The Dynamin-like GTPase DLP1 Is Essential for Peroxisome Division and Is Recruited to Peroxisomes in Part by PEX11*

Xiaoling Li and Stephen J. GouldDagger

From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, November 25, 2002, and in revised form, February 17, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The regulation of organelle abundance involves a complex interplay of organelle synthesis and destruction. In the case of peroxisomes, little is known about how cells control these competing processes. Peroxisome destruction appears to occur by a specialized form of autophagy (pexophagy) which requires most APG/CVT genes. In addition to the APG/CVT genes that are involved in general autophagy, some specific factors are required for pexophagy (1). Peroxisome formation appears to be even more complex. Many lines of evidence suggest that peroxisomes are formed mainly from preexisting peroxisomes, either by budding or fission, and this may be the predominant mechanism for peroxisome formation (2, 3). However, peroxisome synthesis has also been observed in the absence of preexisting peroxisomes (4, 5), indicating that there may be two parallel pathways for peroxisome formation, one from preexisting peroxisome and another de novo pathway for peroxisome formation (4, 6).

Of the many PEX1 genes and products (peroxins) required for peroxisome biogenesis, only PEX11 has been shown to have a conserved role in peroxisome division (7-13). The overexpression of PEX11 promotes peroxisome elongation and subsequent division, whereas loss of PEX11 results in reduced peroxisome abundance (7, 8, 12, 14). There is also evidence for metabolic control of peroxisome division (6, 15, 16), although PEX11 proteins can induce peroxisome proliferation independently of peroxisome metabolism (17). These results indicate that PEX11 proteins may play a positive role in the division process. In human cells, PEX11-mediated peroxisome division involves three steps: first, the import of PEX11 into the peroxisome membrane; second, the elongation of peroxisomes and the segregation of PEX11 proteins away from other peroxisomal membrane proteins, forming PEX11-enriched patches; and third, the division of elongated peroxisome tubules into multiple, small peroxisome vesicles (12, 17). However, the biochemical activities of PEX11 proteins remain obscure, and there is as yet no mechanistic model for PEX11-induced peroxisome division. There may also be species-specific differences in PEX11 function because mammalian cells express at least three distinct PEX11 genes, whereas Saccharomyces cerevisiae has a single PEX11 gene (18).

Recently, Hoepfner et al. (19) reported that the yeast dynamin-related GTPase, Vps1p, is also involved in peroxisome division. Yeast vps1 null mutants contain only one or two large peroxisomes and cannot increase peroxisome abundance under conditions that normally induce PEX11 expression and promote peroxisome division (19). To test whether a similar process exists in mammalian cells and to determine further whether this is related to PEX11-mediated peroxisome division, we studied the role of human DLP1 in peroxisome biogenesis. DLP1 is the human gene most similar to two of three dynamin-like proteins found in yeast, Vps1p and Dnm1p. DLP1 is also known to regulate the dynamics of mitochondria and the endoplasmic reticulum (20, 21). We report here that a reduction in DLP1 activity inhibits peroxisome division and reduces peroxisome abundance. Furthermore, we provide evidence that PEX11 overexpression recruits DLP1 to peroxisomes, providing a potential mechanism for PEX11-induced peroxisome division.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- The plasmids pcDNA3-PEX11beta myc (12), pcDNA3- PMP34myc (22, 23), pPGK1-GFP/PTS1, and pRS425/GAL1-PEX11 (17) have been described. The plasmids expressing COOH-terminal 3×HA-tagged PEX11alpha , PEX11beta , PEX11gamma , and PMP34 were created by inserting above four full-length genes into a modified pcDNA3-3×HA vector (4). The plasmid pcDNA3-PMP34VSVG was cloned by inserting full-length PMP34 gene upstream (in-frame with) of a VSVG epitope in a modified pcDNA3-VSVG vector (4).

The human DLP1 gene was cloned by reverse transcription-PCR against the total RNA of human fibroblasts with a primer (DLP1-RT: 5'-CCACATGAGCAGATTCATACC-3') designed according to the 3'-untranslated region of human DLP1 cDNA for reverse transcription and two DLP1-specific primers (DLP1-5', 5'-CCCCTCGAGGGAGGCGCTAATTCCTGTCATAAAC-3', and DLP1-3', 5'-CACCGCGGCCGCTCACCAAAGATGAGTCTCCCGG-3') for PCR. The resulting PCR products were then digested with XhoI-NotI and cloned downstream of (in-frame with) a c-myc epitope in a modified pcDNA3-Nmyc vector. Plasmids from various colonies were sequenced, and three isoforms of DLP1 (DLP1 a form, b form, and c form, see Fig. 9) have been identified in fibroblasts. The full-length DLP1 gene was cloned by inserting the deleted nucleotides into the DLP1c by PCR. The dominant negative forms of DLP1 (DLP1S39N and DLP1T59A) were created by PCR site-directed mutagenesis using the oligonucleotides DLP1-5' and DLP1S39N-3', 5'-GAAGCAGGTCCCTCCCCACCAGGCTTTCTAGCACTGAGTTCTTTC-3', and DLP1-5' and DLP1T59A-3', 5'-GAGAGGTCTCCGGGCGACAATTCC-3', respectively. The resulting PCR fragments were then digested with XhoI-PpuMI and XhoI-BsaI, respectively, and used to replace their corresponding sequence in pcDNA3- NmycDLP1.

Antibodies-- Two affinity-purified rabbit anti-DLP1 peptide antibodies anti-DLP1MID and anti-DLP1N were provided by Dr. McNiven (Mayo Clinic and Foundation, Rochester, MN) (20). Antiserum against the COOH-terminal 16 amino acids of DLP1 (anti-DLP1C) was generated by injecting rabbits with ovalbumin (Pierce) coupled peptide NH2-CKQGASQIIAEIRETHLW-COOH. Sheep antibodies against PMP70 and rabbit antibodies against PEX13 have been described (4). The monoclonal antibodies to the c-myc epitope were obtained from the tissue culture supernatant of the hybridoma 1-9E10 (24). Polyclonal antibodies to the c-myc epitope and anti-HA monoclonal antibodies coupled to agarose beads were obtained from the Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HA monoclonal antibodies were obtained from Roche Applied Science. Secondary antibodies specific for sheep, rabbit, and mouse antibodies were obtained from commercial sources.

