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Fig. 4
A Capsaicin Gradient Directs rTRPV1-Neutrophil Motility
(A) Injecting capsaicin (red) into surrounding agarose generates an intracellular calcium gradient (green) in neutrophils expressing rTRPV1 channels (blue lines). R indicates the reference point, defined as the initial point source of the gradient. Three experimental groups were imaged: Tg(LysC:dsRed; LysC:GCaMP3) larvae in a capsaicin gradient (dsRed+CAP), Tg(LysC:rTRPV1; LysC:GCaMP3) larvae in an ethanol gradient (rTRPV1+EtOH), or capsaicin gradient (rTRPV1+CAP).
(B) Last frames from Movie S4 show the tracks of individual neutrophils from each experimental group (quantified in D and E) relative to the reference point R at the focal point of the gradient.
(C) (x,y) coordinates for each neutrophil at every frame tracked within the Tg(LysC:rTRPV1) larvae exposed to a capsaicin gradient visually illustrate the directed motion toward the source. The track for each cell was normalized such that the starting (x,y) position was (0,0) for every cell. All units are µm. One larva from the rTRPV1 + CAP group was assessed after exposure to a ventrally centered gradient along the bottom of the animal and the three tracked cells from that animal are highlighted with red arrows.
(D and E) Graphs show the final distance traveled toward the reference point (D), and straightness ratio (E) for tracked neutrophils. Error bars are mean ± SD. (D) Kruskal-Wallis test followed by Dunn’s multiple comparison test: , adjusted p = 0.012 and , adjusted p value = 0.0075. (E) One-way ANOVA followed Tukey’s multiple comparisons test: , adjusted p value = 0.0052 for dsRed+ CAP versus rTRPV1+CAP and , adjusted p value = 0.005 for rTRPV1+ EtOH versus rTRPV1+CAP. For each group, n e 30 cells from e10 larvae.
See also Figure S4 and Movie S4.