Figure 2. The CAP-Gly domain is necessary for the distal enrichment of dynactin in neurons.

(A) Distal ends of DRG neurons expressing GFP were stained at 2 DIV for endogenous p150^Glued or endogenous dynein heavy chain (DHC) and GFP, as a marker of cytoplasmic volume. These images were individually contrast enhanced to display both axonal and tip staining. The raw p150 and DHC data were divided by the corresponding raw GFP signal to create the ratio-image (R^p150/_GFP or R^DHC/_GFP). 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. (B) Line-scan quantification of the distal accumulation. The normalized ratio of endogenous dynactin or dynein 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. Dynactin accumulated significantly more than dynein over the distal 15 μm of the neurite. Mean ± SEM, n≥41 neurite tips from 5–7 neurons per condition, ***P<0.001, two-way ANOVA Bonferroni post test. (C) Images of the distal neurites of DRG neurons expressing myc-tagged full-length wild-type or ΔCAP-Gly p150^Glued and GFP from a bicistronic vector at 2 DIV. Neurites were stained for myc and GFP and images individually contrast enhanced to display both axonal and tip staining. The corresponding ratio-images (R^p150/_GFP) were made from the raw imaging data and a heat map was applied as described in (A). (D) Line-scan analysis from the end of the neurite. The normalized ratio of myc-tagged p150^Glued fluorescence intensity to GFP intensity was determined along the length of the neurite tip. Wild-type p150^Glued accumulated significantly more over the distal 10 μm compared to ΔCAP-Gly p150^Glued. Mean ± SEM, n≥29 neurite tips from 5–6 neurons per condition, ***P<0.001, two-way ANOVA Bonferroni post test. Scale bars: 5 μm. Also see Figure S2.