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First published online July 30, 2004
doi: 10.1242/10.1242/jcs.01268

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Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1

¹ Department of Cell Biology and Cell Pathology, Robert Koch Strasse 6, University of Marburg, Marburg, 35037, Germany
² Department of Anatomy and Cell Biology, Robert Koch Strasse 8, University of Marburg, Marburg, 35037, Germany

View larger version (68K): [in a new window]   Fig. 1. Silencing of DLP1 induces elongation of the peroxisomal compartment. COS-7 cells stably expressing GFP-PTS1 were transfected with DLP1 siRNA duplexes (siRNA) and processed for immunofluorescence (A-E) and immunoblotting (G) with anti-DLP1 antibodies 48 hours after the first transfection. (A,B) Control cells (Con) transfected with luciferase siRNA duplexes. (C-E) COS-7 cells transfected with DLP1 siRNA duplexes. The arrow in C points to a peroxisomal aggregate. (E) A high magnification view of elongated peroxisomes. Note the segmented appearance of the organelles (arrowheads). Arrows point to largely elongated, tubular peroxisomes. N, nucleus. (F) Quantification of peroxisome morphology at different time points after transfection with siRNA. The data are from five to seven independent experiments and are expressed as means ± s.d. (P<0.01 when compared to controls). (G) Immunoblots of homogenates prepared from controls treated with buffer (Con) and cells transfected with luciferase (Luc) or DLP1 siRNA duplexes (D1) using anti-DLP1 and anti-tubulin (Tub) antibodies. Homogenates were separated in a supernatant (Sup) and pellet (P) fraction. Equal amounts of protein (DLP1, 70 µg/lane; Tub, 30 µg/lane) were loaded onto the gels. Anti-tubulin was used to check for equal loading and integrity of the cells after transfection. Bars, 10 µm.

View larger version (40K): [in a new window]   Fig. 3. Peroxisomal constriction can occur independently of DLP1. COS-7 cells stably expressing GFP-PTS1 were transfected with DLP1 siRNA duplexes (siRNA) and processed for immunofluorescence with anti-DLP1 antibodies 48 hours post transfection. (A,B) Co-localization of peroxisomes (green) and DLP1 (red). (A) DLP1 is efficiently silenced in cells that took up the DLP1 siRNA (asterisks). In those cells the remaining DLP1 localizes to a few small spots in the cytoplasm but is not associated with the constriction sites on elongated peroxisomes. Note the untransfected, non-silenced cell on the right, which has a high DLP1 protein level and contains spherical instead of elongated, segmented peroxisomes. (B) Higher magnification view of elongated, segmented peroxisomes (arrows) after efficient silencing of DLP1. Note the absence of co-localization of the remaining DLP1 with constricted peroxisomes. DLP1 is not efficiently silenced in the cell on the left (x). N, nucleus. (C) Immunoblots of peroxisomal fractions isolated from controls treated with buffer (Con) and cells transfected with DLP1 siRNA using anti-PMP70 and anti-DLP1 antibodies. Equal amounts of protein (PMP70, 10 µg/lane; DLP1, 45 µg/lane) were loaded onto the gels. Anti-PMP70 was used as a marker for peroxisomes. (D) Quantitation of peroxisome morphology at different times after transfection with a DLP1-WT construct. Cells were immunostained 24 and 48 hours after transfection by electroporation with antibodies to DLP1, and quantified. The data are from four independent experiments and are expressed as means ± s.d. Con, control cells (untransfected and vector only). For quantitative evaluation, cells were categorized as having elongated (el) (% of total), segmented (seg) (% of cells with elongated peroxisomes) or spherical (sph) (% of total) peroxisomes. Bars, 10 µm.

View larger version (136K): [in a new window]   Fig. 2. Ultrastructure of peroxisomes in (A) controls and (B,C) after silencing of DLP1. HepG2 cells were transfected with buffer (Con) (A) or with DLP1 siRNA duplexes (B,C). After 48 hours the cells were processed for cytochemical visualization of peroxisomal catalase and embedded for electron microscopy. (A) Typical single, spherical peroxisomes (Po). (B,C) Peroxisomes after silencing of DLP1. They are arranged like beads on a string and are constricted but still interconnected (arrows). Bars, 500 nm.

View larger version (92K): [in a new window]   Fig. 4. Perturbation of peroxisome morphology by Pex11pß expression after silencing of DLP1. COS-7 cells stably expressing GFP-PTS1 (GFP) were transfected with (A) buffer (Con) or (B-E) DLP1 siRNA duplexes (siRNA), incubated for 24 hours, transfected with a Pex11pß-myc expression vector and another dose of siRNA duplexes, and processed for immunofluorescence after an additional 24 hours. (A,B) Pex11pß-myc staining with antibodies to the myc epitope tag. (C-E) GFP-PTS1 labeling of peroxisomes in Pex11pß-myc-expressing cells. (D) Higher magnification image of boxed region in C. Note the extreme tubulation of peroxisomes (arrows) in cells treated with DLP1 siRNA/Pex11pß-myc, and the absence of peroxisome segmentation. N, nucleus. Bars, 10 µm.

