CCSer2 depletion results in migration defects in two zebrafish migration models and human cell culture. (A) In situ hybridization showing ccser2a and ccser2b expression at 30 hpf in the zebrafish embryo. (B) Maternal deposition of ccser2a and ccser2b mRNA compared with control β-actin mRNA, which is known to be maternally deposited at high levels. (C) Observed and expected genotypes for ccser2a;ccser2b double heterozygous crosses. (D and E) DAPI (magenta) and neurons (green) of fixed WT 4 dpf zebrafish larva (D) and ccser2a/b crispant (E). High magnification image of pLL nerve end shown below for each. Scale bar = 100 μm in whole trunk image; 10 μm in high magnification image below. (F) Number of pLL neuromasts in WT or ccser2 crispants, n = 8 and 25 zebrafish, respectively. Error bars are the mean ± SEM (chi-square; P < 0.001). (G and H) Schematic showing pLL development (G) and organization of the pLL primordium (H). (I) Measurement axis used for quantification of data in Fig. 3 E. (J) Number of mitotic nuclei for WT of ccser2a/b morphants, n = 13 and 15 embryos, respectively. Error bars are the mean ± SEM (chi-square; P = 0.2514). (K) Number of cells undergoing apoptosis for WT and ccser2a/b morphants, n = 13 and 15 embryos, respectively. Error bars are the mean ± SEM (chi-square; P < 0.005). (L) Fixed fluorescence microscopy images of the fin region of uninjured (uncut) or 6 hpi in WT and ccser2a/b morphants. Injury is denoted by the orange dashed line. DIC imaging is shown in green, and macrophages are labeled in magenta. Scale bar is 50 µm. (M) Quantification of macrophage accumulation at the fin injury site compared with uncut fins. n = 12 for uncut WT and ccser2a/b morphants; n = 18 and 23 for WT 6 hpi and ccser2a/b morphants 6 hpi, respectively. Error bars represent the median and 95% CI, and statistics were determined with Brown–Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons. (N) Representative western blot using whole-cell lysate from WT and CCSer2-KO cell lines. Membranes were probed with α-CCSer2, α-Ndel1, α-DHC, α-Lis1, and α-GAPDH as a loading control. (O) Quantification of western blots in N reporting the ratio of CCSer2, Ndel1, dynein, and Lis1 to GAPDH. n = 4 biological replicates for CCSer2; n = 3 biological replicates for Ndel1, dynein, and Lis1. Error bars are the mean ± SEM, and statistics were determined with Mann–Whitney tests. (P) Results of indel analysis of the CCSer2-KO. Two frameshift (fs) mutations identified in the CCSer2 gene result in early stop codons and truncation of the protein (Ter282 and Ter279). The single base pair insertion occurred in 80% of the alleles sequenced (24/30 alleles), and the 8 base pair deletion mutation occurred in 20% of the alleles sequenced (6/30 alleles). No WT alleles were found in the CCSer2-KO. (Q) Time to anaphase of dividing WT and CCSer2-KO cells is quantified. n = 170 and 102 mitotic cells analyzed for WT and CCSer2-KOs, respectively. Error bars are the median ± interquartile range (Mann–Whitney test; P = 0.0047). (R) Mean speed of individually migrating WT or CCSer2-KO cells on fibronectin over 24 h. SIR-DNA–labeled nuclei were tracked using the TrackMate plugin in FIJI to obtain cell paths. n = 267 and 301 cells tracked for WT and CCSer2-KO cell, respectively. Error bars are the median ± interquartile range. Statistical analysis was determined with a Mann–Whitney test. (S) Plot of the MSD values over the first 300 frames of live wound-healing assay (Fig. 3, L and M). n = 20 fields of view per cell type, across two biological replicates. Error bars are the mean ± SEM. (T) MSD data in S are transformed into a log–log plot of log (MSD) over log (Time(frames)). n = 20 fields of view per cell type, across two biological replicates. Error bars are the mean ± SEM. (U) Linear regressions were applied to the log–log-transformed data in T to extract the µ value (or slope), where MSD(t) ∼ t^µ. Error bars are the median ± interquartile range. Statistical significance was determined with a Mann–Whitney test. n = 20 fields of view per cell type, across two biological replicates. (V) Fluorescence microscopy images of fixed CCSer2-KOs transfected with a GFP vector or CCSer2-SxNN^ALL quantified in Fig. 3 H. Cells were stained with phalloidin to visualize actin (pink), α-GFP to visualize vector and CCSer2-SXNN^ALL (yellow), and DAPI to visualize nuclei (blue). DHC, dynein heavy chain; hpi, hours after injury. Source data are available for this figure: SourceData FS3.