Fig. 5. Semaphorin 3A blocks the formation of axonal F-actin patches that serve as precursors to filopodial and lamellipodial extension. (A) Example of filopodial protrusion from a spontaneously formed axonal F-actin patch in an eYFP-actin expression axon. Numbers in panels reflect seconds. At 15 seconds, a patch forms (arrow) that subsequently gives rise to a filopodium (sec 30-45). By 60 seconds, the filopodium has retracted and the patch has disappeared. As described by Loudon et al., the majority of axonal protrusive events are preceded by patch formation (Loudon et al., 2006), although only approximately 6-7% of F-actin patches give rise to filopodial or lamellipodial protrusion. (B) Determination of the frequency of F-actin patch formation, during 6-minute sampling periods (6-second interframe intervals) in eYFP-actin-transfected axons revealed that semaphorin 3A inhibited the formation of patches. Inhibition of patches occurred with a similar time course to that of growth cone collapse (i.e. during the first 10 minutes of treatment). The effects of semaphorin 3A were blocked by 10 µM y-27632 (P values shown for comparison of semaphorin 3A treatment and controls within time points, Welch t-test, n=6-7 axons per group; y-27632 together with semaphorin 3A frequencies were not different from controls at either time points). (C) Example of axonal F-actin patch formation in a control axon, as previously described by Loudon et al. (Loudon et al., 2006). Patches form spontaneously and increase in size and fluorescence intensity. Double-magnification insets of the patches are shown in the bottom of each panel. Numbers in panels reflect seconds in the time-lapse sequence. Arrowhead in middle panel indicates a briefly detectable eYFP-actin patch. (D) Example of minimal patch-formation, and lack of patch development, in an axon treated with semaphorin 3A for 13 minutes. A small, but detectable, patch of eYFP-actin formed (middle panel) but the patch disappeared at 6 seconds. The rapid disappearance of patches in the semaphorin-3A-treated axon stands in contrast to the much longer live-span of patches observed in control axons (compare seconds elapsed in C and D). Arrowheads in middle panel indicate a briefly detectable eYFP-actin patch. Double-magnification insets of the patches are shown in the bottom of each panel; arrow in 0-second-panel magnification indicates the collapsed growth cone. Numbers in panels reflect seconds in the time-lapse sequence. (E) Growth cone pretreated with y-27632 followed by a 13-minute treatment with semaphorin 3A. Notice that the growth cone is not collapsed (as in D), and continues to undergo morphologic remodeling. Numbers in panels reflect minutes. (F) Example of an eYFP-actin patch (arrowhead) forming and developing in an axon pretreated with y-27632 followed by treatment with semaphorin 3A for 13 minutes. Notice the similarity to patch formation in control axons (in C). Number in panels reflect seconds. (G) Time-lapse sequence from an eYFP-actin-transfected axon treated with semaphorin 3A for 13 minutes (t=0); 72 seconds later, bundle-like eYFP-actin is apparent (arrowheads) and becomes more pronounced at 240 seconds. Image was inverted to increase the contrast of the eYFP-actin signal.