Extended Data Fig. 6: Regulation of the neuronal actin cytoskeleton by neddylation.
From: Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics
a, Neddylation-deficient neurons form enlarged axonal growth cones. Mouse hippocampal neurons treated with DMSO or 1 μM MLN4924 at DIV2 for 24 h, fixed at DIV3 and stained for microtubules (tyr-tubulin) and F-actin (phalloidin). Measurement of axonal growth cone area and actin/microtubule ratio in stage 3 neurons (unpaired, two-tailed t tests, growth cone area, t(22) = 3.25, **P < 0.01 (P = 0.0037); actin/microtubule ratio, t(22) = 2.413, *P < 0.05 (P = 0.0246), n = 11 control, 13 MLN4924 neurons, from one culture). Scale bar, 2 μm. b, Neddylation inhibition increases actin dynamics and retrograde flow. Spinning disk confocal microscopy (every 2 s for 5 min) of axon growth cones of DIV2 rat hippocampal neurons, nucleofected with LifeAct-GFP and treated with DMSO or 1 μM MLN4924 for 3−5 h. Measurement of relative changes in LifeAct-GFP pixel intensity (unpaired, two-tailed t test, t(72) = 2.511, *P < 0.05 (P = 0.0143), n = 38 DMSO and 36 MLN4924 growth cones, from four neuron cultures) and retrograde flow (unpaired, two-tailed t test, t(8) = 3.304, *P < 0.05 (P = 0.0108), each data point represents a single growth cone in which the retrograde flow was measured and averaged in kymographs of three ROIs, each for six events, n = 5 growth cones per condition, from two cultures). Scale bars, 10 μm. Related to Supplementary Video 2. Data for graphs are available as Source data. Data are presented as mean ± s.e.m.
