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. 2016 Oct 29;25(23):5059–5068. doi: 10.1093/hmg/ddw310

Figure 4.

Figure 4.

Axonal mitochondrial transport defects in MNs of FUS-transgenic flies as detected by the mitochondrial imaging in MN axons in the ventral nerve cord (VNC). (A) Representative kymographs of axonal mitochondria labeled by mito-GFP in MNs of control (Ctr) or transgenic flies expressing either Wt- or P525L-mutant FUS. A 40-μm axonal region was photo-bleached before image acquisition and video recorded for 6 min with VNC placed on the left of the acquired images. The anterograde (plus end-directed) and retrograde (minus end-directed) trafficking of mitochondria are marked in red and blue respectively. Scale bar: 5 μm. (B–H) Quantification of mitochondrial transport in MN axons of flies expressing Wt- or P525L-mutant FUS as compared with the control flies. Images were processed and quantified using ImageJ. (B, C) show duty cycles of the mitochondria that exhibit net anterograde movement (AM) and retrograde movement (RM). Duty cycles of individual mitochondria are divided into anterograde run (AR), retrograde run (RR), and stop (ST). A significant decrease in the time spent on moving (AR in B and RR in C, respectively) together with a significant increase in time spent pausing is detected in flies expressing either Wt- or P525L-mutant FUS as compared with the control flies. (D) The velocity of moving mitochondria in the anterograde and retrograde runs (AR and RR respectively). Flies expressing either Wt- or P525L-mutant FUS showed significantly reduced velocity of RR mitochondria, as compared with the control flies. (E, F) Duration of individual mitochondrial running or pausing events in different fly groups. Duration of mitochondrial anterograde movement (AM) in flies expressing the P525L-mutant FUS is reduced (E); whereas the duration of retrograde movement (RM) was decreased in flies expressing either Wt- or P525L-mutant FUS as compared with the control group. (G, H). The frequency of reversal of mitochondrial movement in AM or RM. The reversal of AM mitochondria was increased in flies expressing either Wt- or P525L-mutant FUS; whereas the P525L-mutant FUS group showed a significant increase in the frequencies of both stop and reversal of mitochondrial RM. At least 120 mitochondria from > 10 fly larvae were analyzed for each group. Data were analyzed using one-way ANOVA (*, P < 0.05; ***, P < 0.001). Fly genotypes, Ctr: D42-Gal4/UAS-mitoGFP/UAS-RFP; Wt: D42-Gal4/UAS-mitoGFP/UAS-Wt-hFUS-RFP; P525L: D42-Gal4/UAS-mitoGFP/UAS-P525L-hFUS-RFP.