Cell Lines, Transfection, and Immunofluorescence-- An immortalized wild type human skin fibroblast GM5756-Ti and the wild type and PEX11beta -deficient mouse embryonic fibroblasts (MEFs) (14) were cultured under standard conditions (25). All transfections were performed by electroporation (26). For immunofluorescence, cells were fixed in 3% formaldehyde in Dulbecco's modified phosphate-buffered saline, pH 7.1 (DPBS, Invitrogen), for 20 min and then permeabilized in 25 µg/ml digitonin in DPBS for 5 min. Cells were then incubated with primary antibodies for 30 min, washed extensively in DPBS, incubated with fluorescently labeled secondary antibodies for 10 min, washed extensively in DPBS, and mounted on slides. Peroxisome abundance in fibroblasts was determined as described previously (17).

RNA Interference-- To knock down the expression of DLP1 in fibroblasts by RNA interference, two pairs of DLP1 gene-specific 21-nucleotide small interfering RNA (siRNA) (sense strand: DLP1·siRNA1, 5'-UAGCUGCGGTGAACCCGUGdTdT-3'; and DLP1·siRNA2, 5'-CCAACCACAGGCAACUGGAdTdT-3') and one pair of control PXR2 siRNA (sense strand: PXR2·siRNA, 5'-GCAGGGAAAAGGCUCUAGGdTdT-3') were synthesized by Dharmacon Research (Lafayette, CO) and annealed according to the manufacturer's guidelines. 50 µl of 20 µM siRNA duplexes was then transfected into fibroblasts from one confluent T-75 flask by electroporation (26), and the DLP1 protein level and peroxisome morphology and abundance were analyzed at various time points by immunoblot and indirect immunofluorescence, respectively. To examine the peroxisome proliferating activity of PEX11beta in siRNA duplex-treated cells, fibroblasts were first transfected with DLP1 or PXR2 siRNA duplexes and cultured for 24 h, then transfected with PEX11beta expression vector pcDNA3-PEX11beta myc together with another dose of siRNA duplexes. Cells were then cultured for an additional 48 h before analyzing the peroxisome abundance and morphology.

Immunoprecipitation-- To study the association of DLP1 with peroxisomes, fibroblasts expressing the COOH-terminal 3×HA-tagged or VSVG-tagged PMP34 in combination with mycDLP1aT59A were harvested by scraping in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and CompleteTM protease inhibitor mixture (Roche Applied Science) and lysed by passing through a 22-gauge syringe needle five times. Lysates were then subjected to centrifugation at 25,000 × g for 15 min to pellet large organelles. The resulting supernatant was diluted in TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and CompleteTM protease inhibitor mixture). Immunoprecipitation was initiated by adding 15 µl of a 50% slurry of anti-HA monoclonal antibodies coupled to agarose beads into 500 µl of the above supernatant together with 1% bovine serum albumin. The mixture was then incubated with mixing at 4 °C for 2 h, and anti-HA beads were pelleted by brief centrifugation, washed five times with 1 ml of TBS, and resuspended in SDS-sample buffer. The supernatant and corresponding immunoprecipitate sample were then separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with anti-myc polyclonal antibodies.

To study the possible interaction between PEX11 proteins as well as that between PEX11 and DLP1, fibroblasts expressing the appropriate proteins were lysed by incubation with mixing in TBS buffer plus 1% Triton X-100 or 0.2% digitonin at 4 °C for 1 h. Lysates were then subjected to centrifugation at 100,000 × g for 30 min to pellet cellular membranes. The resulting supernatant was diluted into TBS buffer with detergent and 1% bovine serum albumin. Immunoprecipitation was carried out by adding either 15 µl of a 50% slurry of anti-HA beads or 20 µl of a 50% slurry of protein A-agarose (Sigma) plus 10 µl of 200 µg/ml anti-myc polyclonal antibodies and incubating with mixing at 4 °C for 2 h. The beads were then pelleted, washed extensively 3 × 1 ml with TBS buffer with detergent, 3 × 1 ml with high salt TBS buffer (50 mM Tris-HCl, pH 7.5, 350 mM NaCl, 1 mM EDTA, and CompleteTM protease inhibitor mixture with detergent), and 3 × 1 ml with TBS buffer. The beads were resuspended in SDS-sample buffer, separated together with corresponding supernatant by SDS-PAGE, and probed with either anti-myc polyclonal antibodies or anti-HA monoclonal antibodies.

Peroxisome Purification by Nycodenz Gradient-- To study the association of DLP1 with peroxisomes, fibroblasts expressing PMP34myc or PEX11beta myc were harvested by trypsin digestion, washed with PBS buffer once and with homogenization buffer (0.25 M sucrose, 1 mM Tris-HCl, pH 7.5, and 1 mM EDTA) once. Then the cells were resuspended in 1.2 ml of homogenization buffer with CompleteTM protease inhibitor mixture and homogenized by five passes through a precision ball-bearing homogenizer. Homogenates were centrifuged for 30 s at 1,500 × g to yield a postnuclear supernatant. The resulting postnuclear supernatant was loaded on top of 10.0 ml of linear 15-42% nycodenz gradient with a cushion of 1.5 ml of maxidenz and centrifuged at 4 °C for 25 min at 137,500 × g. 0.75-ml fractions were collected and analyzed for catalase activity and succinate dehydrogenase activity as described (27).

