Imaging of the dynamics of Eph receptors and their ephrin ligands in mature hippocampal neurons

The Eph receptors comprise the largest subfamily of receptor tyrosine kinases (RTKs) with important roles during neuronal development. Unlike other RTKs, these receptors can be activated by their membrane bound ligands, the ephrins. Recent evidence strongly suggests that Eph receptors and their ligands also contribute to synapse formation and synaptic plasticity in the postnatal brain. However, the details of the mechanisms still remain unclear. In order to better understand the role of EphB2 receptors during these processes in living cells, EphB2 receptors were visualized. Therefore, one variant of the enhanced blue, green, or yellow fluorescent protein (E(C/G/Y)FP) was fused to the receptor. For this, ExFP was inserted in one of three different positions of EphB2: the N-terminus (EphB2-N), a site close to the juxtamembrane region (EphB2-C1) and between two functional relevant domains of the cytoplasmic tail (EphB2-C2). The different EphB2-ExFP receptors, EphB2-N, EphB2-C1 and EphB2-C2, were exogenously expressed in cell lines and showed intense fluorescence at the plasma membrane, comparable to other described transmembrane ExFP fusion proteins. Biochemical methods were used to test functionality of the different EphB2-ExFP proteins concerning tyrosine phosphorylation and interaction with known proteins, such as NR1 and GRIP2, and showed no obvious impairment. Surprisingly, however, the cluster behaviour of the EphB2-ExFP variants transiently expressed in neurons was different, only the EphB2- C1 proteins revealed a proper cluster formation after ephrinB stimulation, indicating that the ExFP insertion at the N- and C-terminus impaired the clustering. In order to study the dynamics of trafficking, insertion and cluster behaviour of fluorescently tagged EphB2 receptors in living neurons, hippocampal cultures were transfected with these constructs. EphB2-C1 expressing neurons revealed two pools of fluorescent clusters, one pool was static, representing EphB2 receptors at the plasma membrane, whereas the other pool was trafficking along neurites, presumably reflecting EphB2 proteins in transport vesicles. In addition, the subcellular distribution of these receptors was analyzed, revealing that e.g. EphB2 clusters are present at the tips of filopodia and in growth cones. Filopodia are highly dynamic structures, which explore the environment and therefore have to extend and retract a lot. Our group recently described a new mechanism of how an adhesive Eph-ephrin interaction between filopodia of immature growth cones and EphB2-expressing cell lines can be turned into a retraction response by bi-directional EphB/ephrinB-triggered trans-endocytosis. Intrigued by these findings, we were interested whether bi-directional transcytosis also exists in mature hippocampal cultures, presumably being involved in the dynamics of filopodia. The present thesis could show that after contact of mature neurons with cells, most-likely glial cells, the exogenous expression of fluorescently tagged EphB2 receptors in neurons induces a retrograde transcytosis into the interacting neighbouring cell. This reverse transcytosis of EphB2-C1 proteins was often followed by persistent retraction of the neuronal protrusion. This could have a potential role in axon pruning or in morphological plasticity in mature neurons, thereby adjusting the proper connectivity. In a second approach, thy1-EphB2-C1-EYFP transgenic mice were generated to gain insights into the in vivo behaviour of these fluorescently tagged proteins and to have a closer look in the dynamics and cluster behaviour of EphB2 receptors during synaptogenesis and in spines of more mature neurons. Contrary to our expectations, both the fluorescent intensities and the distribution of the EphB2-C1-EYFP proteins in these transgenic mice were not bright nor sparse enough for conclusive imaging experiments.