Dynamics and organization of actin in axon shafts. (A) Representative kymographs from axons transfected with GFP:Utr-CH to label filamentous actin. Note vertical “on/off” events representing dynamic local assembly (hotspots) and diagonal plumes of fluorescence representing bidirectionally elongating actin polymers (trails). Also note that actin trails often emerge from where the hotspots are situated (some marked by dashed circles; see also Ganguly et al., 2015). (B) Anterograde and retrograde frequency of actin trails (histogram) and polymer elongation rate (top right graph). Note the slightly higher frequency of anterograde trails (58/42%; mean elongation rates were 0.99 ± 0.001 µm/s [mean ± SEM] in both directions; data are from ∼1,000 total events; adapted from Ganguly et al., 2015). (C) Axon from a DIV3 neuron labeled with phalloidin (C′ shows STORM image of region within dashed red box). Note clusters of actin with radiating filaments (arrowheads in zoomed insets below) likely corresponding with actin hotspots seen by live imaging. (D) Axon from a DIV7 neuron labeled with phalloidin (A′ shows STORM image of region within dashed red box). Note clusters of actin and radiating filaments (red arrowheads in zoomed insets below; regions labeled a–e; actin rings marked by small comb in A′). Bars: 10 µm (C and D); 2 µm (A′, C′, and magnifications).