Live imaging of chemokine receptor trafficking in neutrophils. a Constructs used for neutrophil-specific transgenic expression of Cxcr1-FT (Fluorescent Timer) and Cxcr2-FT. Confocal projections of neutrophils in the head of a 3 days post fertilization (dpf) transgenic larva (Tg(lyz:Cxcr1-FT), top; Tg(lyz:Cxcr2-FT), bottom) showing tRFP (tagRFP; magenta) and sfGFP (green) channels. Scale bar = 20 µm. b Anatomical scheme of 3 dpf larva showing the location of the caudal hematopoietic tissue (CHT), the venus circulation (VC, blue), the ventral fin (VF), and the wound site. Below the larva are schemes depicting the area of the wound (W) with neutrophils getting mobilized from the CHT (top) or performing chemotaxis upon entering the ventral fin (bottom). The Caudal Vein plexus (CVP) of the CHT tissue is drawn in blue. Dashed square indicates area imaged in snapshots on the right. c Neutrophils in Tg(lyz:Cxcr1-FT) larvae (sfGFP is shown) upon mobilization from the CHT (top panels) or chemotaxis towards the wound (bottom panels). Arrows show the same cells over time. Time points on the right image are minutes elapsed after image on the left. Scale bar = 10 µm. d Schematic of Cxcr1/2-FT construct behavior in neutrophils. Newly synthesized receptors fluoresce in green due to short residence time at the plasma membrane. Older receptors fluoresce in red and green, and accumulate in intracellular compartments through constitutive turnover. Upon exposure to ligands at wounds, the receptor may internalize and subsequently be degraded or recycled
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