Figure 4.

Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg(mpeg:Lifeact-GFP)^zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg(mpeg:WT-zebrafish Paxillin- EGFP)^zj503, Tg(mpeg:zebrafish Y118E-Paxillin- EGFP)^zj504, and Tg(mpeg:zebrafish Y118F-Paxillin- EGFP)^zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also Video 7. Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis (n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.