Fis1 is associated with highly purified peroxisomes and is induced during peroxisome proliferation. (A) Highly purified peroxisomes were isolated from rat liver. Distribution of peroxisomal and mitochondrial marker enzyme activities after OptiPrep gradient centrifugation of a crude peroxisomal fraction. Relative concentration = (U ×∑ΔV) (∑U ×ΔV)^–1. ΔV represents the volume of a single fraction, U its corresponding enzyme activity, ∑ΔV the total gradient volume, and ∑U the total units recovered from all gradient fractions. CAT, catalase; CytcOx, cytochrome c oxidase. (B) Left, localization of Fis1 in the peroxisomal (Po) and mitochondrial (Mit) fractions. Equal amounts of protein (5 μg/lane) from a single preparation were run on 12.5% acrylamide gels, blotted onto nitrocellulose membranes, and incubated with antibodies to hFis1, PMP70, porin, and ATP synthase α (ATPsyn). In the mitochondrial fraction, an additional band of ∼45 kDa was detected with the hFis1 antibody when higher amounts of protein were applied (20 μg/lane) (immunoblot obtained with another organelle preparation tested negative for mitochondrial contamination). Right, highly purified peroxisomes were separated into a matrix (Ma) and a membrane fraction (Mb). Proteins (PMP70, CAT, 4 μg/lane; hFis1, ATPsyn, porin, 10 μg/lane) were immunoblotted using antibodies to PMP70, catalase (CAT), hFis1, porin, or ATP synthase α. (C) Highly purified peroxisomes from controls (–) (Co) and rats treated with the peroxisome proliferator bezafibrate (+) (Beza) were isolated and immunoblotted using antibodies to peroxisomal acyl-CoA oxidase (AOX), catalase (CAT) or hFis1. Amounts of protein loaded onto the gel: AOX, CAT, 3 μg/lane; hFis1, 6 μg/lane. For hFis1, immunoblots loaded with different amounts of protein (5–35 μg/lane) were quantitated and are expressed as means ± SD. The positions of molecular mass markers (in kilodaltons) are indicated on the right. For acyl-CoA oxidase, only the B subunit (52 kDa) is shown.

Localization of hFis1 to peroxisomes requires an intact C terminus. COS-7 cells were transfected with hFis1-Myc (A and B), with Myc-hFis1-ΔC (C and D), or with hFis1-YFP-TM/C (E and F), and immunostained with antibodies to the Myc tag of hFis1 (A and C) and to PMP70 (B, D, and F). The inset in E shows a mitochondrial aggregate induced by the expression of YFP-hFis1-TM/C. Arrows in F point to peroxisomal aggregates. N, nucleus. Bars, 10 μm.

Inhibition of DLP1 function interferes with peroxisomal fission induced by hFis1 expression. COS-7 cells were cotransfected with Myc-hFis1 and GFP-DLP1-K38A (A–D) and immunostained with antibodies to the Myc tag of hFis1 (A) or to PMP70 (C). The corresponding GFP fluorescence of DLP1-K38A is shown in B and D. Note the pronounced elongation of peroxisomes in DLP1-K38A coexpressing cells (arrows). A quantitative analysis of peroxisome morphology is shown in E. Cells were categorized as cells with tubular (tub) or punctiform (punc) peroxisomes (percentage of total) (see Materials and Methods); *p < 0.01, # p < 0.05 compared with controls. Con, untransfected control. Asterisks, coexpressing cells. N, nucleus. Bars, 10 μm.