Yeast Strains and Culture Conditions-- Yeast strains used in this study were based on S. cerevisiae strain BY4733 (MATa, his3Delta 200, leu2Delta 0, met15Delta 0, trp1Delta 63, ura3Delta 0). The strain XLY1 (pex11Delta ) was generated as described previously (17). The XLY3 strain (vps1Delta ) was generated by one-step PCR-mediated disruption of the VPS1 gene in BY4733 using HIS3 as the selectable maker (28). The strain XLY4 (pex11Delta , vps1Delta ) was generated from XLY1 by one-step PCR-mediated disruption of the VPS1 gene using HIS3 as the selectable maker. Plasmid transformations were performed using the LiOAc procedure (29). All strain were cultured in minimal S medium (0.17% yeast nitrogen base w/o ammonium sulfate (Sigma), 0.5% ammonium sulfate) with 2% glucose, 1% galactose, or 0.2% oleic acid or 0.02% Tween 40 as carbon source. Media were supplemented with amino acids, uracil, and adenine as required (29).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reduced DLP1 Levels Cause Reduced Peroxisome Abundance-- The observation that yeast Vps1p participates in peroxisome membrane division suggested that the human ortholog of Vps1p could have a similar role. However, the human DLP1 gene product, which is the human protein that shows the greatest sequence similarity with S. cerevisiae Vps1p, shares even greater similarity with S. cerevisiae Dnm1p, a yeast protein that regulates mitochondrial division and endosome dynamics but has no discernible role in peroxisome division (19, 30). Furthermore, human DLP1 has been shown previously to regulate mitochondrion division and endoplasmic reticulum dynamics (20, 21), indicating that it might share more functional similarity with Dnm1p than with Vps1p. Therefore, it was essential to empirically determine whether human DLP1 has a role in peroxisome biogenesis.

We first attempted to reduce DLP1 mRNA and DLP1 protein levels using small interfering RNA (siRNA) specific for the DLP1 message. Two pairs of DLP1 siRNA duplexes (DLP1· siRNA1 and DLP1·siRNA2) and a control siRNA of a nonperoxisomal gene PXR2 (PXR2·siRNA) were designed according to Elbashir et al. (31). After annealing, they were transfected into an immortalized human skin fibroblast cell line (5756-Ti) by electroporation. Cells were then incubated at 37 °C and processed for immunoblot and indirect immunofluorescence analyses at various times after transfection. DLP1 is known to have multiple isoforms that are generated by alternative splicing of the initial DLP1 transcript (20, 33). Our immunoblot analysis indicated that at least two different isoforms were present in fibroblasts (Fig. 1A). The immunoblot results also confirmed that both DLP1·siRNA duplexes were able to reduce DLP1 protein level, and the maximal reduction (to 30-40% of normal level) was reached by day 4 after transfection (Fig. 1A). Control PXR2·siRNA, on the other hand, had no effect on the DLP1 protein level (Fig. 1A). Given that the transfection efficiency is typically about 80-90% for the siRNA duplex in these cells, DLP1 protein levels are likely to be even lower than 30% of normal level in those cells that took up the siRNA to DLP1. As expected, peroxisome abundance in untransfected cells (141 ± 46 pps) was similar to that of cells transfected with the control siRNA (138 ± 46 pps) (Fig. 1B). However, cells transfected with the DLP1·siRNA contained fewer peroxisomes (58 ± 23 pps). Furthermore, the morphology of peroxisomes in these cells was quite atypical, with peroxisomes in these cells having a long, tubular morphology (Fig. 1, E and F) compared with the small, punctuate morphology of normal peroxisomes (Fig. 1, C and D). Control experiments with antibodies to the peroxisomal matrix enzyme catalase indicated that peroxisomal protein import was unaffected even in the cells with the most severe reductions of peroxisome abundance (data not shown).


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Fig. 1.   RNA interference of DLP1 results in reduced peroxisome abundance. Wild type human skin fibroblasts 5756-Ti were transfected with PXR2·siRNA or DLP1·siRNA1 duplexes, and cells were then harvested for immunoblot (A) or processed for double indirect immunofluorescence (B-F) at various times after transfection. A, whole cell lysates from different days were separated by SDS-PAGE and probed with DLP1 antibodies (DLP1MID) and PEX13 antibodies. B, peroxisome abundance in 5756-Ti cells, 5756-Ti cells transfected with PXR2·siRNA, and 5756-Ti cells transfected with DLP1·siRNA1. C-F, representative cells treated with PXR2·siRNA (C and D) and DLP1· siRNA1 (E and F). Cells were processed at 48 h after transfection for immunofluorescence with antibodies to PMP70 and catalase (not shown). Samples were examined by confocal fluorescence microscope, and the number of peroxisomes present in at least 30 randomly selected cells was counted. Results in B are presented as the average peroxisome abundance ± 1 S.D.

Dominant Negative DLP1 Mutants Reduce Peroxisome Abundance-- As an independent test of DLP1 involvement in peroxisome division, we expressed wild type and dominant negative DLP1 mutants in human fibroblasts and assessed their effects on peroxisome abundance. DLP1 shares a high degree of sequence similarity with dynamin, especially in the amino-terminal tripartite GTP binding motif. Previous studies have identified two dominant negative dynamin genetic mutants that exhibit reduced GTP binding affinity (S45N) and reduced GTP hydrolysis activity (T65A), respectively (32). We therefore generated the corresponding mutations in the DLP1 cDNA (S39N and T59A, respectively). Amino-terminally myc-tagged forms of wild type DLP1a (one of several DLP1 isoforms generated by alternative splicing (20, 33)) and each of these single substitution mutants of DLP1a were transfected into human fibroblasts. Four days after transfection each cell population was processed for indirect immunofluorescence using antibodies specific for the myc epitope tag and PMP70, an integral peroxisomal membrane protein marker.

