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First published online 31 January 2006
doi: 10.1242/jcs.02776

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Failure of microtubule-mediated peroxisome division and trafficking in disorders with reduced peroxisome abundance

¹ Cell Biology Group, Eskitis Institute for Cell and Molecular Therapies, Griffith University, 170 Kessels Road, Nathan, Brisbane, Queensland 4111, Australia
² School of Biomolecular and Biomedical Science, Griffith University, 170 Kessels Road, Nathan, Brisbane, Queensland 4111, Australia
³ Department of Genetic Medicine, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia, 5006, Australia
⁴ Department of Pediatrics, University of Adelaide, Adelaide, South Australia, 5005, Australia

View larger version (39K): [in a new window]   Fig. 1. PEX14 localizes peroxisomal structures in mammalian cells. (A) Western blot analysis of mouse liver fractions. T, total liver homogenate; P, organelle pellet; S, Post-organellar supernatant. +/+, wild-type mice; -/-, PEX13-null mice. (B) Immunofluorescence microscopy of a normal human skin fibroblast transfected with plasmid expressing EGFP-PTS1 fusion protein. Green, EGFP; red, PEX14. Bar, 20 µm.

View larger version (46K): [in a new window]   Fig. 2. Peroxisomes align with, and are dispersed along, microtubules in cultured fibroblasts. (A) Immunofluorescence microscopy of peroxisomes and microtubules in control HSFs (top) and wild-type MEFs (middle). Bottom panels show confocal laser-scanning microscopy of a HSF. Green, PEX14; red, -tubulin. (B) HSFs (top panels) and wild-type MEFs (bottom panels) treated with 20 µM nocodazole for 20 hours. PEX14 (green); -tubulin (red). (C) Mitochondria (green) and microtubules (red) detected using MitoTracker Green FM dye and -tubulin antibody, respectively, in untreated (-NCZ, top panels) and nocodazole treated (+NCZ, bottom panels) HSFs. Bars, 20 µm (top and middle panel in A; and B and C); 5 µm (bottom panel in A).

View larger version (33K): [in a new window]   Fig. 3. Peroxisome abundance and distribution along peripheral microtubules are perturbed in PEX1-null ZS cells. (A) PEX1-null patient HSFs processed with antibodies against PEX14 (green) and -tubulin (red). Top panels, minimal retraction of remnant peroxisomes; middle panels, pronounced remnant peroxisome retraction, with initial signs of clustering; bottom panels, dramatic clustering of remnant peroxisomes. Arrow indicates MTOC, corresponding to microtubule nucleation/anchor region. (B) Confocal laser-scanning microscopy showing remnant peroxisomes aligning (white arrowhead) or not aligning (white arrows) with microtubules. (C) PEX1-null cells stained for remnant peroxisomes using PEX14 antibody (green), and for lysosomes using LysoTracker Red DND-99 (red). (D) PEX1-null HSF cells stained for mitochondria using MitoTracker Green FM and MitoTracker Red CM-H2XRos dyes. Bars, 20 µm (A,C,D); 5 µm (B).

View larger version (62K): [in a new window]   Fig. 4. Peroxisome abundance and distribution along peripheral microtubules are perturbed in fibroblasts from patients with IRD but not RCDP. Abundance and distribution of remnant peroxisomes along microtubules of cultured patient HSFs from patients with IRD (A) and RCDP (B), assessed by immunofluorescence using PEX14 (green) and -tubulin (red) antibodies. Bars, 20 µm.

View larger version (44K): [in a new window]   Fig. 5. Cultured MEFs and neurons from PEX13-null mice exhibit decreased remnant peroxisome abundance and distribution along peripheral microtubules. (A) PEX13-null MEFs and (B) PEX13 heterozygote (top panel) and PEX13-null (bottom panel) neurons (elongated cells in each case), assessed by immunofluorescence using PEX14 (green) and -tubulin (red) antibodies. Bars, 20 µm.

View larger version (43K): [in a new window]   Fig. 6. The single-enzyme disorder, D-BP deficiency, is also characterized by decreased cellular peroxisome abundance and distribution along peripheral microtubules. (A) Peroxisome abundance and distribution along microtubules in HSFs from a D-BP-deficient patient. (B) Confocal laser-scanning microscopic image of a D-BP-deficient cell showing alignment of residual peroxisomes with microtubules. (C) Abundance and distribution of peroxisomes along microtubules in HSFs from an X-ALD patient. PEX14, green; -tubulin, red. Bars, 20 µm.

View larger version (72K): [in a new window]   Fig. 7. PEX11ß-myc overexpression leads to remnant peroxisome proliferation and redistribution along microtubules in PEX1-null and D-BP deficient cells. (A-C) PEX1-null and (D-F) D-BP-deficient patient HSFs stably expressing human PEX11ßmyc. For epifluorescence microscopy, cells were double-labeled with PEX14 antibody (red) and myc antibody (green) (A,B,D,E). Panels B and E are magnifications of regions shown in overlay panels in A and D, respectively. For confocal laser-scanning microscopy, cells were double-labeled with PEX14 antibody (green) and -tubulin antibody (red) (C and F). Bars, 20 µm (A,C,D,F); 5 µm (B,E).

View larger version (96K): [in a new window]   Fig. 8. PEX11ßmyc overexpression recruits DLP1 to peroxisomal structures in PEX1-null and D-BP-deficient cells. (A) Confocal laser-scanning microscopy of untransfected PEX1-null fibroblasts, and fibroblasts from patients deficient in D-BP double-labeled with PEX14 antibody (red) and DLP1 antibody (green). (B) Confocal laser-scanning microscopy of PEX1-null and D-BP-deficient cells stably expressing PEX11ßmyc, and double labeled with c-myc antibody (green) and DLP1 antibody (red). Bars, 20 µm (A); 5 µm (B).