Figure 6. Attractive Ca2+ signals suppress endocytosis via CaMKII and Cdk5. A, TIRFM image of EGFP-clathrin in a growth cone treated with roscovitine. NP-EGTA was photolyzed by repetitive UV light (3 s interval) to generate attractive Ca2+ signals. The UV-irradiated area and the corresponding area on the opposite side were used as near and far ROIs, respectively (blue circles). Scale bar, 10 μm. B, Schematic representation of the growth cone shown in A. TIRFM images of EGFP-clathrin were acquired every 3 s before (pre) and after the onset (time 0 s) of attractive Ca2+ signals. Each red cross marks the position of a newly formed CCP within the ROIs during the indicated 30 s periods. Scale bar, 10 μm. C–O, Near-to-far ratios of CCP formation. Growth cones expressing EGFP-clathrin (C–G) or mCherry-clathrin (H–O) were analyzed. The y-axis indicates the number of newly formed CCPs per unit area within near ROI divided by that within far ROI, before (pre; −120 to 0 s) and after (UV; 0 to 120 s) the onset of repetitive photolysis that generated attractive (pink; C–I, M–O) or repulsive (light blue; J–L) Ca2+ signals. Each line represents a photolysis-induced change in a single growth cone. Ca2+-induced asymmetry in CCP formation was assessed in the absence (no drug; C) or presence of roscovitine (D, L, N, O), olomoucine (E), KN93 (F), or myr-AIP (G). In addition, the asymmetry was assessed in growth cones that had been transfected with EGFP-tagged wild-type (WT; H) and the dominant-negative form (D144N; I) of Cdk5, Venus-tagged phosphodeficient (T286A; J), and phosphomimetic (T286D; K, L) mutants of CaMKII or EGFP-tagged wild-type (WT; M, N) and phosphomimetic mutant (S649E; O) of PIPKIγ90. *p < 0.05, **p < 0.01, ***p < 0.001; N.S., not significant, paired t test.