Peroxisome abundance in cells expressing wild type DLP1a (123 ± 46 pps) was similar to that in untransfected cells (141 ± 46 pps) (Fig. 2A, compare first two bars, p = 0.194, Student's t test), and the overexpressed, myc-tagged form of DLP1a appeared to be largely cytosolic (Fig. 2, B and C). On the other hand, the peroxisome abundance was reduced (71 ± 37 pps) (Fig. 2A, p < 0.001, Student's t test) in cells expressing the DLP1aS39N mutant, which has a reduced affinity for GTP (32). Similar to the DLP1 RNA interference cells, some of the DLP1aS39N-expressing cells have only a couple of giant, reticular peroxisomes (Fig. 2, D and E). This DLP1a mutant appeared to be primarily cytosolic like wild type protein (Fig. 2D). Overexpression of the DLP1aT59A mutant also reduced peroxisome abundance in these cells (83 ± 26 pps) (Fig. 2A, p = 0.011, Student's t test). However, this DLP1 mutant appeared to be associated principally with small punctuate structures, few if any of which appeared to be peroxisomes (Fig. 2, F and G).


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Fig. 2.   Overexpression of dominant negative mutants of DLP1 reduces peroxisome abundance. A, peroxisome abundance in 5756-Ti cells and 5756-Ti cells overexpressing mycDLP1a, mycDLP1aS39N, or mycDLP1aT59A. B-G, representative cells transfected with pcDNA3- mycDLP1a (B and C), pcDNA3-mycDLP1aS39N (D and E), or pcDNA3- mycDLP1aT59A (F and G). 5756-Ti cells were transfected with appropriate plasmids. 4 days after transfection, cells were processed for indirect immunofluorescence with antibodies to the myc epitope (B, D, and F) and PMP70 (C, E, and G). Samples were examined by confocal fluorescence microscope, and the number of peroxisomes present in at least 30 randomly selected cells was counted. Results in B are presented as the average peroxisome abundance ± 1 S.D.

A Subpopulation of DLP1 Colocalizes with PEX11-induced, Elongated Peroxisomes-- Previous studies of dynamin and DLP1 have indicated that these large GTPases may act as "pinchases," promoting vesicle budding and organelle fission by contracting membrane tubules (34, 35). If applied to peroxisome division, this model would predict that DLP1 physically associates with peroxisomes during their division.

To test for DLP1 association of peroxisomes, we generated rabbit antibodies (DLP1C) against the COOH-terminal 16 amino acids of DLP1 and used them for immunofluorescence analysis. Under normal conditions, the endogenous DLP1 proteins localize in small cytoplasmic vesicles in fibroblasts (Fig. 3, B and E). Although the majority of DLP1-positive vesicles were nonperoxisomal, a small subpopulation of DLP1-positive vesicles also overlapped with the peroxisomal membrane marker protein PMP70 (Fig. 3C, indicated by arrowheads).


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Fig. 3.   A subpopulation of DLP1 colocalizes with peroxisomes. 5756-Ti cells (A, B, and C) and 5756-Ti cells expressing PEX11beta myc (D, E, and F) were processed for double indirect immunofluorescence for DLP1 (green) and peroxisomal membrane marker PMP70 (red) (C) or myc epitope (red) (F). Note that although the majority of DLP1-positive structures are cytoplasmic, a subpopulation of them are associated with peroxisomes (arrowheads in C inset), especially along the elongated peroxisomes in PEX11beta myc-expressed cells (arrowheads in F inset).

To examine the association of DLP1 with peroxisomes during the division process, we took advantage of the fact that overexpression of PEX11 proteins induces peroxisome division. We overexpressed myc-tagged PEX11beta protein in fibroblasts and then used antibodies specific for DLP1 (DLP1C) and confocal immunofluorescence microscopy to detect DLP1 localization. We also used the antibodies to PMP70 and the myc epitope to label peroxisomes. Under these conditions, more DLP1 was colocalized with peroxisomes, and some DLP1 signals were aligned along the elongated peroxisome tubules prior to their division (Fig. 3F, indicated by arrowheads). Given that PEX11-mediated peroxisome division appears to involve multiple stages, particularly peroxisome elongation and subsequent division (12, 17), these observations suggest that DLP1 associates with peroxisomes prior to or during the division process. However, this interaction is likely to be transient because the majority of DLP1 does not overlap with peroxisomes.

We next asked whether we could collect biochemical evidence for peroxisome-associated DLP1. We transfected 5756-Ti cells with a plasmid designed to express mycDLP1T59A, a dominant negative mutant predicted to lack GTPase activity (a similar mutant of dynamin displays increased association with target membrane (32)), and a plasmid expressing HA-tagged PMP34. The lysates were prepared from transfected cells, and then the peroxisomal membranes were immunopurified with anti-HA monoclonal antibodies. We used anti-myc antibodies to detect myc-tagged DLP1T59A and found that DLP1T59A was coimmunopurified with peroxisome membranes (Fig. 4Aa). As a control for the nonspecific binding of peroxisomal membranes to anti-HA beads, parallel experiments were carried out with cells cotransfected with plasmids expressing VSVG-tagged PMP34 and myc-tagged DLP1T59A. Like PMP34-myc, PMP34-VSVG was also targeted into peroxisomes and had no effect on peroxisome abundance in fibroblasts (not shown). This control revealed that coimmunoprecipitation of DLP1 with peroxisomes was specific, with a signal of ~2-fold over background (Fig. 4Ab). This experiment was repeated several times, and each trial yielded essentially the same results. Furthermore, similar results were obtained when other peroxisomal membrane proteins were used for immunopurification of peroxisomal membranes (data not shown).


