Kir7.1 regulates plasma MP gradient during neutrophil chemotaxis. (A) Schematics of the neutrophils being imaged in the zebrafish ventral fin fold when migrating toward an LTB4 gradient. (B) Representative time-lapse imaging of ASAP3 and mCherry-CAAX in fish neutrophils migrating toward 30 nM LTB4 using Tg(lyzC: ASAP3;lyzC: mCherry-CAAX). Ratiometric analysis (ASAP3/mCherry-CAAX) was performed. (C) Cell outlines over time, indicating changes in cell shape during migration. Time was color-coded. Scale bar, 10 μm. Cell outlines were used to quantify the speeds of cell protrusion (green) and retraction (magenta). (D) Protrusion (green) and retraction (magenta) speeds for the cell shown in B. Data are presented as mean ± SD for n = 3 spots in each group. (E) Pearson’s correlation between the protrusion and retraction speeds for the cell in B with the indicated time offset. (F and G) ASAP3 ratio and the correlated migration speed at the retraction and the protrusion of the cell are shown in B. Data are presented as mean ± SD for n = 3 spots in each group. (H) A representative frame of the cell in B and the ratio of ASAP3/mCherry-CAAX are color-coded. Scale bar, 10 μm. (I) Quantification of fluorescence intensity along the indicated line in H. green: ASAP3; red: mCherry-CAAX; blue: ASAP3/mCherry-CAAX ratio. (J) Kymograph plotting the dynamics of the MP gradient change. The time interval between frames is 15 s. Blue triangles indicate the tail retraction phase during neutrophil migration. The ratio indicates ASAP3/mCherry-CAAX. (K) Quantitative analysis of the normalized ratio of ASAP3/mCherry along the axis of neutrophil migration upon Kir7.1 WT or Q153H overexpression compared with mCherry-CAAX control. Data are presented as mean ± SD for n = 15 cells in each group. (L–S) similar analysis of (B, C, H, and I) using Tg(lyzC: ASAP3;lyzC:kcnj13-2A-mCherry-CAAX) or Tg(lyzC: ASAP3;lyzC:kcnj13-Q153H-2A-mCherry-CAAX).
Figure 3.

Kir7.1 regulates plasma MP gradient during neutrophil chemotaxis. (A) Schematics of the neutrophils being imaged in the zebrafish ventral fin fold when migrating toward an LTB4 gradient. (B) Representative time-lapse imaging of ASAP3 and mCherry-CAAX in fish neutrophils migrating toward 30 nM LTB4 using Tg(lyzC: ASAP3;lyzC: mCherry-CAAX). Ratiometric analysis (ASAP3/mCherry-CAAX) was performed. (C) Cell outlines over time, indicating changes in cell shape during migration. Time was color-coded. Scale bar, 10 μm. Cell outlines were used to quantify the speeds of cell protrusion (green) and retraction (magenta). (D) Protrusion (green) and retraction (magenta) speeds for the cell shown in B. Data are presented as mean ± SD for n = 3 spots in each group. (E) Pearson’s correlation between the protrusion and retraction speeds for the cell in B with the indicated time offset. (F and G) ASAP3 ratio and the correlated migration speed at the retraction and the protrusion of the cell are shown in B. Data are presented as mean ± SD for n = 3 spots in each group. (H) A representative frame of the cell in B and the ratio of ASAP3/mCherry-CAAX are color-coded. Scale bar, 10 μm. (I) Quantification of fluorescence intensity along the indicated line in H. green: ASAP3; red: mCherry-CAAX; blue: ASAP3/mCherry-CAAX ratio. (J) Kymograph plotting the dynamics of the MP gradient change. The time interval between frames is 15 s. Blue triangles indicate the tail retraction phase during neutrophil migration. The ratio indicates ASAP3/mCherry-CAAX. (K) Quantitative analysis of the normalized ratio of ASAP3/mCherry along the axis of neutrophil migration upon Kir7.1 WT or Q153H overexpression compared with mCherry-CAAX control. Data are presented as mean ± SD for n = 15 cells in each group. (L–S) similar analysis of (B, C, H, and I) using Tg(lyzC: ASAP3;lyzC:kcnj13-2A-mCherry-CAAX) or Tg(lyzC: ASAP3;lyzC:kcnj13-Q153H-2A-mCherry-CAAX).

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