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. 2019 Nov 14;10:5166. doi: 10.1038/s41467-019-13107-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — 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