Figure 5. The CAP-Gly domain enhances the retrograde flux of cargo from the distal axon.

DRG neurons were imaged at 4 DIV after transfection with siRNA to deplete endogenous p150^Glued and rescued with either wild-type or ΔCAP-Gly p150^Glued. The retrograde flux of LAMP1-RFP cargo from the end of the neurite was measured following photobleaching of a zone 10 μm proximal to the neurite end. 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. (A) Kymographs of the photobleached zone were made prior to and subsequent to photobleaching to assess the retrograde flux of cargo from the distal neurite. Scale bars for the x and y-axes represent 5 μm and 20 seconds, respectively. On the right, for illustrative purposes, the retrograde moving cargos were traced over in blue, the time of photobleaching is marked in red and the point where flux measurements were made is in orange. (B) Retrograde vesicle flux was determined by counting the number of retrograde vesicles that moved at least 3.5 μm into the photobleached zone from the distal neurite. Rescue with ΔCAP-Gly p150^Glued significantly decreased the flux from the distal neurite as compared to either rescue with wild-type p150^Glued or control neurons not treated with siRNA. Mean ± SEM, n>11 neurites from 6–14 neurons per condition, **P<0.01, compared to wild-type, one-way ANOVA Bonferroni post test. (C) Model for the function of the CAP-Gly domain of p150^Glued in neurons. The CAP-Gly domain is necessary to distally enrich p150^Glued in neurite tips and facilitate the flux of cargo from the neurite end. Also see Figure S4.