We used siRNAs to deplete endogenous p150Glued, and achieved 60% knockdown as compared to neurons treated with scrambled control siRNAs (Figures S1A–S1C). Depletion of p150Glued did not significantly disrupt neurite outgrowth, selleckchem similar
to knockdown of dynein (He et al., 2005), likely due to the gradual loss of the target proteins. We used LAMP1-RFP to monitor lysosome dynamics in DRG processes, which have a uniform MT polarity with plus ends oriented distally as assessed by EB3 imaging (Figure S1D). Quantitative analysis indicated that the motility of LAMP1-RFP-labeled organelles was not different from that of organelles labeled with LysoTracker (data not shown). Depletion of p150Glued resulted in a significant decrease in the motility of both anterograde and retrograde cargos, with a corresponding increase in the non-motile fraction compared to scrambled siRNA-treated neurons (Figures 1D and 1E). These data show that the p150Glued subunit of the dynein-dynactin complex is necessary
for the bidirectional motility of lysosomes along the axon, consistent with previous studies demonstrating the reciprocal dependence of dynein and kinesin motors (Hendricks et al., 2010, Martin et al., 1999 and Waterman-Storer et al., 1997). Next, we asked if expression of p150Glued lacking the CAP-Gly domain, ΔCAP-Gly, could rescue the arrest in motility caused by the knockdown of endogenous p150Glued, as compared to rescue with the full-length protein. We used a bicistronic vector to simultaneously and independently express both siRNA-resistant p150Glued Talazoparib and GFP, a transfection marker. Expression of either wild-type or ΔCAP-Gly p150Glued fully
rescued the disruption in motility caused by the knockdown of p150Glued. No significant differences in the fraction of anterograde, retrograde or nonmotile events were observed among the scrambled control, wild-type, and ΔCAP-Gly rescue experiments (Figures 1D and 1E; Movie S2). Analysis of individual tracks from the kymographs showed no difference in mean instantaneous velocities in either the anterograde or retrograde direction between wild-type and ΔCAP-Gly-expressing neurons, nor did we Mephenoxalone observe a significant difference in the number of pauses per track or the number of motility switches per track (Figures S1E–S1G). Additionally, we observed no change in the total number, apparent size or distribution of the lysosomes in the axon. Together, our data demonstrate that while dynactin is required, the CAP-Gly domain of p150Glued is not necessary for processive motility along the axon in primary neurons. Since the CAP-Gly domain of p150Glued does not contribute to the processive motility of cargos along the axon, we investigated other possible functions of the domain. In fungi, dynein and dynactin are enriched at hyphal tips (Lenz et al., 2006).