DHA restores peroxisome morphogenesis in AOx- and D-BP-defective fibroblasts. (A) Fibroblasts defective in AOx (Aa–Ad) or D-BP (Ae–Ah) were cultured in the absence (Aa,Ae) or presence of 50 μM DHA for 12 hours (Ab,Af), 24 hours (Ac,Ag) or 48 hours (Ad,Ah), and then stained with an antibody to Pex14p. (B) Peroxisome abundance per cell following treatment with 50 or 150 μM DHA for the indicated times was determined as described in Materials and Methods. (C) AOx-deficient fibroblasts were cultured with 50 μM DHA for 0, 3, 6, 9, 12 and 24 hours. Peroxisome morphology was verified using an antibody to Pex14p (left panel). Total DHA levels were analyzed as described for Table 1 (right panel). Values are mol% of total DHA in control fibroblasts. (D) Control (Da,Dc) or PEX10-deficient (Db,Dd) fibroblasts were cultured in the absence (Da,Db) or presence (Dc,Dd) of 150 μM DHA for 48 hours and then stained with an antibody to Pex14p. (E) Fibroblasts from control and individual patients defective in PEX10 and PEX2 were treated with DHA as in A and peroxisome abundance determined. Insets are higher-magnification images of the boxed regions. Scale bars: 10 μm and 2 μm (insets). Data represent the means ± s.d. of three independent experiments; *P<0.05.

Functional role of DLPI in peroxisome proliferation. (A) AOx-deficient fibroblasts were cultured for 16 hours in the absence (Aa,Ab) or presence (Ac,Ad) of 150 μM DHA and then treated for an additional 8 hours with DMSO (Aa,Ac) or 40 μM dynasore (Ab,Ad). Cells were stained with an anti-Pex14p antibody. (B) Peroxisome abundance per cell was determined as in Fig. 2B. (C) AOx-deficient fibroblasts were treated for 48 hours with a control dsRNA (left panel) or DLP1 #2 dsRNA (right panel). Cells were cultured for an additional 24 hours in the absence (Ca,Cb) or presence (Cc,Cd) of 150 μM DHA and then stained with an anti-Pex14p antibody. (D) AOx-deficient fibroblasts were treated for 48 hours with a control dsRNA or two different test dsRNAs (DLP1 #1 and DLP1 #2). DLP1 levels were verified by immunoblot analysis using an anti-DLP antibody. Actin was used as a loading control. (E) Peroxisome abundance per cell was determined as in Fig. 2B. (F) Control (Fa–Fc) and AOx-deficient fibroblasts (Fd–Fi) were cultured for 24 hours in the absence (Fa–Ff) or presence (Fg–Fi) of 150 μM DHA. Cells were stained with antibodies to Pex14p (Fa,Fd,Fg) and DLP1 (Fb,Fe,Fh); the merged view of the two different proteins is shown in Fc, Ff, and Fi. Insets are higher-magnification images of the boxed regions. Scale bars: 10 μm and 2 μm (insets). Data represent the means ± s.d. of three independent experiments; *P<0.01.

DHA induces peroxisome elongation and division in a Pex11pβ-dependent manner. (A) AOx-deficient fibroblasts were treated with a control dsRNA (upper row) or PEX11β #2 dsRNA (lower row) for 48 hours. Cells were cultured for an additional 12 hours (Ab,Ae) and 24 hours (Aa,Ac,Ad,Af) in the absence (Aa,Ad) or presence (Ab,Ac,Ae,Af) of 150 μM DHA and then stained with an anti-Pex14p antibody. Insets are higher-magnification images of the boxed regions. Scale bars: 10 μm and 2 μm (insets). (B) AOx-deficient fibroblasts were treated with control dsRNA or two different test dsRNAs (PEX11β #1 and PEX11β #2) for 48 hours. Pex11pβ levels were verified by immunoblot analysis using an antibody to Pex11pβ. Actin was used as a loading control. (C) AOx-deficient fibroblasts were treated as in A with control, PEX11β #1, PEX11β #2 or PEX11β #1 plus PEX11α #1 dsRNAs. Cells were cultured for an additional 24 hours in the absence or presence of 150 μM DHA. Peroxisome abundance per cell was determined as in Fig. 2B. Data represent the means ± s.d. of three independent experiments; *P<0.01. (D) AOx-deficient, control, and PEX10-deficient fibroblasts were cultured for 20 hours in the absence (left panels) or presence (right panels) of 150 μM DHA and then treated for 30 minutes with 1 mM DSP. Cell lysates were fractionated by sucrose density gradient ultracentrifugation and each fraction analyzed by immunoblot using antibodies to Pex11pβ, DLP1 and Fis1. Upward arrowhead indicates the higher molecular-mass oligomer of Pex11pβ.