View larger version (66K): [in a new window]   Fig. 5. Effects of DLP1 silencing on the morphology of other intracellular organelles in COS-7 cells. COS-7 cells stably expressing GFP-PTS1 were transfected with buffer (Con) (A,B,E,G,I,K) or with DLP1 siRNA duplexes (siRNA) (C,D,H,J,L) and processed for immunofluorescence 48 hours after the first transfection. (A,C) Visualization of peroxisomes by GFP-PTS1 (GFP). (B,D) Golgi staining with anti-p115 antibodies. (E, F) Co-localization of DLP1 and p115 in untransfected COS-7 cells (not expressing GFP-PTS1). For rER labeling (G-J) cells were transfected with DLP1 siRNA duplexes, incubated for 24 hours, transfected with a Marburg virus glycoprotein (MbVGP) expression vector and another dose of siRNA duplexes, and processed after an additional 24 hours. Cells were incubated with anti-MbVGP antibodies. (I,J) Higher magnification images of boxed regions in G,H. (K,L) Staining of mitochondria with antibodies to MnSOD. Note the largely elongated peroxisomes (C) and mitochondria (L) after silencing of DLP1 compared to the appropriate controls. N, nucleus. Bars, 10 µm.

View larger version (150K): [in a new window]   Fig. 6. Ultrastructure of the Golgi complex in (A) controls and (B) after silencing of DLP1. COS-7 cells were transfected with buffer (Con) (A) or with DLP1 siRNA duplexes (B). Cells were fixed and prepared for electron microscopy 48 hours post transfection. (A) Control cell with a modest concentration of Golgi stacks (arrows). (B) Cells silenced for DLP1 display many clusters of Golgi stacks. Classic cisternae and an extensive array of associated tubules and vesicles are visible (arrows). Bar, 500 nm.

View larger version (82K): [in a new window]   Fig. 7. Influence of the microtubule cytoskeleton on peroxisomal morphology and dynamics after silencing of DLP1. COS-7 cells stably expressing GFP-PTS1 (GFP) were transfected with (A) buffer (Con) or (B-G) DLP1 siRNA duplexes (siRNA) and processed for immunofluorescence (A-D) or live-cell imaging (E-G). Cells in C,D,G were treated with nocodazole to depolymerize microtubules. (A,B,D) Immunofluorescence of COS-7 cells using antibodies to tubulin (Tub). (C) Visualization of peroxisomes labeled with GFP-PTS1 after transfection with DLP1 siRNA and treatment with nocodazole. Arrows point to peroxisomal aggregates. (E-G) Images of the motile behavior of individual, elongated peroxisomes in vivo after silencing of DLP1. Numbers in the top right of the panels show time elapsed in seconds. In E, an elongated peroxisome (arrow) moves to the right of the image (0 to 30 seconds) before it continues to move on a circular track (50 to 65 seconds). In F, an elongated peroxisome (arrows) with a globular structure at one end (*) moves from the left to the right of the image (0 to 5 seconds). Afterwards it emanates a tubular projection and moves to the left of the image (30 seconds), presumably to interact with another peroxisome, before it retracts and collapses into a circular structure (65 seconds). In G, the dynamic behavior of elongated peroxisomes after depolymerization of microtubules with nocodazole is shown. Arrows mark the positions of elongated peroxisomes and arrowheads point to segmented organelles. N, nucleus. Bars, 10 µm (A-D), 5 µm (E-G).

View larger version (28K): [in a new window]   Fig. 8. Schematic model for the morphogenesis of mammalian peroxisomes, based on observations reported here and previously (Schrader et al., 1996; Schrader et al., 1998b; Schrader et al., 2000). Recent evidence suggesting the involvement of the ER in the de novo formation of peroxisomes (Titorenko and Rachubinski, 2001; Eckert and Erdmann, 2003; Lazarow, 2003) is included. The majority of matrix and membrane proteins are synthesized on free ribosomes in the cytosol and imported post-translationally into pre-existing peroxisomes. Peroxisomes multiply by budding and segmentation from pre-existing ones. Under induced conditions (e.g. cultures at low density, growth factors, fatty acids, free radicals), highly elongated peroxisomes are formed that undergo segmentation and fission, forming spherical peroxisomes. Pex11pß is involved in the elongation/tubulation of peroxisomes (Schrader et al., 1998b), whereas DLP1 mediates peroxisomal fission (Koch et al., 2003; Li and Gould, 2003). Proteins mediating the constriction of peroxisomes are presently unknown. Proper intracellular distribution of the formed peroxisomes requires microtubules and a functional dynein/dynactin motor (Schrader et al., 2003). In yeast and plants peroxisomes are distributed via the actin cytoskeleton (Hoepfner et al., 2001; Mathur et al., 2002).

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