Biology Reference
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Figure 4.2 Ephrin-A5 causes collapse of Chick RGC growth cones via activation of Eph
receptors. (A) Ephrin-A5-fc dimers were clustered with an anti-human antibody and added
to the bathing medium of cultured RGCs. Times indicated are times after addition of
ephrin. (B) Addition of soluble ephrin-A5 leads to phosphorylation of Eph receptors. The
anti-pEph receptor antibody (right-hand panels) recognizes the phosphorylation of two
highly conserved juxtamembrane tyrosines in the Eph receptor intracellular tail. Times
shown are times after ephrin addition. (C) Isolated and purified RGCs show
phosphorylation of Eph receptors after 15 min stimulation with soluble clustered ephrin
A5. Tubulin is shown as a loading control
In the presence of a specific inhibitor of Rho associated kinase, Y27632,
soluble ephrin-A5-Fc induces the loss of lamellae seen in the control situation
(compare Figure 4.2A and 4.3A). However, the collapse of filopodia and the
dramatic axon retraction are not observed. RGC growth cones fixed and
stained with phalloidin can be placed into one of three categories according to
their morphology and filamentous actin distribution (Figure 4.3B). We
consider a full growth cone to have lamella and filopodia and a partial growth
cone to have no lamellae but at least three or more filopodia. Full collapse is
defined as loss of both lamellae and filopodia. Although the proportion of
growth cones which have fully collapsed in response to ephrin-A5 stimulation
is returned to near control levels by the presence of Y27632, the majority of
growth cones show a partially collapsed morphology with no lamellae
(Figure 4.3B). Since inhibition of Rho associated kinase prevents the ephrin-
induced collapse of filopodia and dramatic axon retraction, but not loss of
lamella, it seems the Rho-Rho kinase pathway must not be the only one
activated during ephrin-A5 induced growth cone collapse.