In vitro Pex11pβ oligomerization assay. (A) DHA levels in proteoliposomes. SL were prepared by mixing PC and PE. DL were prepared by mixing PDPC and PDPE, and OL by mixing POPC and POPE. DHA levels were determined as described in Materials and Methods. (B) Introduction of an MBP–Pex11pβ fusion protein into liposomes. Liposomes were solubilized with 1% Triton X-100 and then fractionated by ultracentrifugation in a 1–30% glycerol density gradient. Each fraction was analyzed by immunoblot using an anti-MBP antibody. Upward arrowheads indicate the higher molecular mass oligomers of MBP–Pex11pβ.

DHA induces the formation of Pex11pβ-rich regions on elongated peroxisomes. (A) AOx-deficient fibroblasts were cultured in the absence (Aa–Ac) or presence (Ad–Ai) of 150 μM DHA for 12 hours and then stained with antibodies to Pex14p (Aa,Ad,Ag) and Pex11pβ (Ab,Ae,Ah); the merged view of the two proteins is shown in Ac, Af and Ai. Insets are higher-magnification images of the boxed regions. Scale bars: 10 μm and 1 μm (insets). Arrowheads indicate regions enriched with Pex11pβ compared with Pex14p. The numbers 1, 2 and 3 correspond to those in panel B. (B) Mutually distinct peaks of Pex11pβ and Pex14p were apparent upon in-line scanning of the transecting line shown in Ag–Ai. The intensity of Pex11pβ signals was significantly increased at regions (arrowheads) that did not contain Pex14p.

PMP70 and catalase colocalize with Pex14p. (A) AOx-deficient fibroblasts were cultured in the absence (Aa–Ad) or presence (Ae–Al) of 150 μM DHA for 12 hours and then stained with antibodies to Pex14p (Aa,Ae,Ai), PMP70 (Ab,Af,Aj), and catalase (Ac,Ag,Ak); the merged view of the proteins is shown in Ad, Ah and Al. Scale bars: 10 μm and 1 μm (insets). (B) Line-scanning evaluation of the transecting line shown in Ai–Al. Note the similar peaks for Pex14p, PMP70 and catalase. Insets are higher-magnification images of the boxed regions.

Real-time imaging of peroxisome division. (A,B) AOx-deficient fibroblasts were transfected with an expression vector for HA₂-EGFP-AKL. After 4 hours, fibroblasts were supplemented with 150 μM DHA for 20 hours and then analyzed by time-lapse fluorescence microscopy. Images show division (A) and the sequence of steps involved in peroxisome elongation (B). Scale bar: 1 μm.

Association of peroxisomes with microtubules is required for proper distribution but not division. (A) AOx-deficient fibroblasts were cultured for 4 hours in the absence (Aa,Ab) or presence of 150 μM DHA (Ac–Af) followed by the addition of DMSO (Aa–Ad) or 10 μM nocodazole (Ae,Af) for 20 hours. Cells were stained with antibodies to Pex14p (Aa,Ac,Ae) and α-tubulin (Ab,Ad,Af). Arrows indicate peroxisomal clusters. Scale bars: 20 μm. (B) Microtubules are not involved in peroxisomal elongation. AOx-deficient fibroblasts were treated with 10 μM nocodazole for 1 hour and then cultured for 12 hours in the presence of 150 μM DHA. Cells were stained with antibodies to Pex14p (Ba) and α-tubulin (Bb). Insets are higher-magnification images of the boxed regions. Scale bars: 10 μm and 2 μm (inset).