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.
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The central nervous system has a very limited capacity to regenerate damaged tissue. Therefore, regeneration strategies focus on transplantation of neural stem cells or differentiated neural cells. In order to make such a treatment effective, it is important to understand the mechanisms that enable cell differentiation. It is well known that besides biochemical cues, also mechanical and geometric properties of the cell environment, such as topography, curvature and stiffness, can influence the process, which has been studied mostly in 2D. In order to conduct relevant cell studies in vitro, it is therefore important to mimic the 3D structure of the in-vivo cell environment. Many different approaches have been adopted to create scaffolds for neuronal cells, such as freeze-drying, electrospinning and stereolitography. The main drawbacks of these methods are the limited resolution and the constraints in terms of achievable geometries. Two-photon polymerization overcomes these problems by using a laser to polymerize a photosensitive material in extremely confined volumes, achieving a submicrometric resolution. In this study, we fabricated 3D microscaffolds made of an acrylate polymer called IP-Dip by employing two-photon polymerization in order to study the effect of curved versus straight lattice geometries on the differentiation of mouse embryonic stem cells into neural progenitor cells. First, feasibility studies were carried out with HeLa cancer cells and the effect of curvature on these cells was investigated on 2.5D structures. We established a workflow for conducting these experiments from the fabrication up until the analysis. By employing confocal imaging, image stacks were obtained and then analysed to obtain the volumetric cell occupancy of the scaffolds and identify the location of the cells within the scaffolds. We concluded that mESCs could successfully grow and differentiate within the 3D scaffolds without a specific preference for a curved over a straight lattice structure.