![Figure 1. pxdA is required for the distribution and movement of peroxisomes, but not EEs or nuclei. (A) Representative micrographs of WT and pxdAΔ hyphae expressing fluorescent proteins that label peroxisomes (mCherry-PTS1), EEs (GFP-RabA/5a) or nuclei (Histone H1–TagGFP). (B) Cartoon of an A. nidulans hypha (top). Dynein moves away from the hyphal tip, whereas kinesin-3 moves in the opposite direction. Peroxisome (magenta) and EE (green) distribution in hyphae was quantified from line scans of fluorescence micrographs and displayed as the mean (solid lines) ± SEM (shading) fluorescence intensity as a function of distance from the hyphal tip. Peroxisome distribution near the hyphal tip was significantly different between WT and pxdAΔ (P < 0.001, two-way analysis of variance, Bonferroni post hoc test significant between 0.65 and 3.25 µm from hyphal tip; n = 40 hyphae [WT] and n = 44 [pxdAΔ]). EE distribution was not significantly different between WT and pxdAΔ (P = 0.99, two-way analysis of variance; n = 51 hyphae [WT] and n = 33 [pxdAΔ]). Nuclear distribution was quantified by measuring the distance from the tip-proximal nucleus to the hyphal tip. The mean (blue vertical lines) ± SEM (blue shading) was 8.47 ± 0.39 µm in WT and 7.66 ± 0.42 µm in pxdAΔ hyphae. The means were not significantly different between WT and pxdAΔ (P = 0.1073, Mann-Whitney test; n = 31 hyphae [WT] and n = 31 [pxdAΔ]). (C) Representative kymographs generated from movies of peroxisomes in WT and pxdΔ hyphae. (D) Bar graph of the flux of peroxisome movements in WT and pxdAΔ hyphae, calculated as the number of peroxisomes crossing a line drawn perpendicular and 10 µm away from the hyphal tip during a 1-min time-lapse movie. Peroxisome movements were 7.17 ± 0.79 (SEM) per minute in WT and 0.52 ± 0.10 per minute in pxdAΔ hyphae (*, P < 0.0001, Mann-Whitney test; n = 44 hyphae for both genotypes). (E) Histogram of instantaneous velocities of moving peroxisomes in WT versus pxdAΔ strains. Mean velocities were 1.75 ± 0.99 (SD) µm/s in WT and 0.97 ± 0.68 µm/s in pxdAΔ hyphae (P < 0.001, Kolmogorov-Smirnov test; n = 1,195 instantaneous velocities [WT] and n = 136 [pxdAΔ]). (F) Kymographs generated from movies of EEs in WT and pxdAΔ hyphae. (G) Bar graph of the flux of EE movements during a 10-s movie calculated as in D. EE movements were 9.34 ± 0.78 (SEM) per min in WT and 7.60 ± 0.48 per min in pxdAΔ hyphae (P = 0.0920, Mann-Whitney test; n = 29 hyphae [WT] and n = 47 hyphae [pxdAΔ]). (H) Histogram of instantaneous velocities of EEs in WT versus pxdAΔ strains. Mean velocities are 2.40 ± 0.99 (SD) µm/s in WT and 2.24 ± 0.93 µm/s in pxdAΔ hyphae (P = 0.0274, Kolmogorov-Smirnov test; n = 1,602 instantaneous velocities [WT] and n = 332 [pxdAΔ]).](https://cdn.rupress.org/rup/content_public/journal/jcb/212/3/10.1083_jcb.201512020/6/jcb_201512020_fig1.jpeg?Expires=1773527188&Signature=Q6cCKAjtunN~oPocvC7gG~ZvBzmtoV0xc0GiV~GA-8lW1YqfOWqB4hZ2bTI1KOgQwAwPvG1K7MCKkiIsJI8tLcuqMNhecJHJVSqN4UWzyMfqlCEbjHkUOJ2qS4gexqpjIvqMVQXz7GmRmAdH2oiXz-hBlMt~vgIZcwvkVhlCZ1JKPlG-QyqqfhZ6oWYyB4Gq9o-fYlMxjulFbJ3~jREeOheituBlkKLC~9rlPO7tR0p2sXU5m7R4ABFJn5IFbZd2tIPRa7VscmgVn5gt5yunaiH5gJBcXKyuXWKLXIeMnZ679PqWBGBC3m9zzlDWqpv60sHhP4DO-4YQiZcHDcmo1Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
