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of Rac-mediated Rho activation (Ridley et al., 1992). This we have confirmed
by co-injecting Eph receptor expressing cells with C3 transferase (a specific
inhibitor of Rho) and observing a failure of actin stress fibre formation
without inhibition of lamellipodial protrusions (data not shown).
Role of Rho GTPases in ephrin induced growth cone collapse
Several different model systems have demonstrated a role for members of the
Rho family proteins in axon guidance. From the classic studies of Rho
GTPase manipulation in fibroblasts, it might be predicted that activation of
the small GTPases Rac and Cdc42 would mediate growth cone responses to
attractive cues, whereas Rho would likely underlie growth cone collapse and
axon retraction events via Rho-associated-kinase signalling and consequent
actin-myosin based contraction events (Dickson, 2001). A novel guanine
nucleotide exchange factor for Rho GTPases named ephexin has been
identified as an EphA receptor interacting protein through a two-hybrid
screen (Shamah et al., 2001). Transfection of a dominant negative mutant
version of ephexin into RGCs inhibited ephrin-A induced growth cone
collapse, implying a link between Eph receptors, Rho GTPases and the actin
cytokeleton. While it has been shown that growth cone collapse induced by
another repulsive cue, Semaphorin3A, requires Rac activity (Jin and
Strittmatter, 1997; Kuhn et al., 1999; Vastrik et al., 1999), studies of ephrin-
induced RGC growth cone collapse have demonstrated a reduction in the
levels of active Rac in the neurons after ephrin treatment, concurrent with an
increase in Rho activity (Wahl et al., 2000). Indeed, Mueller and colleagues
(Wahl et al., 2000) have demonstrated that inhibiting Rho or Rho associated
kinase (a Rho effector molecule) prevents ephrin-induced growth cone
collapse in RGCs.
We have repeated this experiment on cultured RGCs, by stimulating with
soluble ephrin molecules. RGC axons growing on laminin have a characteristic
growth cone, consisting filopodia (Figure 4.2A first panel, arrowheads) with a
veil-like lamella between them (Figure 4.2A, arrow). Addition of 1mg/ml
ephrin-A5 to the bathing medium of cultured RGCs causes rapid collapse of
the growth cone lamella and filopodia, followed by axon retraction within
5min (Figure 4.2A). Using a phosphorylation-specific Eph receptor antibody
we observe that Eph receptors on the RGC growth cone and along the length
of the axon rapidly become phosphorylated in response to ephrin-A5
(Figure 4.2B, right-hand panels). This antibody reveals a single band after
immunoblotting whole cell lysates from RGCs stimulated with ephrin-A5,
confirming that there is a significant increase in Eph receptor phosphorylation
in these cells in response to ephrin-A5 (Figure 4.2C).
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