Formation and processing of Eph/ephrin complexes at cell-contact interfaces 41

interfaces 

To establish a suitable strategy for studying Eph/ephrin clustering in detail, it is highly beneficial to gain a general insight in surrounding processes leading to, and governing the formation and processing of Eph/ephrin complexes. The most feasible and physiological way to sample cluster formation and surrounding processes is by performing a co-culture assay with two populations of cells, one expressing Eph receptors, the other expressing ephrin ligands [238,245]. HeLa cells were chosen because they show a high mobility on laminin substrate, morphological flexibility and responsiveness, and thereby nicely mimic the in vivo modality of Eph/ephrin signaling as observed in axon growth cone collapse or boundary formation processes.

Figure 3.1 depicts the formation and processing of Eph/ephrin complexes after cell contact. At the cell-contact interface, signals from fluorescence (FL) protein tagged Ephs/ephrins were formed as seen by the presence of entities, i.e. aggregates with higher FL intensity. As a control, co-expressed myr-mCherry FL remained diffuse at these sites (Fig. 3.1B-I) indicating that Ephs/ephrins were specifically clustered upon contact. These FL entities at cell edges were then rapidly processed into Eph+ and ephrin+ cells (Fig. 3.1B-IV/V,C,D). Entities emerging after cell-cell contact at the cell-edge were subsequently broken apart (Fig. 3.1C, D) over time with little fluorescent particles pinching off from the initial large complex. Entities, which were located in cut-off filopodia and/or not in direct contact to the cell’s membrane surface did not undergo processing (Fig. 3.1 B-V-VII). In Eph+ _{cells, the stimulation by}

ephrin ligand led to a signaling response resulting in retraction of the cell periphery (Fig. 3.1B, dotted black line, compare I with VI), similar to growth cone collapse in axons. In ephrin+ cells, this cellular collapse response could not be observed. However, in contrast to the Eph+ cell, due to the lack of cell retraction, the processing of FL particles away from the cell-edge became more evident (Fig. 1D). Little fluorescent particles were constitutively pinching off from a bigger surface-trapped FL entity and were moving towards the inner of the cell, presumably being internalized into endocytic vesicles for degradation. In Eph+ _cells,

this process could not be so readily observed because of the simultaneous retraction of the cell periphery.

Taken together, I conclude that live-cell imaging based co-culture assays are a justifiable method to display physiological Eph/ephrin complex formation and functionality on the cellular level. However, this general insight into the processes surrounding Eph/ephrin cluster formation emphasizes two major aspects that severely complicate the investigation of the role

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of cluster formation in Eph/ephrin signaling. This not only holds true for co-culture assays but also for other Eph/ephrin single cell assays in general:

1) Tangibility of the Eph/ephrin cluster. FL entities or aggregates are usually described as Eph/ephrin clusters based on their association to the experimentally proven structural feature of oligomerization. Yet the depiction of these FL entities as clusters is rather inaccurate and misleading since the limit of spatial resolution in epifluorescent imaging does not permit this degree of analysis. Upon formation, single FL entities could simultaneously be large, single clusters and assemblies of multiple Eph/ephrin complexes. Moreover, Eph/ephrin complexes are subject to rapid internalization processes, which in its initial steps, are accompanied by invagination processes of the membrane. These processes may very well lead to a crowding of multiple fluorescent complexes into one observed single FL entity. Presumably, FL entities observed at later stages after initiation of complex formation represent internalized Eph/ephrin in vesicles.

Since we do not know the exact composition and origin of these FL entities, it is not possible to extrapolate observed FL Eph/ephrin entities from a mechanistic to functional way. Therefore, an alternative experimental strategy implementing a direct clustering readout to specifically address Eph/ephrin complex formation is required.

Fig. 3.1 Formation and processing of Eph/Ephrin complexes at cell-contact interfaces.

(A) Co-culture situation of HeLa cell populations one transiently expressing YFP-wtephrinB2, the other wtEphB2-CFP in low resolution. The cartoon delineates the physiological event of receptor/ligand co-

clustering at cell-contact interfaces. (B) Insets in higher resolution to outline the co-culture situation

from (A) in time course. Co-culture of HeLa cell populations, one transiently expressing YFP- wtephrinB2, the other wtEphB2-CFP. Cell-contact events were imaged in time-lapse 35 min after seeding of ephrin expressing cells. Sequential time-lapse images show the processing of fluorescent (FL) entities over time. Dotted line serves as orientation for site of initial contact at start of imaging.

During the time course of contact the Eph+ cell gradually moved away from the ephrin+ cell. (I) Arrows

in blue indicate co-clustering of ligand and receptor but not myr-mCherry. (II) Arrows in green indicate

an Eph/ephrin surface cluster on the Eph+ _{cell. (III) Arrows in red indicate an Eph/ephrin surface}

cluster on the ephrin+ _{cell. (IV) Arrows in green indicate an Eph/ephrin surface cluster beginning to be}

processed into the Eph+ cell. (V) Arrows in red indicate an Eph/ephrin surface cluster beginning to be

processed into the ephrin+ cell. (V-VII) Red circles accentuate clusters at filopodia which are not

subject to processing, presumably because they are not in contact to the active cell surface. (C) Image

sequence starting from (B-II) outlines the processing of high-fluorescence entities into the Eph+ _cell.

Graph highlights the size degradation of one FL entity (red) into multiple particles (entities X) (D)

Image sequence starting from (B-III) outlines the processing of FL entities into Eph+ cells. Image

panels and graph (I) depict the size degradation of one FL entity (entity 1) into two particles (entities 2 + 3). Image panels and graph (II): pinched off FL particles were moving towards the center of the cell (displayed as distance [µm] to cell edge) whereas entity 1 remained stable at cell edge. Scale bar, 10 µm.

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2) Multi-parameter impact on cluster formation, processing and signaling. Another big issue is the handling of multiple parameters, which have, at least in parts, a significant influence on cluster constitution and processing. In addition to the amount and significance of parameters governing other RTK signaling systems, Eph/ephrin signaling is also subject to additional controls due to their unique features, such as cell-contact dependent receptor/ligand engagement. Furthermore, in consideration of temporal aspects, only few parameters can be seen as static (related to the time frame of observation), such as receptor/ligand density. Most parameters are also strongly influenced by temporal aspects e.g. the cell-contact interface, which varies over time. Eventually the nature of Eph/ephrin signaling also comprises spatial control on the sub-cellular level. Overall, parameters could be grouped in Eph/ephrin-specific and cell-specific determinants. A list of possible parameters that may be involved in governing clustering, processing, and signaling of Ephs/ephrins is shown in Fig. 3.3A, without claim to be complete.

3.1.2 Generating an instant imaging‐readout for Eph clustering in living cells