Slit induces filopodium elongation. (A) DIC images of DRG growth cones 15 s before (i and iii) and 10 min after addition of 1.5 collapsing units (CU) of either Slit (ii) or Sema3A (iv). One CU is the ligand concentration which induces ∼50% growth cone collapse (Slit2, 400 ng/ml; Sema3A, 500 ng/ml). Note the elongation of filopodia after stimulation with Slit (ii, white arrowheads mark filopodium tips), but not with Sema3A (iv, red arrowheads mark retraction fibers). See Videos 1 and 2. Growth cone collapse (solid lines) and filopodium length were measured over a concentration range of Slit2 (v, blue) or Sema3A (vi, green); for ease of comparison, concentrations are given as CU. Greater than 40 growth cones were scored at each concentration per condition. ***, P < 0.0001; n.s., P > 0.05; one-way ANOVA. (B) Live-cell DIC images of a growth cone during stimulation with 1.5 CU Slit. Filopodium tips are marked at 10s before (orange arrowheads) and at 170s after (blue arrowheads) Slit stimulation. Montages show time-lapse images of single filopodia, with the far-right panel showing an immunofluorescent image of the same filopodium after fixation at 180s post-Slit (phalloidin [F-actin], red; Mena, green). See Video 3. Bars, 10 µm. (C) Spontaneous and Slit-induced filopodium elongation (Elong.) kinetics were measured from DIC images captured every 5 s for 10 min before and after Slit addition. (i) Depicts length, rate, lifetime, and extension period measurements shown in panels ii–v (ii–v: preslit, n = 638 filopodia; postslit, n = 560 filopodia; 15 biological replicates). ***, P < 0.0001; n.s., P > 0.05; two-tailed t test.