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(n ΒΌ 24). The response of the Y27632 treated RGC to the ephrin-expressing
fibroblast contrasts with the response to soluble ephrin. The lamella of
Y27632 treated RGC is lost within 5 minutes after stimulation with soluble
ephrin-A5 (see Figure 4.3A). Following contact with the fibroblast cell the
lamella of Y27632 treated RGC is retained for longer and the growth cone
continues to advance across the substrate (Figure 4.4B). These contrasting
results might reflect a difference in concentration or complement of ephrin-A
ligands that stimulate RGC Eph receptors. Soluble ephrin-A5 causes
activation of Eph receptors all over the growth cone and along the axon
(Figure 4.2B) whereas only the receptors at the leading edge of the growth
cone will be activated by direct contact with the fibroblast cell, which may
express ephrin-A ligands other than ephrin-A5.
Inhibiting Rho associated kinase does not prevent the repulsive response of
the ephrin-expressing fibroblast cell after interaction with the RGC
(Figure 4.4B). This suggests that Rho kinase may not be involved in the
ephrin-signalling pathway.
It is clear that, in vitro, ephrin stimulation of Eph receptor expressing cells has
a profound effect on the actin cytoskeleton, and that this in turn can influence
the dynamic behaviour of the cells involved. In fibroblast cells we see two
temporally distinct actin based responses: first a protrusive response in which
lamellae and/or filopodia are induced, and later the formation of contractile
actin stress fibres which are associated with a cell's withdrawal or retraction.
In neuronal axons in culture, soluble and membrane bound ephrin causes
axonal retraction, which is mediated by Rho associated kinase. It is therefore
possible that contractile actin filaments analogous to fibroblast stress fibres
are assembled in the neuronal axon following ephrin stimulation. In the co-
culture assay it seems that the growth cone may continue to advance a small
distance across the substrate after contact with the ephrin-expressing cell. It
will be interesting to analyse this further to determine whether the full
compliment of actin responses seen in the Eph receptor expressing fibroblast
cells are reflected in the neuronal response to ephrins; both the early
protrusive and the later retraction behaviours.
In the case of stimulation with membrane-bound ligand, not only does
inhibiting Rho associated kinase prevent axonal retraction, but also the loss of
lamella is significantly delayed and the axon continues to advance across the
substrate. The differences in response to soluble versus membrane bound
ligand may reflect differences in the complement of ephrin molecules
presented, or the level of neuronal Eph receptor activation. Ongoing work
involves introducing ephrin-A molecules into cells which do not endogenously
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