Figure 3.

Ca²⁺ influx through MS channels activates calpain in growth cones. a, The average rates of neurite outgrowth over 15 min periods before treatment (white bars), after the addition of inhibitors (gray bars), and after the subsequent addition of inhibitors with 10 μm GsMTx4 (black bars). Inhibitors of Ca²⁺ effectors included the CaMKII inhibitor KN62 (5 μm), the calcineurin inhibitor CsA (10 nm), and the calpain protease inhibitors (1 μm CPI;10 μm ALLM). b, A schematic diagram illustrates the Ca²⁺ signaling pathway (black) and inhibitors (red) used throughout this figure. Dominant-negative protein inhibitors are in parentheses. c–e, A fluorogenic calpain substrate, t-BOC, was used to measure protease activity during activation and inhibition of MS channels. c, Pseudocolored fluorescence images of growth cones loaded with t-BOC for 30 min under indicated conditions. Scale bar, 10 μm. d, The intensities of t-BOC fluorescence (12-bit scale) plotted over 30 min with or without AGAs in 0 or 2 mm Ca²⁺ (n ≥ 83 for each condition). e, t-BOC fluorescence intensity values measured at 30 min after t-BOC addition and the baseline is normalized to control conditions in the presence of AGAs. Removing AGAs increases t-BOC fluorogenesis in the presence of 2 mm Ca²⁺, but this is prevented in 0 mm Ca²⁺ and with MS channel blockers, with inhibition of calpain and in by growing neurons on flexible FN (PDMS). f, The rate of outgrowth was measured in the presence of inhibitors of Src family kinases included PP2 (1 μm) and SU6656 (1 μm), and in neurons expressing KD-Src. Control data are transferred from a for comparison. g, The rate of outgrowth was quantified for 30 min before (black) and after (gray) the removal of AGAs in each condition. The removal of AGAs reduces the rate of outgrowth in wild-type and eGFP-Talin overexpressing neurons, but is blocked by the addition of 1 μm CPI or expression of the calpain-resistant talin mutant, eGFP-Talin-L432G. *p < 0.05 and ***p < 0.001 using a Kruskal–Wallis test with a Dunn's post test.