CLASP2–MT interactions mediated by GSK3β inactivation contribute to lamella MT dynamics. (A) Migrating HaCaT keratinocyte expressing EGFP-CLASP2(340–1,362). (B) HaCaT cell expressing 9×S/A EGFP-CLASP2(340–1,362) containing mutated GSK3β phosphorylation sites. (A and B) Control HaCaT cells. (A′ and B′) HaCaT cells additionally expressing constitutively active mRFP-GSK3β(S9A) (insets). (C) Dynamics of EGFP-CLASP2(340–1,362) and mCherry-paxillin in focal adhesions in the leading lamella of a migrating HaCaT cell. Elapsed time is indicated in minutes. (D) Quantification of ectopic EGFP-CLASP2(340–1,362) association along cell body MTs of WT or the nonphosphorylatable 9×S/A construct as a function of cytoplasmic EGFP fluorescence intensity. Red symbols represent cells with plus end tracking only, and black symbols represent cells in which the CLASP construct is clearly detectable along cell body MTs. (E) Quantification of migration rates of control HaCaT cells and cells expressing GSK3β(S9A) alone or in combination with 9×S/A CLASP2(340–1,362). Open symbols represent individual cells, and closed symbols represent means. n = 42 cells. (F) Plot of the correlation coefficient between image regions in the lamella of cells expressing the indicated EGFP-CLASP2(340–1,362) or EGFP-CLASP(340–1,084) constructs as a function of the time interval between images. n = 6 cells. (G) Analysis of lamella MT polymerization dynamics by direct manual tracking of EGFP-CLASP2(340–1,362)–decorated MTs. Open symbols represent parameters calculated from individual cells, and closed symbols represent means of measurements from all six cells analyzed. The bar graph summarizes time MTs spent growing (g), pausing (p), or shortening (s). AU, arbitrary unit. Error bars indicate 95% confidence intervals. Bars, 10 µm.