FIGURE 4.

Requirement of intact Gas7 for Gas7-induced membrane protrusion in Neuro-2a. A, a schematic representation of Gas7 truncates with various internal deletions is shown. Gas7, Gas7ΔWW, Gas7ΔFCH, and Gas7Δcoiled were tagged with His-Myc peptides at the C terminus. B, Gas7 truncates were transiently expressed in Neuro-2a cells, and cell morphology was examined. Neuro-2a cells transfected with either Gas7ΔWW/Myc, Gas7ΔFCH/Myc, or Gas7Δcoiled/Myc failed to form processes. Note that Gas7 and Gas7ΔWW predominantly localized in the submembrane region, whereas Gas7ΔFCH and Gas7Δcoiled were evenly distributed in the cytosol. Scale bar, 5 μm. C, shown is the fraction of Neuro-2a cells transfected with either Gas7/Myc, Gas7ΔWW/Myc, Gas7ΔFCH/Myc, or Gas7Δcoiled/Myc successfully extending membrane protrusions. Data are the means ± S.E. of 3 independent determinations using 100–130 cells. Significant differences (paired Student's t test) in group means compared with the control group (Gas7 transfection) are indicated; *, p < 0.05; **, p < 0.01. D, extracts from COS-7 cells co-transfected with HA/N-WASP and either Gas7/Myc (positive control), Gas7ΔWW/Myc, Gas7ΔFCH/Myc or Gas7Δcoiled/Myc were subjected to co-IP to examine in vivo interactions of HA-N-WASP with various Gas7 internal deletions. Only Gas7ΔWW failed to interact with N-WASP. E, the effect on Gas7-induced membrane protrusions of treatment with an actin-depolymerizing reagent is shown. Gas7/Myc-transfected Neuro-2a cells were treated with 2 μm latrunculin B for 30 min and immunostained for actin and Gas7. Gas7-expressing Neuro-2a cells had similar membrane protrusions after latrunculin B treatment as before. Scale bar, 10 μm.