Background Cell adhesion molecules (CAMs) are membrane-bound proteins that mediate cell-cell interactions through trans-cellular protein complexes. In the context of the neuronal synapse, studies of CAMs have revealed their roles from neuronal recognition and neuronal wiring to synaptic plasticity. CAMs form macromolecular complexes via cis- and trans-interactions; however, identifying the specific proteins in these assemblies is challenging. Their interactions are dynamic and transient, making them difficult to capture, and their hydrophobic transmembrane domains complicate extraction from biological samples. New method Here, we present a protocol to pulldown interacting partners of a Teneurin-3-GFP bait protein, as a representative CAM, from minimal mouse brain lysate. Comparison with existing methods Affinity purification of a bait protein from a biological sample, followed by mass spectrometry to identify captured prey proteins is a widely used, unbiased approach, though it usually requires large amounts of material. We show that our refined approach detects known Teneurin interactants while substantially reducing the animal tissue required. We further compared detergents used for lysate preparation and found that the total of CAM species enriched in Teneurin-3 samples relative to control varied considerably. Finally, we evaluated different normalization workflows to aid dataset interpretation. Conclusion This protocol provides an accessible approach for studying CAM interactions with limited animal tissue, enabling refined insights into the complex protein networks underlying synaptic connectivity.

There are 100 billion neurons in the brain, and each of them forms thousands of connections with other neurons. Despite this immense complexity, neural connections are established in a highly specific manner to build functional circuits. The molecular mechanisms underlying synapse formation are therefore intricate and finely tuned. Key players in this process are cell adhesion molecules (CAMs), which assemble into trans-synaptic complexes across the synaptic cleft to link two neurons. Understanding the molecular features that determine their compatibility is crucial for elucidating neuronal recognition before synapse formation.

In the first chapter, I provide a more detailed introduction to the importance of specificity in neural connectivity and discuss the role of CAMs in this process. I then explore two protein-modifying mechanisms that expand the diversity beyond the genetic code: glycosylation and alternative splicing. Examples of how these mechanisms regulate CAM function at the synapse are presented. Finally, I introduce Teneurins, a family of synaptic CAMs, and discuss their roles in organizing brain circuits across different animal models.

In the second chapter, I describe a pulldown protocol specifically optimized for CAMs to establish their interactome from mouse brain tissue. This protocol significantly reduces the amount of animal material required. Representative data are presented using Teneurin-3 as bait, providing an overview of its interactome. I also examine the effects of testing different detergents during brain lysate preparation and of applying different normalization strategies to the resulting mass spectrometry data - two methodological aspects rarely discussed in similar studies. An extensive, user-friendly protocol for brain lysate preparation, pulldown, and data analysis is provided in the supplementary methods.

In the third chapter, I investigate how alternative splicing of Teneurin-3 influences its interactions with other CAMs. I first present results from mouse brain pulldowns (as detailed in Chapter 2) using the four splice variants of Teneurin-3 as bait. Next, I apply the AlphaFold Multimer pipeline to predict the likelihood of interactions between these variants and potential binding partners. Finally, I experimentally test the predicted interactions using a cell-clustering assay, which specifically measures the ability of two proteins to form trans-synaptic complexes. These results reveal an alternative-splicing dependent mechanism regulating Teneurin-3 interactions.

In the fourth chapter, I systematically characterize the effects of Teneurin-3 N-glycosylation on its expression and stability. To this end, I generated a glycomutant library of Teneurin-3 extracellular domain constructs, each carrying a mutation at a distinct N-glycosylation site that prevents glycan attachment. These data are compared to mass spectrometry-based characterization of the Teneurin-3 glycosylation profile, which identifies the types of glycan chains present at individual sites. Beyond providing structural insights, the establishment of this library offers a strong foundation for future research.

In the fifth chapter, I address the unusual N-glycosylation profiles typically observed for brain proteins as an emerging issue in understanding the molecular basis of synapse formation. Drawing on recently published data, I propose that this atypical glycosylation profile may shift following synaptic activation, acting as a molecular mechanism that helps stabilize functional synapses, including through effects on CAMs.