CCSer2 knockout results in reduced microtubule polarization during migration and defects in retrograde integrin trafficking. (A and B) Fluorescence microscopy images of WT (A) and CCSer2-KO (B) cells fixed 4 h after wounding and stained for microtubules (gray), centrosome (magenta), and the nucleus (DAPI). Cells are migrating toward the top of the image. The rose histogram plots to the right of the image display the probability of finding the centrosome 360° around the nuclear centroid. 90° on the rose plots is perpendicular to the angle of the wound. (C) Quantification of the relative integrin enrichment at FAs (paxillin puncta) over the mean total cellular intensity as a ratio (from data in Fig. 4 F). n = 74 cells analyzed, across three biological replicates. Error bars are the median ± interquartile range, and statistics were performed with a Mann–Whitney test. (D) Quantification of the relative integrin enrichment outside of FAs (paxillin puncta) over the mean total cellular intensity as a ratio (from data in Fig. 4 F). n = 74 cells analyzed, across three biological replicates. Error bars are the median ± interquartile range, and statistics were determined with a Mann–Whitney test. (E) Representative western blot from whole-cell lysate of WT and CCSer2-KO cells, blotting for α-β1-integrin and α-GAPDH as a loading control. (F) Quantification of the β1-integrin levels in WT and CCSer2-KO cells normalized to GAPDH levels. n = 3 biological replicates. Error bars shown are the mean ± SD, and the statistical significance was determined with a Mann–Whitney test. (G) Quantification of EB1 plus-end growth in WT and CCSer2-KO cells. n = 30 cells analyzed across three biological replicates per sample. Significance was determined with a Mann–Whitney test, and error bars are the mean ± SD. (H) Fluorescence microscopy images of fixed WT (top) and CCSer2-KO (bottom) cells in a nocodazole washout experiment, stained for microtubules, FAs, and nuclei with α-tubulin (green), α-paxillin (magenta), and DAPI (blue), respectively. Nocodazole-treated cells are fixed after 0 min (left panels) or 15 min (right panels) of washing out with nocodazole-free media. (I) Quantification of the FA size at 0 min using thresholding and particle analysis functions in the paxillin channel in FIJI. n = 20 fields of view for WT and CCSer2 KO cells, across two biological replicates. Error bars are the median ± interquartile range. Statistical analysis was performed with a Mann–Whitney test. (J) Quantification of the FA size at 15 min, normalized to the average size at 0 min in I for both WT and CCSer2-KO cells, respectively. n = 20 fields of view for WT and CCSer2 KO cells, across two biological replicates. Error bars are the median ± interquartile range. Statistical analysis was performed with a Mann–Whitney test. (K) Displacement of the averaged anterograde and retrograde mCherry-integrin tracks per cell of WT and CCSer2-KOs. n = 31 and 33 cells analyzed for WT and CCSer2-KO cells, respectively, across three biological replicates. (L) Mean speed of the averaged anterograde and retrograde integrin tracks per cell of WT and CCSer2-KOs. n = 31 and 33 cells analyzed for WT and CCSer2-KO cells, respectively, across three biological replicates. Error bars are the median ± interquartile range. Statistical analysis was performed with a Kruskal–Wallis test with Dunn’s multiple comparisons test. (M) Three example fluorescence microscopy images of fixed WT and CCSer2-KO cells, 4 h after wounding, stained with phalloidin and DAPI. (N) Fractional distribution of actin intensity from the centroid of the nucleus to the cell edge for WT and CCSer2-KO cells. n = 45 cells analyzed per sample across three biological replicates. Error bars are the mean ± SEM. Statistical analysis was performed with multiple Mann–Whitney tests and a false discovery rate of 1%. Source data are available for this figure: SourceData FS4.