Figure 5

FRAP analysis reveals that hyperosmotic stress conditions impact GFP-DCX dynamics of association with MTs. COS-1 cells were transfected to express (A,B) GFP-DCX WT or (C,D) GFP-DCX ΔC. Cells were exposed to sorbitol (0.5 M, 1 h) to induce hyperosmotic stress. A FRAP protocol with a small bleach area and measuring recovery into the bleached area was utilized (A–D). A small ROI (indicated by the white rectangle in each cell image) was photobleached in (A) and (C) and the fluorescence recovery subsequently monitored at 3 s intervals for 60 s. Plots of the recovery of fluorescence in small area of bleach in DCX WT (B) and DCX ΔC (D) constructs in the presence and absence of sorbitol treatments are shown. Regression values for the accuracy of the respective curve fits are shown. Insets represent the recovery of fluorescence in bleach area for first three time-points (0–6 s of post-bleach) with the line of best fit used for the calculation of initial rate. (E) Equivalent expression of the GFP-DCX constructs was detected in the presence and absence of sorbitol by immunoblotting for GFP (upper panel), with equivalent protein loading detected by immunoblotting for total α-tubulin (lower panel). Scale bars represent 10 µm. See Fig. 6 for quantitative pooled data.