Eph receptor activation by soluble ephrins rapidly stimulates
the assembly of filamentous actin structures in fibroblast cells
To analyse early changes in actin organization following activation of Eph
receptor kinases, we microinjected eukaryotic expression vectors encoding
EphB2 and EphA7 into the nuclei of quiescent, serum-starved Swiss 3T3 cells.
These fibroblast cells are frequently used to study cellular cytoskeletal changes
induced by growth factors (Nobes and Hall, 1995; Kozma et al., 1995; Ridley
and Hall, 1992), since serum starvation induces loss of filamentous actin
structures such as lamellipodia and stress fibres.
We find a diffuse surface localization of EphB2 or EphA7 can be detected
2 h after microinjection of constructs encoding these receptors (data not
shown). Expression of EphB2 or EphA7 alone does not induce actin filament
assembly within this time (data not shown).
Activation of EphB2 by the addition of soluble, dimeric ephrin-B1-Fc
results in a rapid increase in tyrosine phosphorylation (data not shown) and a
concomitant accumulation of polymerized actin in broad sheet-like lamelli-
podia (Figure 4.1A) beginning within 5-7 minutes of ligand presentation.
Lamellipodial activity is downregulated after 30min and EphB2 receptors
internalized (data not shown).
Activation of EphA7 leads to a pattern of actin assembly distinct from that
stimulated by activating EphB2, with cells forming localized filopodial
protrusions accompanied by associated lamellipodial spreading, within
5-7min of stimulation by dimeric ephrin-A4-Fc (Figure 4.1B). The phospho-
tyrosine immunofluorescence pattern is also different for the two receptors,
with activated EphA7-expressing cells displaying small clusters of phospho-
tyrosine localized to zones of filopodial and lamellipodial activity (data not
shown). Following the initial increase in actin accumulation at the cell
periphery, actin stress fibres are frequently observed in both EphB2 and
EphA7 expressing cells, beginning 10-15min after stimulation with ephrin
(Figure 4.1A and 4.1B).
EphB2 and EphA7 induced lamellipodial protrusion is
mediated by the small GTPase Rac
Small GTPases of the Rho family - the three best characterized being Rho,
Rac and Cdc42 - control the assembly of filamentous actin structures and are
implicated in the regulation of axon guidance. Cdc42 induces actin filament
assembly leading to filopodia protrusion; Rac triggers protrusion of
lamellipodia, and Rho regulates the assembly of contractile actin:myosin