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Fig. 4.   A small population of DLP1 is associated with peroxisomes. A, 5756-Ti cells expressing PMP34-3×HA and mycDLP1aT59A (a) or PMP34-VSVG and mycDLP1aT59A (b) were lysed by thorough mixing in hypotonic buffer, and the lysates were cleared by centrifugation at 25,000 × g for 15 min. The resulting supernatant was processed for immunoprecipitation using anti-HA monoclonal antibodies. The supernatant and corresponding immunoprecipitate sample were then separated by SDS-PAGE and probed with anti-myc polyclonal antibodies (DLP1T59A). DLP1as, amount of DLP1aT59A associated with peroxisomes; DLP1in, amount of DLP1aT59A in input supernatant. Please note that although immunoprecipitates of a and b were loaded equally, and the supernatants of a and b were also loaded equally, the immunoprecipitate (IP) and corresponding supernatant for each sample were not loaded equally (supernatant is only ~4% of the immunoprecipitate sample) because of the detection limit of the immunoblot. Therefore, the relative amount (DLP1as/DLP1in) can only be used for the relative comparison between samples a and b. B, fibroblasts expressing PMP34myc (c) or PEX11beta myc (d) were homogenized and centrifuged. The resulting postnuclear supernatant was fractionated into peroxisome, mitochondrion, and cytosol fractions. The peak of the peroxisome fraction was then separated by SDS-PAGE and probed with anti-DLP1MID and anti-PEX13 antibodies. C, 5756-Ti cells expressing PMP34-3×HA and mycDLP1a (e) or PEX11beta -3×HA and mycDLP1a (f) were lysed in hypotonic buffer and cleared by centrifugation at 25,000 × g for 15 min. The resulting supernatant was processed for immunoprecipitation using anti-HA monoclonal antibodies. The supernatant and corresponding immunoprecipitate sample were then separated by SDS-PAGE and probed with anti-myc polyclonal antibodies (DLP1a). DLP1as, the amount of DLP1a associated with peroxisomes; DLP1in, the amount of DLP1a in the input supernatant. The relative amount (DLP1as/DLP1in) is defined the same as in A. The densitometry results of A-C show that DLP1 is associated with peroxisomes (a and b) and that peroxisome-associated DLP1 is increased in PEX11beta -expressing cells (c-f).

As independent measurements of DLP1 association with peroxisomes, we used nycodenz gradient centrifugation to purify peroxisomes from human fibroblasts that were transfected with PMP34myc (Fig. 4Bc) or PEX11beta myc (Fig. 4Bd). Peak peroxisome fractions were then separated by SDS-PAGE and blotted with anti-DLP1MID, an antibody that recognizes a DLP1 peptide from the middle region of this protein (20), and antibodies specific for PEX13, a peroxisomal membrane protein marker (Fig. 4B). The relative level of peroxisome-associated DLP1 was ~2-fold higher in cells expressing PEX11beta compared with those expressing PMP34, suggesting that DLP1 is recruited to peroxisomes during division. A PEX11beta -mediated increase of peroxisome-associated DLP1 was also detected with immunopurification of peroxisome membranes (Fig. 4C). We coexpressed mycDLP1a together with PMP34-3×HA (Fig. 4Ce) or PEX11beta -3×HA (Fig. 4Cf) in 5756-Ti cells, then lysed the cells and purified peroxisome membranes with anti-HA monoclonal antibodies. The copurified mycDLP1 were detected by anti-myc antibodies. Again, the relative level of peroxisome-associated DLP1 was about 1.6-fold higher in cells expressing PEX11beta than in control cells (Fig. 4C).

Inhibition of DLP1 Blocks PEX11-mediated Peroxisome Proliferation-- The apparent recruitment of DLP1 to peroxisomes by PEX11beta raises the possibility that DLP1 is required for PEX11-medidated peroxisome proliferation. To address this issue we reduced DLP1 in human skin fibroblasts with the DLP1·siRNA, then transfected these cells with the PEX11beta myc expression vector to induce peroxisome division. Overexpression of PEX11beta myc induced a 4-fold increase in peroxisome abundance in untransfected cells or cells pretransfected with the control siRNA (PXR2·siRNA) for 24 h (Fig. 5A). In contrast, PEX11 overexpression was unable to induce peroxisome proliferation in cells pretransfected with DLP1·siRNA for 24 h (Fig. 5A). In the cells pretransfected with DLP1·siRNA, PEX11beta protein still stimulated peroxisome elongation but did not induce peroxisome division (Fig. 5D). Interestingly, PEX11beta overexpression still led to the segregation of peroxisome membrane into subregions that were either rich in PEX11beta proteins or were enriched in other peroxisomal membrane proteins (Fig. 5, compare D and E, the merged image is shown in the inset of D). Similar results were also observed when dominant negative DLP1 mutants were expressed (data not shown).


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Fig. 5.   Inhibition of DLP1 blocks the PEX11beta -mediated peroxisome proliferation. A, peroxisome abundance in 5756-Ti cells, 5756-Ti cells overexpressing PEX11beta myc, 5756-Ti cells treated with PXR2·siRNA/PEX11beta myc, and 5756-Ti cells treated with DLP1·siRNA1/PEX11beta myc. 5756-Ti cells were transfected with the control PXR2·siRNA or DLP1·siRNA1, incubated for 1 day, transfected with the PEX11beta myc expression vector and another dose of siRNA duplexes. After an additional 2 days cells were processed for immunofluorescence microscopy using antibodies to the myc epitope tag (B and D) and PMP70 (C and E). B-E, representative cells transfected with PXR2·siRNA and PEX11beta myc expression vector (B and C), and DLP1·siRNA1 and PEX11beta myc expression vector (D and E). The inset in D is the merged image of the boxed area in D and E, indicating the segregation of PEX11beta myc (green) from PMP70 (red) along the elongated peroxisomes in DLP1·siRNA-treated cells. Bar, 1 µm.

