Phosphorylation site mutations rescue CLASP2–MT binding in cells expressing constitutively active GSK3β(S9A). (A) Representative examples of HeLa cells expressing WT or mutated EGFP-CLASP2(512–650) containing only the plus end tracking domain MT#1 in combination with mRFP-GSK3β(S9A) (insets). WT EGFP-CLASP2(512–650) is completely cytoplasmic in cells expressing constitutively active GSK3β(S9A), whereas the 9×S/A construct robustly tracks MT plus ends. (B) Scatter plots of cells with different expression levels of EGFP-CLASP2(512–650) phosphorylation site mutants and of constitutively active mRFP-GSK3β(S9A) categorized by whether the CLASP2 construct was detectable on MT plus ends or not. Each symbol represents the mean cytoplasmic EGFP and mRFP fluorescence intensities of an individual HeLa cell. Gray symbols indicate cells in which plus end tracking was barely detectable. (C) Representative examples of HeLa cells expressing mutated EGFP-CLASP2(340–1,084) containing both MT#1 as well as MT#2 required for CLASP association along lamella MTs in combination with mRFP-GSK3β(S9A) (insets). Mutation of the first GSK3β motif, 3×S/A, completely rescues plus end tracking but has little effect on binding along MTs. Mutation of all GSK3β sites, 9×S/A, is necessary to rescue binding along MTs, and this construct shows almost no preference for plus ends. (D) Scatter plots of cells with different expression levels of EGFP-CLASP2(340–1,084) phosphorylation site mutants and of constitutively active mRFP-GSK3β(S9A) categorized by whether the CLASP2 construct was only in the cytoplasm, on MT plus ends, or bound along MTs. Each symbol represents the mean cytoplasmic EGFP and mRFP fluorescence intensities of an individual HeLa cell. Gray symbols indicate cells with only weak binding along MTs. All images were taken at identical illumination and exposure settings. The axes on all graphs are scaled identically. AU, arbitrary unit. Bar, 10 µm.