Neither the Perry syndrome (G71R, Q74P) nor HMN7B (G59S) mutations showed any distal enrichment at the neurite tip compared to expression of wild-type p150Glued (Figures 8A and 8B). Significant differences in the accumulation of wild-type p150Glued compared to the Perry syndrome and
HMN7B mutations occur over the first 14 μm from the neurite tip; however, expression of the mutants did not alter neurite morphology. These data further support the conclusion that both the Perry syndrome and HMN7B mutations disrupt CAP-Gly function. For the HMN7B mutation, however, it is unclear if the decreased distal accumulation is caused by a decreased affinity for EBs, or is due to bidirectional inhibition of transport caused by expression of this protein, Z-VAD-FMK in vitro as anterograde transport is also required to establish the distal dynactin pool (Figure 3). The accumulation of distal dynactin increases the efficient initiation of transport from the distal neurite (Figure 5). Therefore, the decreased distal accumulation caused by expression of the Perry syndrome mutations suggests that this will in turn cause decreased cargo efflux from
the neurite tip. We tested this by photobleaching a region 10 μm proximal to the end of the neurite and observed the retrograde flux into the photobleached BYL719 zone (Figure 8C). Expression of the G71R Perry syndrome mutation had a dominant-negative effect and significantly disrupted retrograde flux, as compared to overexpression of wild-type p150Glued (Figure 8D). These data suggest that the primary pathogenic mechanism either in Perry syndrome is a decrease in the efficiency of retrograde transport from the distal axon (Figure 8E). We have demonstrated a required function of the conserved CAP-Gly domain of dynactin in facilitating the efficient initiation
of transport from the distal axon. We show that the CAP-Gly domain of p150Glued is necessary to enrich dynactin in distal neurites and that this enrichment promotes the flux of cargo out of the neurite tip. Kinesin-1 delivers dynactin to the distal neurite, while EBs retain dynactin distally and may also promote the initiation of transport by recruiting dynactin onto the MT plus end. Once transport is initiated, the CAP-Gly domain is not necessary for transport of cargo along the axon. The identification of the CAP-Gly motif of dynactin as an independent MT-binding domain initially suggested that it might act to enhance the processivity of the dynein motor (Hendricks et al., 2010, King and Schroer, 2000, Ross et al., 2006 and Waterman-Storer et al., 1995).
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