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

Annett Koch 1 Gabriele Schneider 1 Georg H. Lers 0 Michael Schrader ) 1 0 Department of Anatomy and Cell Biology , Robert Koch Strasse 8 , University of Marburg , Marburg, 35037 , Germany 1 Department of Cell Biology and Cell Pathology , Robert Koch Strasse 6 - The mammalian dynamin-like protein DLP1 belongs to the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes. We have previously shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells. In this study, we conducted RNA interference experiments to knock down the expression of DLP1 in COS-7 cells stably expressing a GFP construct bearing the C-terminal peroxisomal targeting signal 1. The peroxisomes in DLP1-silenced cells were highly elongated with a segmented morphology. Ultrastructural and quantitative studies confirmed that the tubular peroxisomes induced by DLP1-silencing retained the ability to constrict their membranes but were not able to divide into spherical organelles. Co-transfection of DLP1 siRNA with Pex11pb , a peroxisomal membrane protein involved in peroxisome proliferation, induced further Peroxisomes are ubiquitous eucaryotic organelles that contribute to important metabolic processes, including hydrogen peroxide metabolism, the b -oxidation of fatty acids and the biosynthesis of ether lipids (van den Bosch et al., 1992). An interesting feature is their ability to proliferate and multiply, or be degraded in response to nutritional and environmental stimuli. The prevailing model of peroxisome biogenesis, proposed by Lazarow and Fujiki (Lazarow and Fujiki, 1985), predicts that peroxisomes grow by uptake of newly synthesized proteins from the cytosol and multiply by division. A key question in the field is, whether this is the predominant mechanism of peroxisome formation, or are there alternative modes of peroxisome formation which may involve the ER or other kinds of endomembranes (South and Gould, 1999; Titorenko and Rachubinski, 2001; Faber et al., 2002; Geuze et al., 2003). At present, little is known about the proteins required for growth and division of the peroxisomal compartment. The peroxisomal membrane protein Pex11p has been proposed to function in peroxisome division in a variety of species (Erdmann and Blobel, 1995; Marshall et al., 1995; Sakai et al., 1995; Passreiter et al., 1998; Schrader et al., 1998b; Lorenz et elongation and network formation of the peroxisomal compartment. Time-lapse microscopy of living cells silenced for DLP1 revealed that the elongated peroxisomes moved in a microtubule-dependent manner and emanated tubular projections. DLP1-silencing in COS-7 cells also resulted in a pronounced elongation of mitochondria, and in more dispersed, elongated Golgi structures, whereas morphological changes of the rER, lysosomes and the cytoskeleton were not detected. These observations clearly demonstrate that DLP1 acts on multiple membranous organelles. They further indicate that peroxisomal elongation, constriction and fission require distinct sets of proteins, and that the dynamin-like protein DLP1 functions primarily in the latter process. al., 1998). Overexpression of Pex11p promotes peroxisome elongation and subsequent division, whereas loss of Pex11p results in reduced peroxisome number (Erdmann and Blobel, 1995; Marshall et al., 1995; Schrader et al., 1998b; Li et al., 2002). A striking increase in elongated forms of peroxisomes upon expression of Pex11p has been observed in all organisms studied, indicating that tubule formation may be an important aspect of peroxisome division (Schrader et al., 1996; Schrader et al., 1998b). Mammalian cells express at least three distinct Pex11 genes (Pex11pa , b , g ), whereas Saccharomyces cerevisiae has a single Pex11 gene (Li et al., 2002). However, recent findings indicate that Pex25p and the novel peroxin Pex27p are Pex11p-related proteins, which are involved in the regulation of peroxisome size and number in S. cerevisiae (Smith et al., 2002; Rottensteiner et al., 2003; Tam et al., 2003). The biochemical properties of Pex11p are still a matter of debate and there is currently no mechanistic model for peroxisome division. Evidence for a metabolic control of peroxisome division has also been presented (Poll-The et al., 1988; Sacksteder and Gould, 2000; Smith et al., 2002) and might be mediated by signals derived from the b -oxidation of fatty acids (Chang et al., 1999; van Roermund et al., 2000). However, Pex11p can induce peroxisome proliferation independently of peroxisome metabolism (Li and Gould, 2002). Other proteins involved in peroxisome division are members of the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes (McNiven, 1998; Danino and Hinshaw, 2001). The dynamin-related protein Vps1p mediates peroxisome division in S. cerevisiae (Hoepfner et al., 2001), whereas the dynaminlike protein DLP1 has recently been shown to be required for peroxisomal fission in mammalian cells (Koch et al., 2003; Li and Gould, 2003). Mammalian DLP1 and its homologues Dnm1 (S. cerevisiae) and DRP-1 (C. elegans) are also suggested to be involved in the control of mitochondrial morphology and division (Yoon et al., 1998; Labrousse et al., 1999; Smirnova et al., 2001). We have recently shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells (Koch et al., 2003). In this study, we have performed RNA interference experiments which were combined with protein expression, ultrastructural and live cell studies, to examine the effects of reduced DLP1 protein levels on peroxisome morphology and division as well as on other intracellular organelles in more detail. Materials and Methods Plasmid pVgRXR was obtained from Invitrogen (Groningen, The Netherlands) and plasmid pGHL97, for expression of a fusion protein of the green fluorescent protein with a peroxisomal targeting signal (GFP-PTS1), was described earlier (Luers et al., 2003). Wild-type DLP1 (DLP1-WT) and DLP1 fused to GFP (DLP1-WT-GFP) were described previously (Pitts et al., 1999). The C-terminally tagged version of Pex11pb myc in pcDNA3 was described by Schrader et al. (Schrader et al., 1998b). MBGV-GP, encoding the glycoprotein GP of the Marburg virus, as well as MBGV-GP-specific polyclonal antibodies were kindly provided by Dr S. Becker (University of Marburg, Germany) (Becker et al., 1996). Rabbit polyclonal antiPMP70 (peroxisomal membrane protein 70) antibodies (Luers et al., 1993) were a gift from Dr A. Vlkl (University of Heidelberg, Germany). The rabbit polyclonal anti-DLP1 antibody was described previously (Yoon et al., 1998). The following monoclonal antibodies were used: anti-p115 (Transduction Laboratories); anti-myc epitope 9E10 (kindly provided by Dr M. Eilers, University of Mar (...truncated)