To assess further the connection between DLP1 and PEX11-mediated peroxisome division, we expressed the myc-tagged wild type DLP1a or dominant negative mutant DLP1aS39N into wild type and PEX11beta -/- mouse embryonic fibroblasts. The peroxisomes in the transfected cells were then examined with immunofluorescence microscopy using antibodies for the myc epitope (Fig. 6, A, C, E, and G) or PMP70 (Fig. 6, B, D, F, and H). In wild type cells, the expression of DLP1a had no significant effect on either peroxisome abundance or morphology (Fig. 6, A and B). Expression of DLP1aS39N, on the other hand, caused reduction of peroxisome abundance and peroxisome elongation (Fig. 6, C and D). As described previously (14), peroxisomes of PEX11beta -/- MEFs were reduced in abundance by about 50% (Fig. 6, E and F). Expression of DLP1aS39N in PEX11beta -/- MEFs exacerbated this phenotype (Fig. 6, G and H).


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Fig. 6.   Dominant negative mutant of DLP1 (DLP1S39N) reduces peroxisome abundance and induces tubular peroxisomes in PEX11beta -/- MEFs. Wild type (WT) and PEX11beta -/- MEFs were transfected with pcDNA3-mycDLP1a or pcDNA3-mycDLP1aS39N and processed for indirect immunofluorescence with antibodies for myc epitope (A, C, E, and G) and PMP70 (B, D, F, and H). Note that in wild type MEFs, the expression of DLP1aS39N induced fewer and elongated peroxisomes (D). In PEX11beta -/- MEFs, expression of DLP1aS39N induced more elongated peroxisomes than PEX11beta deficiency alone, and the peroxisome abundance decreased further.

Yeast Vps1p Is Required for PEX11-mediated Peroxisome Proliferation-- It was reported previously by Hoepfner et al. (19) that peroxisome abundance was reduced significantly in a vps1 null mutant of yeast. To improve our understanding of the roles of Vps1p and Pex11p in peroxisome division, we examined the peroxisome morphology in yeast mutants lacking either or both genes. GFP-PTS1 was used to mark the peroxisomes. Also, we measured peroxisome abundance in these strains during growth on glucose, which tends to repress peroxisome abundance, and oleic acid, which induces peroxisome abundance (in yeast, peroxisomes are the sole sites of fatty acid beta -oxidation and are required for growth on fatty acids such as oleic acid (36)).

As noted previously, the abundance of peroxisomes in the S. cerevisiae strain BY4733 is extremely heterogeneous (17). This was observed under a variety of growth conditions and complicated the quantification of peroxisome abundance. However, even with this variability, it is clear that the peroxisome abundance increased when cells were shifted from glucose-containing medium to oleic acid-containing medium (Fig. 7, A and B). As reported many times in previous studies, loss of PEX11 (XLY1) eliminated the increase in peroxisome abundance which occurs in response to fatty acids (Fig. 7D). However, it caused only a very slight reduction in basal peroxisome abundance during growth on glucose (Fig. 7C). Loss of Vps1 (XLY3), in contrast, severely reduced basal peroxisome abundance (Fig. 7E) and also eliminated fatty acid-induced peroxisome proliferation (Fig. 7F), as shown by the earlier study by Hoepfner et al. (19). Even though there were very few peroxisomes in vps1Delta mutants, peroxisome abundance appeared to be reduced even further by the loss of both PEX11 and VPS1 (XLY4) (Fig. 7, G and H). We also tested whether artificial overexpression of PEX11 might be able to drive peroxisome division in the absence of Vps1p. For this we transformed the vps1Delta strain with two different plasmids: an empty vector with a GAL1 promoter or the same vector with the PEX11 open reading frame driven by the GAL1 promoter, which was able to induce peroxisome proliferation in the XLY1 strain (Fig. 7I). After 24-h growth in galactose medium, we found that peroxisome abundance was similar in both strains (Fig. 7, J and K), providing further evidence that the PEX11-mediated peroxisome proliferation requires Vsp1/DLP1.


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Fig. 7.   Yeast Vps1p is required for PEX11-mediated peroxisome proliferation. Various S. cerevisiae strains were cultured under different conditions, and the peroxisome number was counted under a fluorescence microscope in 120 cells for each sample. Shown here are the frequency distributions of cells with different peroxisome numbers. A and B, BY4733 cells transformed with pPGK1-GFP/PTS1 and pRS425/GAL1 were precultured in S minimal medium with 2% glucose to midlog phase (A), shifted to minimal medium containing 0.2% oleic acid and 0.02% Tween 40, and cultured for 24 h (B). C and D, XLY1 (pex11Delta ) cells carrying pRS425/GAL1 were precultured in minimal S medium with 2% glucose to midlog phase (C) and shifted to minimal medium containing 0.2% oleic acid and 0.02% Tween 40 (D) for 24 h. E and F, XLY3 (vps1Delta ) cells carrying pRS425/GAL1 were precultured in minimal S medium with 2% glucose to midlog phase (E) and shifted to minimal medium containing 0.2% oleic acid and 0.02% Tween 40 (F) for 24 h. G and H, XLY4 (pex11Delta , vps1Delta ) cells carrying pRS425/GAL1 were precultured in minimal S medium with 2% glucose to midlog phase (G) and shifted to minimal medium 0.2% oleic acid and 0.02% Tween 40 (H) for 24 h. I, J, and K, galactose-induced XLY1 cells transformed pRS425/GAL1-PEX11 (I), XLY3 cells (J), and XLY3 cells transformed with pRS425/GAL1-PEX11 (K). The desired yeast strains were cultured in glucose to midlog phase, shifted to minimal medium containing 1% galactose, and cultured for 24 h. Ola, oleic acid.

