GSK3β phosphorylation modulates MT affinity of the CLASP2 plus end tracking domain. (A) Representative examples of HeLa cells expressing WT or mutated EGFP-CLASP2(512–650) as indicated. Images were taken at identical illumination and exposure settings. Insets show MT plus ends at a higher magnification. (B) Normalized mean EGFP-CLASP2(512–650) fluorescence profile along MT plus ends showing that mutation of GSK3β phosphorylation sites does not affect the decay of CLASP2(512–650) binding with increasing distance from plus ends. Fluorescence intensities are normalized to the maximum MT-bound fluorescence intensity (1) and the background in the surrounding cytoplasm (0). Three MTs were measured per cell. The number of cells per condition is indicated on the graphs. The measurement error for the 8×S/D construct is large because MT binding of this construct was very weak. d_1/2 indicates the distance from the MT plus end at which half of the EGFP-CLASP construct has dissociated from the growing plus end as determined by least-square curve fitting to a single exponential decay function (solid lines). (C) Absolute EGFP-CLASP2(512–650) fluorescence profile along MT plus ends in cells with similar expression levels (the same cells as in A). Although the fluorescence decay constant is similar, the absolute amount of plus end–bound 9×S/A is significantly larger than of WT EGFP-CLASP2(512–650). (D) Integrated fluorescence intensity from absolute fluorescence profiles as in C plotted against the mean cytoplasmic fluorescence, indicating quantitative differences in MT binding of the different mutants. This represents the same dataset as in B, but profiles were not normalized. Each circle represents the mean of three MTs per cell. Arrows indicate the cells shown in A. Solid lines show least-square fits to a hyperbolic binding isotherm. AU, arbitrary unit. Error bars indicate 95% confidence intervals. Bar, 10 µm.