CCSer2 supports the retrograde trafficking of early endosomes and promotes dynein localization to the cell periphery. (A) Fluorescence microscopy images of fixed WT and CCSer2-KO cells stained with phalloidin to visualize actin (magenta), α-EEA-1 to visualize early endosomes (green), and DAPI to visualize nuclei (blue). (B) Fractional distribution of EEA-1 endosomes from the centroid of the nucleus to the cell edge. n = 37 cells analyzed for both samples across three biological replicates. (C and D) Fluorescence microscopy images of fixed CCSer2-KO cells transfected with CCSer2^WT (C) or CCSer2^∆CC (D) and stained with phalloidin to visualize actin (magenta), α-EEA-1 to visualize early endosomes (green), α-GFP to visualize CCSer2^WT or CCSer2^∆CC (yellow), and DAPI to visualize nuclei (blue). (E) Fractional distribution of EEA-1 endosomes from the centroid of the nucleus to the cell edge for CCSer2-KO cells transfected with vector, CCSer2^WT, or CCSer2^∆CC. n = 44 cells analyzed per sample across three biological replicates. (F) Fluorescence microscopy images of fixed WT and CCSer2-KO cells treated with EGF-555, shown in green. Cells were stained with phalloidin to visualize actin (magenta) and DAPI to visualize nuclei (blue). (G) Fractional distribution of EGF-555 intensity from the centroid of the nucleus to the cell periphery in WT and CCSer2-KOs. n = 89 and 85 cells analyzed for WT and CCSer2-KOs, respectively, across three biological replicates. (H–K) Fractional distribution from nuclear centroid to cell edge for ER intensity (stained with α-PDI) (H), mitochondria (stained with α-TOM20) (I), Golgi apparatus (stained with α-GM130) (J), and lysosomes (stained with α-LAMP1) (K) in WT or CCSer2-KO cells. n = 34 cells for ER, 35 for mitochondria, 37 for Golgi, and 37 for lysosomes, obtained from three biological replicates. Error bars shown are the mean ± SEM (B, E, G, and H–K). Significance was determined with multiple Mann–Whitney tests using a false discovery rate of 1% (B, E, G, and H–K). (L and N) Fluorescence microscopy images of WT and CCSer2 KO cells fixed 30 min after wounding and stained with α-DHC (L) or α-Ndel1 (N). The wound is positioned so that the cells are migrating toward the top of the image. (M and O) Quantification of dynein (M) or Ndel1 (O) localization at the leading edge. The mean intensity of dynein or Ndel1 signal in one-micron band at the leading edge was divided by the mean intensity 6.5 μm into the cell. n = 75 cells analyzed for both WT and CCSer2-KOs across three biological replicates. Error bars represent the median with interquartile range. Significance was determined with a Mann–Whitney test. DHC, dynein heavy chain.