both the remaining technical challenges of cryo-EM, and to the advancement of biological insight through the application of cryo-EM to a motility machinery called a gas vesicle. As an overarching theme, the thesis is titled ’Imaging Life at the Molecular Scale using Electrons’. Chapter 1 provides an introduction into methods to study structure and function in biology at the molecular scale, and into the development of cryo-EM since its inception. A comprehensive review of previous structural work on gas vesicles follows.
Technological improvements in electron detectors have significantly enhanced the quality of protein images obtainable. However, predicting the success of cryo-EM structure determination still poses a challenge, as it is heavily dependent on the unique properties of the sample and its behavior during the sample preparation process. Proteins are typically forced into an thin liquid layer, which is frozen by quick immersion into a cryogen. The large air-water interface can cause proteins to aggregate, denature or adopt preferred orientation, hindering structure determination. The thickness of the layer is also hard to control - too thick layers lead to poor image contrast, too thin layers damage the protein.....

This doctoral thesis stands on three pillars that emerged from following my scientific interests: i) biophysics, ii) synthetic biology, and iii) biosecurity. The former, biophysics, reflects my desire for deep understanding of biological systems and my affinity for experimental work. The latter, synthetic biology and biosecurity, were born from the conviction that the understanding obtained in pursuing science offers the most fulfillment when applied to the benefits of society.

The main part of this thesis explores methodologies for the extraction, and characterization of large-scale DNA, with a particular focus on the megabase-pair length DNA from bacterial sources. The research aims to bridge the gap between in vivo chromosome studies and in vitro single-molecule techniques by developing approaches that enable the investigation of chromosome structure and dynamics at a more relevant genomic scale. The following chapters detail the experimental approaches, results, and conclusions drawn from this work.

The human body is composed of about 3−4×1013 (30-40 trillion) cells [1]. These cells are all functioning consistently, and working elegantly together, to sustain the organism. Not only humans, but all other living things on earth (from plants to parrots) are composed of cells. Cells are the smallest living building blocks of plants and animals, and in fact some organisms are built from a single cell, such as bacteria. To be able to sustain life, cells are dynamic entities that need to grow, divide, and interact with their environment. They accomplish this by a number of complex and dynamic processes. For instance, to perform vital functions such as cell division, a key factor of life, cells need to dramatically change their shape. In addition to shape changes, the internal cellular organization needs to be tightly controlled to properly function. For example, cells need to establish a front-back polarity to drive directional migration, like immune cells that hunt for intruders. Furthermore, the proper functioning of brain cells (also named neurons), which depends on finding and connecting to other neuronal cells, is closely related to their internal organization and cellular shape. For cells, essentially small bags filled with proteins, to change their shape and internal organization, they depend on an internal filamentous scaffold named the cytoskeleton. Unlike the name might suggest, this ’cellular skeleton’ is actually very dynamic, with constant assembly and disassembly of the constituent filaments and changes in filament organization. In addition to organizing the cellular interior, this cytoskeleton provides mechanical support for cells and allows them to generate forces. Two main cytoskeletal components are microtubules and actin filaments. They are usually studied as separate systems, despite a growing body of work indicating their functions are closely intertwined and interdependent. This thesis studies how these two cytoskeletal components influence each other. More specifically, we focus on the question how actin and microtubules co-organize and affect each other via proteins that physically link them to each other, named cytolinkers. To study how cytolinkers impact cytoskeletal crosstalk, we move away from the complex environment of the cell, where many other proteins are present and different processes take place. We took the cytoskeletal building blocks and cytolinking proteins out of the cell, rebuilt a cytoskeleton from these building blocks and characterized the effects of the cytolinkers on cytoskeletal co-organization by fluorescence microscopy. In addition to natural cytolinkers, we engineered our own cytolinkers to better understand how these proteins influence microtubule/actin coordination and in the absence of illdefined regulatory processes in the cell. This isolated context is a powerful tool to study cellular functions, as the simplification allows us to tightly control all variables and identify the underlying mechanisms.