pxdA is required for the distribution and movement of peroxisomes, but not EEs or nuclei. (A) Representative micrographs of WT and pxdAΔ hyphae expressing fluorescent proteins that label peroxisomes (mCherry-PTS1), EEs (GFP-RabA/5a) or nuclei (Histone H1–TagGFP). (B) Cartoon of an A. nidulans hypha (top). Dynein moves away from the hyphal tip, whereas kinesin-3 moves in the opposite direction. Peroxisome (magenta) and EE (green) distribution in hyphae was quantified from line scans of fluorescence micrographs and displayed as the mean (solid lines) ± SEM (shading) fluorescence intensity as a function of distance from the hyphal tip. Peroxisome distribution near the hyphal tip was significantly different between WT and pxdAΔ (P < 0.001, two-way analysis of variance, Bonferroni post hoc test significant between 0.65 and 3.25 µm from hyphal tip; n = 40 hyphae [WT] and n = 44 [pxdAΔ]). EE distribution was not significantly different between WT and pxdAΔ (P = 0.99, two-way analysis of variance; n = 51 hyphae [WT] and n = 33 [pxdAΔ]). Nuclear distribution was quantified by measuring the distance from the tip-proximal nucleus to the hyphal tip. The mean (blue vertical lines) ± SEM (blue shading) was 8.47 ± 0.39 µm in WT and 7.66 ± 0.42 µm in pxdAΔ hyphae. The means were not significantly different between WT and pxdAΔ (P = 0.1073, Mann-Whitney test; n = 31 hyphae [WT] and n = 31 [pxdAΔ]). (C) Representative kymographs generated from movies of peroxisomes in WT and pxdΔ hyphae. (D) Bar graph of the flux of peroxisome movements in WT and pxdAΔ hyphae, calculated as the number of peroxisomes crossing a line drawn perpendicular and 10 µm away from the hyphal tip during a 1-min time-lapse movie. Peroxisome movements were 7.17 ± 0.79 (SEM) per minute in WT and 0.52 ± 0.10 per minute in pxdAΔ hyphae (*, P < 0.0001, Mann-Whitney test; n = 44 hyphae for both genotypes). (E) Histogram of instantaneous velocities of moving peroxisomes in WT versus pxdAΔ strains. Mean velocities were 1.75 ± 0.99 (SD) µm/s in WT and 0.97 ± 0.68 µm/s in pxdAΔ hyphae (P < 0.001, Kolmogorov-Smirnov test; n = 1,195 instantaneous velocities [WT] and n = 136 [pxdAΔ]). (F) Kymographs generated from movies of EEs in WT and pxdAΔ hyphae. (G) Bar graph of the flux of EE movements during a 10-s movie calculated as in D. EE movements were 9.34 ± 0.78 (SEM) per min in WT and 7.60 ± 0.48 per min in pxdAΔ hyphae (P = 0.0920, Mann-Whitney test; n = 29 hyphae [WT] and n = 47 hyphae [pxdAΔ]). (H) Histogram of instantaneous velocities of EEs in WT versus pxdAΔ strains. Mean velocities are 2.40 ± 0.99 (SD) µm/s in WT and 2.24 ± 0.93 µm/s in pxdAΔ hyphae (P = 0.0274, Kolmogorov-Smirnov test; n = 1,602 instantaneous velocities [WT] and n = 332 [pxdAΔ]).
