Figure 8. Perry syndrome mutant disrupts flux from the distal neurite.

(A) DRG neurons were stained at 2 DIV for myc-tagged wild-type (WT), HMN7B (G59S) or Perry syndrome (G71R & Q74P) p150^Glued and GFP, a marker of cytoplasmic volume, expressed from a bicistronic vector. These distal neurite images were individually contrast enhanced to display both axonal and tip staining. The raw myc-p150 data was divided by the corresponding raw GFP signal to create the ratio-image (R^p150/_GFP), which shows 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. Scale bar: 5 μm. (B) Line-scan analysis from the neurite tip. The normalized ratio of myc-p150^Glued to GFP fluorescence intensity was determined along the length of the neurite. Wild-type p150^Glued accumulated significantly more over the first 14 μm as compared to mutant p150^Glued. The WT-p150 data was replotted from Figure 2D. Mean ± SEM, n≥29 neurite tips from 4–6 neurons per condition, ***P<0.001 compared to wild-type, two-way ANOVA Bonferroni post test. (C) DRG neurons expressing wild-type or G71R p150^Glued were imaged at 2 DIV. The retrograde flux of LAMP1-RFP from the neurite tip was measured following photobleaching of a zone 10 μm proximal to the end of the neurite. Entry of cargos from the distal tip into this bleach zone was assessed with time-lapse imaging. Images were acquired at 2 frames per second for 5 seconds pre-photobleaching and 120 seconds post-photobleaching. Kymographs of the photobleached zone were made prior to and subsequent to photobleaching to assess the retrograde flux of cargo from the distal neurite. (D) Retrograde vesicle flux was determined by counting the number of retrograde vesicles that moved at least 3.5 μm into the photobleached zone. The Perry syndrome (G71R) mutation dominantly decreased retrograde flux from the distal neurite. Mean ± SEM, n=14–16 neurites from 8 neurons per condition, **P<0.01 Student's t-test. (E) Model of how the Perry syndrome and HMN7B mutations differentially disrupt dynactin function. The wild-type model is redrawn from Figure 5C. The Perry syndrome mutations decrease distal dynactin accumulation and reduce the flux of cargo from the neurite tip. The HMN7B mutation destabilizes dynactin, which decreases the association between dynein and dynactin and disrupts transport throughout the axon.