Correlation of axonal F-actin dynamics and stationary endosomes. (A) Kymographs from neurons transfected with GFP:Utr-CH (to label F-actin) and pHRodo (a pH-sensitive endosomal marker that largely labeled stationary endosomes in axons, see Materials and methods), simultaneously visualized by live imaging. Note colocalization of F-actin hotspots (green) with pHrodo (red, overlay on right). A′ shows a zoomed ROI from overlay highlighting two actin trails originating precisely from where two stationary endosomes are situated (marked by asterisks). (B) Kymographs from neurons transfected with GFP:Utr-CH (to label F-actin) and Rab5:mCherry (to label early endosomes). Note colocalization of F-actin hotspots with early endosomes. (C, left) Quantification of colocalization data. The mean frequency of F-actin hotspots that overlapped with stationary endosomes labeled with pHRodo, Rab5-mRFP, and Lamp1-mCherry was 46.81 ± 2.76% (n = 13 axons), 29.04 ± 3.75% (n = 10 axons), and 9.58 ± 3.61% (n = 6 axons), respectively. (right) F-actin dynamics also correlated with number of pHRodo-positive endosomes in axons (n = 16 axons). (D and E) Neurons weretransfected with GFP:Utr-CH (to label F-actin) and treated with Brefeldin-A (BFA) to deplete vesicles in axons (Fig. S4 B). Kymographs show that F-actin dynamics in axons were greatly attenuated upon BFA treatment and restored upon washout of the drug; quantified in E. All values represent means ± SEM. ***, P < 0.001, one way analysis of variance followed by Dunnett’s post hoc test. For detailed statistics, see Table S1.