Fis1 localizes to peroxisomes and mitochondria in mammalian cells. Normal mitochondrial (A) and peroxisomal (B) morphology in COS-7 control cells processed for immunofluorescence microscopy. Mitochondria in A are visualized by expression of a Mito-GFP construct, whereas peroxisomes (B) are stained with antibodies to PMP70, a peroxisomal membrane protein. GFP-tagged (C–G) and Myc-tagged (H–J) hFis1 protein (GFP-hFis1, Myc-hFis1) colocalizes with peroxisomes. COS-7 cells were transfected with either GFP-hFis1 (C–G) or Myc-hFis1 (H–J) and processed for immunofluorescence microscopy using antibodies to PMP70 (D, F, and I) and the Myc epitope tag (H). Note that the expression of GFP-hFis1 causes aggregation of mitochondria around the nucleus (C), whereas expression of Myc-hFis1 induces their fragmentation (H). (E–G) Higher magnification view of an area close to the nucleus in a COS-7 cell transfected with GFP-hFis1. The asterisks in E and G mark the beginning of a mitochondrial aggregate. (G) Overlay (merge) of E and F. (J) Overlay and higher magnification view of boxed region in H and I. Myc-hFis1/TRITC (red), PMP70/FITC (green). (K and L) Detection of endogenous Fis1 (L) in COS-7 cells expressing a GFP-PTS1 construct (K). Arrows highlight some regions of colocalization. N, nucleus. Bars, 10 μm (A–D, H–L); 5 μm (E–G).

hFis1 promotes peroxisomal fission. COS-7 cells were transfected with Myc-hFis1 (A–E) and immunostained with antibodies to Myc (A and E) and PMP70 (B–E). Cells expressing Myc-hFis1 contained fragmented mitochondria (A). Note that in A, contrast and brightness have been optimized for the visualization of mitochondrial morphology and that therefore not all Myc-hFis1 positive peroxisomes are visible. Arrows in A and B point to some regions of colocalization. Peroxisomes were observed to segment and to separate into numerous punctiform organelles in Myc-hFis1expressing cells (C, D, and F). Note the normal-sized peroxisomes in the untransfected cell on the left (C). (D) Higher magnification view of separating peroxisomes (arrows). (E) Colocalization of Myc-hFis1 and PMP70 on tubular peroxisomes with beginning segmentation. (F) Quantitative evaluation of peroxisome morphology. Cells were categorized as cells with segmented (seg) and punctiform (punc) peroxisomes (% of total) (see Materials and Methods). Note the high frequency of small, punctiform peroxisomes in cells expressing Myc-hFis1. p < 0.01 compared with controls. Con, control (untransfected and vector only). Asterisks in C highlight a transfected cell. N, nucleus. Bars, 10 μm (A–D); 5 μm (E).

Silencing of Fis1 induces elongation of peroxisomes. COS-7 cells were transfected with Fis1 siRNA duplexes (siRNA) and processed for immunofluorescence (A–F) and immunoblotting (H) with anti-hFis1 antibodies. (A and B) Mitochondrial morphology in control cells (Con) expressing Mito-GFP (A), and in cells silenced for Fis1 (B). (C–F) Peroxisome morphology in control cells (Con) expressing GFP-PTS1 (C), and in cells silenced for Fis1 (E). Note the elongated, segmented peroxisomes in E (arrows). (D and F) Staining of Fis1 in control cells (D) and after transfection with Fis1 siRNA duplexes (F). (G) Quantification of peroxisome morphology after silencing of Fis1. Cells were categorized as cells with spherical (sph) or elongated (elong) peroxisomes (percentage of total) (see Materials and Methods); p < 0.01 compared with controls. (H) Immunoblots of homogenates prepared from controls (Con) and transfected cells (siRNA) using anti-hFis1 and anti-tubulin (Tub) antibodies. Equal amounts of protein (hFis1, 50 μg/lane; Tub, 20 μg/lane) were loaded onto the gels. Anti-tubulin was used to check for equal loading and integrity of the cells after transfection. N, nucleus. Bars, 10 μm.

Coexpression of hFis1 and Pex11pβ influences the intracellular distribution of peroxisomes. COS-7 cells were cotransfected with Mito-GFP and Pex11pβ-Myc (A and B), with Mito-GFP and untagged hFis1(hFis1-UT) (C and D), with Mito-GFP, Pex11pβ-Myc and untagged hFis1(hFis1-UT) (E and F), or with GFP-hFis1 and Pex11pβ-Myc (G and H). Twenty-four hours after transfection, cells were immunostained with anti-Myc (B, F, and H) or anti-PMP70 (D) antibodies. Note the juxtanuclear clustering of peroxisomes and their association with mitochondria in cells coexpressing hFis1 and Pex11pβ (E–H). N, nucleus. Bars, 10 μm.