Fig. 3. PTP-PEST regulates the formation of membrane protrusions. CHOK1 cells were transiently transfected with expression plasmids encoding wild-type PTP-PEST, a catalytically inactive mutant PTP-PESTC231S, or a mock vector encoding GFP. 24 hours after transfection, cells were trypsinized and replated onto FN-coated coverslips in serum-free medium for indicated times. PTP-PEST or PTP-PESTC231S-transfected cells were fixed and co-stained for PTP-PEST with the KT3 mAb and for actin with Texas-Red-phalloidin. GFP-transfected cells were co-stained for actin only. MOCK-transfected cells spread on a FN substrate extending prominent lamellipodia and form membrane ruffles. WT PTP-PEST-transfected cells are inhibited in spreading. These cells are able to attach but have a rounded morphology. They do not extend protrusion or form ruffles. Catalytically inactive PTP-PEST(C231S) enhances membrane ruffling at early times of adhesion (30-60 min). At later times after plating (2 hours) these cells extend numerous protrusions in all directions.