Figure 3
In the presence of Syns actin waves move slower and contain higher concentration of p-cofilin 1.
(a) Time-lapse images of a 2 DIV embryonic hippocampal neuron showing an actin wave that proceeds from the cell body to the growth cone. Neurons were imaged at 1 frame every 2 min for 3 h. (b) Fluorescence analysis of neuron at 2 DIV stained with phalloidin (F-actin) and antibodies to tubulin and cofilin 1. Actin and cofilin 1 are present in the wave (arrows) while tubulin is excluded. (c) Quantification of actin waves velocity calculated on neurons treated with or without wt or A30P Syn for 2 days and imaged as in (a). (d) Representative immunoblots showing levels of proteins indicated on the right in cell homogenates from neurons treated with or without wt or A30P Syn. Actin is shown as an internal standard. (e) Quantification of the ratio between p-cofilin 1 and cofilin 1 bands, analyzed by densitometry and normalized on actin level, for each experimental condition shown in (d). (f) Fluorescence analysis of neurons incubated for 2 days with or without wt or A30P Syn and processed for cofilin 1 and p-cofilin 1. In the right panels are shown images derived from the subtraction of the area stained for p-cofilin 1 from the area stained for cofilin 1. (g) Quantification of the velocity of actin waves calculated on neurons treated with or without 2 μM LatA for 20 min or 12 nM Jaspla for 20 min. In (c, e, g) data are expressed as mean values ± SEM. In c, ctrl, n = 59; wt Syn, n = 60; A30P Syn, n = 76, from 5 independent cultures. In (e), n = 3 independent experiments. In (g), ctrl, n = 74; LatA, n = 20; Jaspla, n = 23, from 3 independent cultures. Statistical significance was determined by one-way ANOVA followed by Dunnett’s test for multiple comparison, *P < 0.05; **P < 0.01. (c) P = 0.0012, alpha value: 0.050:0.890; (e) P = 0.0086, alpha value: 0.050:0.477; (g) P = 0.0012, alpha value: 0.050:0.897). Bars in (a, f) 10 μm, in (b) upper row, 10 μm, lower row, 2 μm.
