FIGURE 2.
Movement of GFP-Gαs in living cells. A–C, PC12 cells were transfected with GFP-Gαs and then treated with 50 ng/ml NGF for 48 h. Images were captured with epifluorescence and deconvolved. Sections were imaged every 200 nm. Analysis of images from a z series reveal that GFP-Gαs concentrates at the leading edge of newly formed cellular protrusions and lamellipodia (A, arrow) and tips of filopodia (B, arrow). A, a series of volume-projected images with maximum intensities from the time-lapse recording of GFP-Gαs movement. Time-lapse imaging was done for 1.5 h every 15 min. Time points (hours:minutes) are selected to illustrate the intermediates of neurite formation. Scale bar = 15 μm. B, GFP-Gαs was transported along the formed cellular processes, concentrating at neurite tips. After NGF treatment, it is present at the leading edges of filopodia (arrow) as well. Scale bar = 15 μm. C and D, ratiometric analysis of images of cells transfected with GFP-Gαs against GFP alone (C, right panel). NGF-treated cells were transfected with either GFP alone or GFP-Gαs. C, fluorescence intensity was measured in six randomly selected regions of the cell body (CB) and growth cone (GC). D, fluorescence intensity of cells transfected with GFP alone or GFP-Gαs. The mean intensities from the cell body and growth cone were calculated from 6 cells/group. Two-way ANOVA was used for statistical analysis. Significance was determined using paired Student's t test. **, p <0.01 between cells that were transfected with GFP alone and cells that were transfected with GFP-Gαs. All data are mean ± S.D.