Speed and trajectories analysis

Actin trajectories recorded by sptPALM were computed and analysed using custom-made algorithms written as a MetaMorph plug-in as described in ref. ⁶³. Single-molecule localization was performed using a wavelet-based algorithm, and trajectories were computed using a simulated annealing algorithm⁶⁵. The trajectory duration, which corresponds to the time during which single mEOS2 fluorophores emit red light upon 561 nm laser illumination, follows an exponential distribution strongly shifted to short values. Only trajectories longer than seven frames in regions of interest were kept, which yielded a median of around nine frames. The proportion of trajectories with more than seven points is 80% in control and 78% Gpsm2 cKO growth cones respectively. The MSD function was computed for each trajectory over time, and fit by the power law MSD=4Dt^α using Kaleidagraph 4.1, where t is the time, D is an adjustable coefficient and the exponent α (values between 0 and 2) reflects the curvature of the MSD function and the type of movement. For highly directed trajectories, the MSD is a quadratic function of time, thus α is close to 2 (ref. ^{64, 66}). The numbers of trajectories analysed were 1344 (controls) and (1121) mutants.

Statistical analyses were carried out using Prism statistical package (GraphPad). Normality of distribution and homogeneity of variance were validated and statistical significance between means was calculated using unpaired Student’s t-test or Mann–Whitney test when normality test failed. P<0.05 was considered significant.