No Evidence for Physical Interaction between DLP1 and PEX11-- We have shown that DLP1 participates in PEX11-mediated peroxisome proliferation, possibly by mediating peroxisome division. Next we set out to determine whether there is a physical association between DLP1 and PEX11 proteins. We first tested conditions for solubilizing PEX11 proteins from the peroxisome membrane. PEX11alpha and PEX11beta were efficiently solubilized by both 1% Triton X-100 and 0.2% digitonin (Fig. 8A). We then tested whether PEX11 proteins solubilized under these conditions retained the ability to interact with other proteins. Specifically, we transfected cells with plasmid pairs expressing the following proteins: PEX11alpha -3×HA and PEX11alpha myc (a), PEX11alpha -3×HA and PEX11alpha (b), PEX11beta -3×HA and PEX11beta myc (c), PEX11beta -3×HA and PEX11beta (d), PEX11alpha -3×HA and PEX11beta myc (e), or PEX11alpha -3×HA and PEX11beta (f). After a 2-day incubation, each cell population was lysed with either 1% Triton X-100 or 0.2% digitonin, processed for immunoprecipitation using anti-myc polyclonal antibodies, and the immunoprecipitates were separated by SDS-PAGE and probed with anti-HA antibodies (Fig. 8A). Although the PEX11 proteins were solubilized by either detergent, we only observed PEX11alpha -PEX11alpha and PEX11beta -PEX11beta interactions in cells homogenized with 0.2% digitonin. No protein-protein interactions were detected between PEX11alpha and PEX11beta . These findings are in agreement with our previous observation that Triton X-100 impairs the detection of PEX11 proteins by immunofluorescence microscopy (12), implying that PEX11 might be one of the rare proteins whose structure is sensitive to Triton X-100.


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Fig. 8.   DLP1 does not physically interact with PEX11. A, 5756-Ti cells expressing PEX11alpha -3×HA and PEX11alpha myc (a), PEX11alpha -3×HA and PEX11alpha (b), PEX11beta -3×HA and PEX11beta myc (c), PEX11beta -3×HA and PEX11beta (d), PEX11alpha -3×HA and PEX11beta myc (e), or PEX11alpha -3×HA and PEX11beta (f) were lysed in TBS buffer with 1% Triton X-100 or 0.2% digitonin, and the lysates were cleared by centrifugation at 100,000 × g for 30 min. The resulting supernatant was processed for immunoprecipitation (IP) using anti-myc polyclonal antibodies. The supernatant and corresponding immunoprecipitation sample were then separated by SDS-PAGE and probed with anti-HA monoclonal antibodies. Samples b, d, and f are the negative controls of samples a, c, and e, respectively. B, 5756-Ti cells expressing PEX11alpha -3×HA and mycDLP1a (a), PEX11beta -3×HA and mycDLP1a (b), PEX11gamma -3×HA and mycDLP1a (c), or PMP34-3×HA and mycDLP1a (d) were lysed in TBS buffer with 0.2% digitonin, and the lysates were cleared by centrifugation at 100,000 × g for 30 min. The resulting supernatant was processed for immunoprecipitation using anti-HA monoclonal antibodies. The supernatant and corresponding immunoprecipitate sample were then separated by SDS-PAGE and probed with anti-myc polyclonal antibodies.

Using the conditions that allow detection of homophilic interaction between PEX11 proteins, we next tested whether DLP1 physically associated with PEX11. Fibroblasts were transfected with plasmids expressing PEX11alpha -3×HA and mycDLP1a (a), PEX11beta -3×HA and mycDLP1a (b), PEX11gamma -3×HA and mycDLP1a (c), or PMP34-3×HA and mycDLP1a (d), incubated for 24 h, lysed in 0.2% digitonin, and subjected to immunoprecipitation using anti-HA antibodies. The immunoprecipitate samples were then fractionated by SDS-PAGE and blotted with anti-c-myc antibodies. No mycDLP1 was detected in any of these samples (Fig. 8B). Additional immunoprecipitation experiments using cross-linkers and nonhydrolyzable GTP analogs also failed to show any evidence of PEX11-DLP1 interactions (data not shown). Finally, we used a yeast two-hybrid system to test for possible interactions between these DLP1 and PEX11, but this too failed to show any evidence for PEX11-DLP1 interaction (data not shown).

Splice Variants of DLP1 Differ in Their Effects on Peroxisome Dynamics-- Multiple forms of DLP1 are generated by alternative splicing of the initial DLP1 transcript, and it has been proposed that some of these may have different activities in vivo (20, 33). We tested whether any of the four splice variants altered the activity of DLP1 vis à vis peroxisome dynamics. These four forms of DLP1 differ as shown in Fig. 9A, with the top form (isoform a) being the one used in preceding experiments. When expressed in human fibroblasts, isoform a had no significant effect on peroxisome abundance (Fig. 9, B-D). Isoform b also had no discernible effect on peroxisome abundance (Fig. 9, B, E, and F). By contrast, isoforms c and full-length DLP1 appeared to inhibit peroxisome division (Fig. 9, B and G-J). The S39N mutants of all four isoforms all inhibited peroxisome division (Fig. 9B).


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Fig. 9.   Splice variants of DLP1 differ in their effects on peroxisome dynamics. A, schematic summary of the alternative splicing region of DLP1. Numbers indicate amino acid positions. Three different isoforms, DLP1a, DLP1b, and DLP1c, are cloned from fibroblasts. B, peroxisome abundance in 5756-Ti cells, and 5756-Ti cells expressing four different isoforms of DLP1 and their corresponding S39N mutants. C-J, representative cells transfected with pcDNA3-mycDLP1a (C and D), pcDNA3-mycDLP1b (E and F), pcDNA3-mycDLP1c (G and H), or pcDNA3-mycDLP1F (I and J). 5756-Ti cells were transfected with appropriate plasmids. 4 days after transfection, cells were processed for indirect immunofluorescence with antibodies to the myc epitope (C, E, G, and I) and PMP70 (D, F, H, and J).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Until recently, the only factors implicated in peroxisome division were those of the PEX11 family of peroxisomal membrane proteins. The levels of PEX11 proteins correlate roughly with peroxisome abundance in the cell, and overexpression of PEX11 alone is sufficient to accelerate peroxisome division and greatly increase peroxisome abundance. However, PEX11 does not share any homology to proteins involved in other organelle formation processes, making it difficult to develop hypotheses for PEX11 function. PEX11 overexpression induces peroxisome division, but loss of PEX11 does not block peroxisome division entirely and results in only a partial reduction in peroxisome abundance, in both mammalian and yeast cells (7, 8, 14). Thus, it is likely that PEX11 proteins play facilitative roles in peroxisome division rather than a core, conserved role in the biochemistry of peroxisome division.

