Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA - PubMed (original) (raw)
Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA
John Salogiannis et al. J Cell Biol. 2016.
Abstract
Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast with this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we found that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging, we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport in which peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.
© 2016 Salogiannis et al.
Figures
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 _pxdA_Δ 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 [_pxdA_Δ]). (H) Histogram of instantaneous velocities of EEs in WT versus _pxdA_Δ 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 [_pxdA_Δ]).
Figure 2.
Microtubule-based movement of PxdA colocalizes with moving peroxisomes. (A) Representative kymograph generated from a movie of PxdA-GFP. (B) Histogram of velocities calculated from PxdA-GFP and PxdA-mKate kymographs. Mean velocity is 2.32 ± 0.88 (SD; n = 328). (C) Bar graph of the number of PxdA-mKate movements calculated as the number of puncta crossing a line drawn perpendicular to the hyphal long axis during a 10-s movie. PxdA puncta moved 5.48 ± 0.62 (SEM) per hyphae in the DMSO control (C; n = 21 hyphae), 0.33 ± 0.16 per hyphae in the presence of benomyl (Ben; n = 15), and 4.81 ± 0.57 per hyphae in the presence of latrunculin A (Lat A; n = 16). Benomyl but not latrunculin A treatment was significantly different than the control (one-way analysis of variance, Bonferroni post hoc test, *, P < 0.001). (D) Representative kymographs generated from simultaneous time-lapse movies of peroxisomes (Pex) and PxdA-GFP. 65.6% (n = 29) of moving peroxisomes overlapped with PxdA. Bars: (x axis) 5 µm; (y axis) 5 s. Left panels correspond to
Video 5
. (E) Stills from
Video 6
of peroxisomes (Pex) and PxdA-GFP. White arrows point to a co-migrating PxdA puncta and peroxisome. Bar, 5 µm. (F) Normalized intensity line scans of peroxisomes and PxdA during comigrating runs (n = 11).
Figure 3.
EEs colocalize with PxdA and moving peroxisomes. (A) Representative kymographs generated from simultaneous time-lapse movies of PxdA-mKate and GFP-RabA/5a–labeled EEs. (B) Bar graph quantifying the colocalization of PxdA-mKate and EEs. EEs colocalized with 54.8% ± 3.8% (SEM) PxdA puncta (n = 20 kymographs from 10 cells). PxdA colocalized with 89.7% ± 2.0% EEs (n = 20 kymographs from 10 cells). (C) Representative micrographs of PxdA-mKate distribution in WT versus hookAΔ hyphae. (D) Normalized line scans of WT and hookAΔ hyphae (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.64 and 1.92 µm; n = 5 hyphae per genotype). (E) A kymograph generated from a simultaneous time-lapse movie of peroxisomes (pex) and EEs. 71.4% of moving peroxisomes (n = 28) overlapped with EEs. Bars: (x axis) 5 µm; (y axis) 5 s. (F) Stills from
Video 8
of peroxisomes (Pex) and EEs. White arrows point to a co-migrating EE and peroxisome. Bar, 5 µm. (G) Normalized intensity line scans of peroxisomes and EEs during comigrating runs (n = 11).
Figure 4.
Coiled-coil region of PxdA is necessary and sufficient for peroxisome motility and endosomal hitchhiking. (A) Schematic of PxdA protein constructs used in this analysis relative to FL PxdA. UR1 (white) and UR2 (yellow) are on the N and C terminus, respectively. Predicted regions of coiled-coil (CC1, 2, and 3) are indicated in orange. All strains contain a C-terminal fluorescent protein tag (either mKate or TagGFP). (B) Normalized line scans of peroxisome distribution from the hyphal tip for FL, C-terminal deletion (ΔCT), N-terminal deletion (ΔNT), and ΔUR2 PxdA strains (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.51 and 3.71 µm for FL vs. ΔCT; n = 51–54 hyphae per genotype). (C) Bar graph of the flux of peroxisome movements in FL, ΔCT, and ΔUR2 hyphae. Peroxisome movements were 3.69 ± 0.47 (SEM)/min for FL, 0.44 ± 0.14/min for ΔCT, and 3.10 ± 0.50/min for ΔUR2 (P < 0.0001, one-way analysis of variance; *, Bonferroni post hoc test significant for FL vs. ΔCT; FL vs. ΔUR2 was not significantly different; n = 29–35 hyphae per genotype). (D) Normalized line scans of peroxisome distribution along hyphae expressing either FL or CC1–3 PxdA (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 1.19 and 3.03 µm from hyphal tip; n = 78 [FL] and n = 68 [CC1–3]). (E) Normalized line scans of peroxisome distribution along hyphae expressing either FL or ΔCC2/3 PxdA (P < 0.0001, two-way analysis of variance, Bonferroni post hoc test significant between 0.87 and 4.11 µm from hyphal tip; n = 28 [FL] and n = 32 [ΔCC2/3]). (F) Bar graph of the flux of peroxisome movements in FL and ΔCC2/3 hyphae. Peroxisome movements were 3.69 ± 0.47 (SEM) per min for FL (this is the same FL data depicted in C) and 0.51 ± 0.13 per min for ΔCC2/3 (P < 0.0001, one-way analysis of variance; *, Bonferroni post hoc test significant for FL vs. ΔCC2/3; n = 29–35 hyphae per genotype). (G) Representative kymographs generated from simultaneous time-lapse movies of EEs with FL or ΔCC2/3 PxdA. Colocalization represents the number of EEs that colocalize with PxdA. FL is 54.8% ± 3.8% (Fig. 3 B) and ΔCC2/3 3.2% ± 1.3% (n = 10 kymographs). (H) Model for peroxisome (P) hitchhiking on EEs mediated by PxdA. HookA recruits the transport machinery to EEs. PxdA is required to tether EEs to peroxisomes and may have distinct binding partners (x) on each organelle. The arrow depicts the direction of movement.
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