Figure 4. EBs are necessary to maintain the highly stable pool of distal dynactin.

(A) FRAP time-series of the distal tip of DRG neurons at 2 DIV expressing either EGFP or EGFP-p150^Glued. The yellow box demarcates the photobleached region. Each image in the time-series was contrast enhanced equally. Also see Movie S3. (B) Mean FRAP recovery curve ± SEM, n=10–11 neurites per condition. Photobleaching occurred at time=0. (C) The mobile fraction for each individual FRAP trace was determined by fitting the trace to a single exponential equation. The mean mobile fraction was determined for EGFP and EGFP-p150^Glued expressing neurons ± SEM, n=10–11 traces per condition, ****P<0.0001, Student's t-test. (D) Quantification of EB1 binding as percent bound relative to wild-type. WT and ΔCAP p150^Glued fragments were synthesized in vitro and incubated with either EB1-conjugated or empty beads. Mean ± SEM, n=4 independent experiments, *P<0.05, Student's t-test. Also see Figure S5. (E) The number of EB3 comets was counted from time-lapse movies of DRG neurons at 2 DIV expressing mCherry-EB3. Distal neurite is defined as the distal 10 μm of the neurite while the midaxon is defined as a 10 μm region of the axon >50 μm proximal to the neurite end. Mean ± SEM, n=21 movies per condition, **P<0.01, Student's t-test. (F) Lysate from DRG neurons at 4 DIV after treatment with either control siRNAs or siRNA directed against EB1 and EB3 was probed for EB1, EB3 and β-catenin. (G) Quantification by western blot of EB1 and EB3 levels after siRNA knockdown showed approximately 80% and 100% knockdown of EB1 and EB3, respectively. Western blot values were normalized to β-catenin as a loading control. (H) Distal ends of DRG neurons at 4 DIV stained for endogenous p150^Glued and GFP after transfection with GFP and either control siRNAs or siRNAs against EB1 and EB3. These images were individually contrast enhanced to display both axonal and tip staining. The ratio-images (R^p150/_GFP) were calculated from the raw imaging data by dividing the raw p150 data by the corresponding raw GFP signal. These images show the distal accumulation relative to GFP. These ratio-images were contrast enhanced to the same level and a heat map was applied to show the relative intensities of the ratio. The warmer colors represent a higher ratio, while cooler colors represent a lower ratio. (I) Line-scan quantification of the distal accumulation. The normalized ratio of endogenous dynactin fluorescence intensity to GFP intensity was determined along the length of the neurite tip. Expression as a ratio to soluble GFP controls for changes in cytoplasmic volume. Knockdown of EB1 and EB3 significantly reduced the distal accumulation of dynactin over 7.8 μm from the distal axon. Mean ± SEM, n≥49 neurite tips from 9–12 neurons per condition, ***P<0.001, two-way ANOVA Bonferroni post test. Scale bars: 5 μm.