CCSer2 regulates cell migration. (A) Fluorescence microscopy image of fixed 4 dpf WT and ccser2a/b zebrafish morphants stained for DAPI (magenta) and neurons (green). Arrowheads indicate the end of the pLL, and asterisks mark neuromasts. Three individual 10X images were taken with 10% overlap and projected using standard deviation z-projection. Images were then stitched together using tissue landmarks. (B) Number of neuromasts in WT and ccser2a/b morphants as shown in A, n = 24 and 32 zebrafish, respectively. Error bars are the mean ± SEM (chi-square; P < 0.001). (C) Percentage of WT of ccser2a/b morphants with truncated lateral lines, as shown in A, n = 24 and 32 zebrafish, respectively (chi-square; P < 0.0001). (D) DAPI (magenta) and neurons (green) in the single plane image of the pLL primordium at 30 hpf for WT embryos or ccser2a/b morphants. Arrows indicate apoptotic nuclei. The cropped primordium area was determined by tissue elevation and placed on a black background for visualization. (E) pLL length at 30 hpf for WT or ccser2a/b morphants, n = 21 and 23 embryos, respectively. Error bars are the mean ± SEM (Mann–Whitney test; P < 0.0001). (F) pLLp area for WT or ccser2a/b morphants, n = 13 and 14 embryos, respectively. Error bars shown are the mean ± SEM (Mann–Whitney test; P < 0.0001). Scale bar = 100 mm in A; 10 mm in D. (G) Fluorescence microscopy images of fixed U2OS WT, CCSer2-KO, and CCSer2-KO with exogenous expression of CCSer2^WT or CCSer2^∆CC, stained with phalloidin to visualize actin (pink), α-GFP to visualize CCSer2^WT and CCSer2^∆CC (yellow), and DAPI to visualize nuclei (blue). (H) Average circularity of cells in a field of view for WT and CCSer2-KO cells (left of line). n = 73 fields of view analyzed across three biological replicates for WT and CCSer2-KO cells. Statistical analysis was performed with a Mann–Whitney test. Average circularity of CCSer2-KO cells transfected with vector, CCSer2^WT, CCSer2^∆CC, or CCSer2-SxNN^ALL (right of line). n = 40 fields of view analyzed across three biological replicates for CCSer2-KO expressing vector, CCSer2^WT, CCSer2^∆CC, or CCSer2-SxNN^ALL. Significance was determined with a Kruskal–Wallis test with Dunn’s multiple comparisons. All error bars are the median ± interquartile range. (I) Maximum length of each projection (protrusion that is thinner than 10 µm and lasts for at least 18 min) formed in WT and CCSer2-KO cells over a period of 24 h. n = 64 and 127 for WT and CCSer2-KO cells, respectively. (J) Percentage of WT or CCSer2-KO cells that formed projections within the field of view. n = 13 fields of view analyzed for both WT and CCSer2-KO cells across two biological replicates. (K) Speed of collective wound closure for WT and CCSer2-KO cells. n = 20 and 23 fields of view, respectively, across three biological replicates. (L) Directionality ratio (net displacement/total distance) of individual tracks of WT or CCSer2-KO cells migrating during a wound-healing assay. SIR-DNA–labeled nuclei were used as a fiducial for tracking. n = 20 fields of view analyzed for both WT and CCSer2-KOs across two biological replicates with two technical replicates (P = 0.012). Error bars are the median ± interquartile range for (I–L). Statistical analysis was performed with a Mann–Whitney test for (I–L). (M) Representative migratory tracks of WT and CCSer2-KO nuclei as cells migrate to fill a wound at the top of the image. Each color designates an individual cell trajectory. pLLp, pLL primordium.