In this study we extended the previous observation by Hoepfner et al. (19) that Vps1p, a dynamin-like protein of yeast, also plays a critical role in peroxisome division. Like so many interesting observations, those of Hoepfner et al. (19) raised many questions. Were similar proteins required for peroxisome division in other organisms? Do these proteins associate with peroxisomes? Does PEX11 recruit these proteins to peroxisomes? How do dynamin-like proteins mediate peroxisome division? In this report we have established that DLP1 plays an essential role in peroxisome division in human cells and does associate with peroxisomes. Most importantly, however, our data suggest a model of peroxisome division in which elevated levels of PEX11 appear to induce peroxisome division by recruiting DLP1 to peroxisomes.

We observed previously that there are two phases to PEX11-induced peroxisome division in human cells (12, 17). The first phase involves peroxisome elongation and the segregation of PEX11 from other components of the peroxisome membrane. The second phase involves the division of these elongated, tubular peroxisomes into numerous small peroxisomes scattered throughout the cell. We detected small amounts of DLP1 on peroxisomes under normal conditions, but overexpression of PEX11 nearly doubled the level of peroxisome-associated DLP1. Furthermore, immunofluorescence experiments revealed that many of the elongated peroxisomes induced by PEX11 overexpression had small foci that stained for DLP1. These results suggest that PEX11 induces peroxisome division, at least in part, by recruiting DLP1 to peroxisome membranes. Consistent with this hypothesis, we found that loss of DLP1 blocks PEX11-mediated peroxisome division but does not affect PEX11-mediated elongation of peroxisomes or the formation of PEX11-enriched patches at foci along the length of the elongated peroxisomes. It should be noted, however, that PEX11 is unlikely to be the sole mediator of DLP1 recruitment to membranes because loss of PEX11 does not result in as severe a defect in peroxisome abundance as loss of DLP1 in human cells or loss of VPS1 in yeast.

Although the data appear to favor a model of peroxisome division in which PEX11 recruits DLP1 to peroxisome membranes, perhaps at sites of PEX11 enrichment, we found no evidence for recruitment via direct protein-protein interactions between PEX11 and DLP1. Such negative data might reflect experimental artifacts that are not physiologically relevant. However, these results at least raise the possibility that PEX11 acts indirectly to facilitate DLP1 recruitment. Lipid moieties have been implicated in dynamin recruitment to membranes (37-39), and the relative levels of PEX11 might affect peroxisomal membrane composition or the presence of lipid microdomains of distinct composition which could act as sites of recruitment. Although DLP1 lacks the known lipid binding domain of dynamin, DLP1 might interact with lipids in a fundamentally different but equally important manner. An indirect mechanism for PEX11-mediated DLP1 recruitment is also easier to reconcile with our observation of PEX11-independent, DLP1-dependent peroxisome division by allowing other factors to modulate the recruitment of DLP1 to peroxisomes. As for the kinetics of the DLP1-peroxisome interaction, a few lines of evidence suggest that it is transient. First, the steady-state proportion of DLP1 which is associated with peroxisomes is very low and rises only slightly (1.5-fold) by pronounced PEX11 overexpression. Second, DLP1 is detected on only a small number of peroxisomes under normal conditions, and after PEX11 overexpression it is detected preferentially on elongated peroxisomes prior to division. Third, when we locked DLP1 on peroxisomes by fusion to the cytoplasmic tail of an integral peroxisomal membrane protein, peroxisome division was severely inhibited (data not shown), indicating that DLP1 release is an important step in peroxisome division. A transient interaction model for DLP1 is similar to that proposed for dynamin in the formation of clathrin-coated vesicles, where dynamin is recruited to the membrane just prior to vesicle formation and is released immediately thereafter (40).

As for how DLP1 actually catalyzes peroxisome division, our paper offers little in the way of new insight into the function of dynamin-like proteins. Dynamin has been proposed to act as a pinchase, constricting the neck of nascent vesicles to form a membranous tube, which presumably promotes fusion of the tightly apposed membrane bilayers (35, 41). However, dynamin does not appear to be sufficient for completing the vesicle scission event, and other factors, such as endophilin, might contribute essential membrane/lipid modifying activities that mediate the final step in vesicle formation (42-44). A similar mechanism might explain the role of DLP1 in peroxisome division. Regardless of the actual mechanism, it is clear that further advances in this field will require the development of techniques that allow the measurement of peroxisome division by real time video microscopy. Extensive efforts on our part to perform such experiments have yet to yield success. The problems in detecting peroxisome division are related to the relatively small size of peroxisomes, their movement, and the relatively low rate at which they divide. Based on our experience, we feel that successful monitoring of peroxisome division might require the identification of inhibitory drugs that can be used to synchronize peroxisomes in the predivision, elongated state, allowing investigators to follow a wave of peroxisome division once such inhibitors were removed.

    ACKNOWLEDGEMENTS

We thank Dr. Paul Watkins for help in peroxisome purification by nycodenz gradient and Dr. Mark A. McNiven for providing anti-DLP1 antibodies.

    FOOTNOTES

* This work was supported by National Institutes of Health grants (to S. J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3424; Fax: 410-955-0215; E-mail: sgould@jhmi.edu.

Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M212031200

    ABBREVIATIONS

The abbreviations used are: PEX, peroxin; DLP1, dynamin-like protein 1; DPBS, Dulbecco's modified phosphate-buffered saline; GFP, green fluorescent protein; HA, hemagglutinin; MEF(s), mouse embryonic fibroblasts; PMP, peroxisomal membrane protein; pps, peroxisomes per section; siRNA, small interfering RNA.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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