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.