This thesis is about a little molecule called guanosine tetraphosphate. ppGpp. Consider it the bacterial brain, at the core of the coordination and regulation of bacterial growth. For over half a century, it has haunted microbiologists as it appears involved in every aspect of microbial physiology, yet incredibly difficult to study due to its fast dynamics, chemical instability and pleiotropic effects. Like the human brain, it cannot simply be removed to show its true nature. In contrast to the pronunciation of its name, ppGpp is a rather simple molecule, and built from two of the most abundant substrates in the bacterial cell (ATP and GTP). The enzymes that make or break ppGpp are highly efficient, such that at any moment, the bacteria can decide to instantly 100-fold increase ppGpp concentrations, or virtually remove all of it. Thanks to this intelligent system, E. coli can decide to arrest growth, protecting itself against any threats, or to rapidly feast upon the sparse nutrients it may be tossed, within the order of minutes.

Biological membranes are selective soft barriers that compartmentalize internal structure of a cell into organelles and separate them as a whole from the external environment. Due to their innate feature of being able to undergo constant reshaping, cellular membranes spatially attain diverse shapes ranging from simple spherical vesicles to more peculiar structures like the interconnected network of tubes found in the endoplasmic reticulum. Membranes are not only composed of lipids, but also host an enormous number of inclusions like proteins. Recent studies of biological membranes have revealed that such inclusions play a key role in diverse biological processes through either sensing or inducing perturbations to the membrane shape. In this dissertation, we studied the interplay between the shape of membrane and the spatial organization of attached curvature inducing objects using mathematical tools and numerical simulations in highly curved spherical and cylindrical geometries.
First, we investigated the interaction between inclusions of different shapes embedded in/adhered to tubular membranes. Our combined theoretical analysis and numerical simulation results evinced that tubular membranes, in contrast to their planar counterpart, transmit an attractive force between inclusions, stemming from their closed and curved geometry. We then elucidated that collective interaction between proteins results in the formation of line-like and ring-like clusters, depending on the their intrinsic shape (Chapters 2–4). We further showed how curvature sensing crescent-like proteins in high densities can constrict tubular membranes and facilitate their splitting, demonstrating that both the curvature-sensing and curvature-inducing property of proteins are two sides of the same coin. Moreover, we used our simulation results to explain how mitochondorial machinery triggers, facilitates and drives membrane fission in its tubular network to avoid entanglements (Chapter 3).
Next, we examined the interaction of spherical proteins adhered to closed vesicles. Our simulation results – supported by recent experimental evidence – revealed membrane curvature as a common physical origin for interactions between any membrane deforming objects, from nanometre-sized proteins to micrometre-sized particles (Chapter 5). Our further simulations unraveled how introducing curvature variation on the surface of a closed vesicle can be exploited by inanimate particles to regulate their pattern formation (Chapter 6).
Finally, through theoretical calculations,we analyzed the interplay between the shape of a cell and the rearrangement of attached microtubules (Chapter 7). Our results particularly suggested that the commonly reported parallel structure and bundling of microtubules can be induced by membrane mediated interactions.

Cells of the most common organisms like plants and animals are filled with polymeric networks that fulfil important functions of the cell. There is however no analytically solvable model that describes diffusion in such a cell. This thesis presents a model for diffusion in polymeric environments, and some predictions about the behaviour of the model are made and confirmed by simulations.
Furthermore, the Fokker-Planck equation of this problem is studied, in order to solve the problem. Certain approximations are presented and solved, and it is investigated when the approximations are sound. Moreover, a method is described that can derive a solution to an equation with certain boundary conditions, from a solution to the same equation with different boundary conditions. This thesis also shows how this novel method can be applied to this model to find a non-approximated solution to the equation, where this was not
possible without this method. Finally, it is described how a multitude of partial differential equations that are linked to each other via the boundary conditions can be solved, can be solved using the method described.

In this thesis we took the challenge to in vitro reconstitute a minimal phenomenon essential for live: Cell polarity. This is an ubiquitous phenomenon that allows cells to define a direction for migration, growth or division. Our study focussed on microtubulebased establishment of polarity taking S. pombe as a model organism. In this rod-shaped unicellular organism, microtubules deposit polarity factor proteins to the poles of the cell, leaving only there cues for initiating the cascade for cellular growth. Such pattern formation is very robust and is believed to rely on feedback mechanisms. In our study, we hypothesized what would be the minimum components needed for the establishment of a polarized cortical pattern of proteins and developed an in vitro system that fulfils those requirements. Those where:
• Microtubule-based transport of polarity factors.
• Elongated cell shape.
• Cortical receptor for the polarity factors.