Figure 4.

Spatiotemporal Dynamics of IP₃ in Growth Cones Responding to Localized IP₃ Signals or NGF Gradients

(A) Pseudo-color IP₃ images in WT and R3KO growth cones 0.5 min before and 1 min after the onset of repetitive FLIP of caged IP₃. Each growth cone was transfected with the FRET-based IP₃ sensor IRIS-2.3 containing EGFP and HaloTag-TMR, and the ratio of EGFP emission compared with TMR emission (F_EGFP/F_TMR, defined as R_FRET) was determined. Shown are relative changes in R_FRET (R_FRET/R_FRET-base, defined as R′) used as a measure of IP₃ levels. The red crosshair indicates the site of laser irradiation. The near (red) and far (blue) ROIs were defined as described in Figure 2. Scale bar, 5 μm.

(B) Time course changes in R′ in the near (red line) and far (blue line) ROIs positioned on WT (dotted line) and R3KO (solid line) growth cones.

(C) The mean amplitude of R′ over the last 30 s of repetitive FLIP shown in (B). ∗∗p < 0.01; ∗∗∗p < 0.001, Wilcoxon matched pairs signed rank test.

(D) Pseudo-color IP₃ images in WT and R3KO growth cones 0.5 min before and 2.5 min after the onset of repetitive NGF ejection from the direction indicated by the white arrows. Scale bar, 5 μm.

(E) Time course changes in R′ in the near (red line) and far (blue line) ROIs positioned on WT (dotted line) and R3KO (solid line) growth cones.

(F) The mean amplitude of R′ over the last 1 min of repetitive NGF ejection. ∗p < 0.05; paired t test.

In (B and E), data are represented as mean ± SEM. In (C and F), each gray line connecting two dots represents data from the near and far ROIs of a single growth cone, and each colored bar represents the mean.