Fig. 3. 6ß4 ligation is required for sustained activation of Rac1, lamellipodia formation and RhoA independent chemotaxis. (A) Effect of 6ß4 expression and ligation upon Rac1 and Cdc42 GTPase activation by EGF. Growth factor-starved cells were stimulated with 2 ng/ml EGF for the indicated times. Cells were lysed, and incubated with GST-PAK and glutathione-sepharose beads. Beads were washed and bound proteins separated on a 12% SDS-polyacrylamide gel. GST-PAK pull down blots were initially probed for Rac1 before stripping and reblotting for Cdc42. Control gels show relative quantities of GTPases present in total lysates. Numbers under each profile represent average optical density of pull down lanes from at least 2 separate blots. (B) Effect of 6ß4 expression and ligation upon RhoA GTPase activation by EGF. Growth factor starved cells were stimulated with 2 ng/ml EGF for the indicated times. Cells were lysed, and incubated with GST-RBD and glutathione-sepharose beads. Beads were washed and bound proteins separated on a 12% SDS-polyacrylamide gel. Control gels show relative quantities of GTPases present in total lysates. Numbers under each profile represent average optical density of pull down lanes from at least 2 separate blots. (C) Effect of EGF treatment upon lamellipodia formation. ß4(-), ß4(+) and ß4(AD) cells were starved of growth factors for 16 hours and stimulated with 2 ng/ml EGF for the indicated times before fixing with 3.4% formaldehyde in PBS and stained with TRITC-phalloidin to identify filamentous actin. The area of lamellipodial projections was measured by tracing around membrane extensions on digital images. The lamellar area was then calculated using NIH image software. The graph shows total lamellar area for ß4(-) cells (open triangles), ß4(+) cells (black triangles) and ß4(AD) cells (black circles). (D) Effect of ß4 inhibitory antibody ASC-8 upon lamellipodia formation. Growth factor-starved NHKs incubated with 10 µg/ml IgG or ASC-8 were stimulated with 2 ng/ml EGF and lamellipodial area calculated as described. The graph shows lamellar area for NHK plus IgG (black circles) and NHK plus ASC-8 (open circles). (E) Effect of GTPase inhibition upon 6ß4-dependent chemotaxis. ß4(+) cells were retrovirally transduced with control LacZ or inhibitory GTPase constructs, N17Cdc42, N17Rac1 or N19RhoA. Transwell chemotaxis experiments were performed with collagen IV-coated transwells and 2 ng/ml EGF stimulation. Actual induction, ß4(+)LacZ 106.4±9.0 cells/field, ß4(+)N17Rac1 23.8±3.7 cells/field, ß4(+)N19RhoA 121.1±3.0 cells/field, ß4(+)N17Cdc42 11.8±9.1 cells/field.

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