Dynamin-like Protein 1 Is Involved in
Peroxisomal Fission*
Annett
Koch
,
Meinolf
Thiemann§,
Markus
Grabenbauer§,
Yisang
Yoon¶,
Mark A.
McNiven¶, and
Michael
Schrader
From the
Department of Cell Biology and Cell
Pathology, University of Marburg, Robert Koch Str. 5, D-35037 Marburg,
Germany, the § Department of Anatomy and Cell Biology,
Division of Medical Cell Biology, University of Heidelberg, Im
Neuenheimer Feld 307, D-69120 Heidelberg, Germany, and the
¶ Department of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, Minnesota 55905
Received for publication, November 19, 2002, and in revised form, December 13, 2002
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ABSTRACT |
The mammalian dynamin-like protein 1 (DLP1), a
member of the dynamin family of large GTPases, possesses
mechanochemical properties known to constrict and tubulate membranes.
In this study, we have combined two experimental approaches, induction
of peroxisome proliferation by Pex11p
and expression of
dominant-negative mutants, to test whether DLP1 plays a role in
peroxisomal growth and division. We were able to localize DLP1 in spots
on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis
revealed the presence of DLP1 in highly purified peroxisomal fractions
from rat liver and an increase of DLP1 after treatment of rats with the
peroxisome proliferator bezafibrate. Expression of a dominant negative
DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in
combination with Pex11p
caused the appearance of tubular peroxisomes
but had no influence on their intracellular distribution. In
co-expressing cells, the formation of tubulo-reticular networks of
peroxisomes was promoted, and peroxisomal division was completely
inhibited. These findings were confirmed by silencing of DLP1 using
siRNA. We propose a direct role for the dynamin-like protein DLP1 in
peroxisomal fission and in the maintenance of peroxisomal morphology in
mammalian cells.
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INTRODUCTION |
Peroxisomes are ubiquitous subcellular organelles that participate
in a variety of important catabolic and anabolic functions, including
hydrogen peroxide metabolism, the
-oxidation of fatty acids, and the
biosynthesis of ether phospholipids (1). Like other subcellular
organelles, peroxisomes have the capacity to proliferate and multiply
or to be degraded in response to nutritional and environmental stimuli
(2). According to the "growth and division model" of Lazarow and
Fujiki (3), new peroxisomes form by division and fission of preexisting
ones after the import of newly synthesized proteins from the cytosol.
Although accumulating evidence suggests that vesicles that originate
from the endoplasmic reticulum or other kinds of endomembranes
might be involved in the de novo formation of peroxisomes in
a multistep process (4-6), the "growth and division model" has
been broadly accepted (7). The peroxisomal membrane protein Pex11p
appears to be directly involved in the regulation of peroxisomal growth
in size and number and, thus, in peroxisomal division. Pex11p-deficient
yeast cells contain a small number of "giant" peroxisomes, whereas
overexpression results in a high degree of peroxisome proliferation (8,
9). In mammals, three Pex11p molecules, Pex11p
(10, 11), Pex11p
, and Pex11p
, have been described, which are supposed to control peroxisome proliferation under induced and basal conditions,
respectively (12-14). When overexpressed, Pex11p
induces a
pronounced peroxisome proliferation through a multistep process
involving peroxisome elongation and segregation of Pex11p
from other
peroxisomal membrane proteins, followed by peroxisome division (13).
Tubulation and fission processes of elongated peroxisomes have also
been observed under conditions of rapid cellular growth or stimulation
of cultured cells with defined growth factors, fatty acids, or
free radicals and have been proposed to contribute to peroxisome
proliferation (15-17). At present, however, little information is
available on the exact function of such complex tubular or reticular
peroxisomal structures, their dynamic behavior, and the molecular
machinery required for their formation and division (18).
Proteins of the dynamin family are large GTPases, which have been
implicated in tubulation and fission events of cellular membranes,
either as a molecular switch or as a pinchase-like mechanoenzyme
(19-22). Recent in vitro studies have indicated that conventional dynamin has the ability to tubulate spherical liposomes and, upon GTP hydrolysis, constrict, deform, or sever membrane tubules
into discrete vesicles (23-26). The dynamin-like proteins Dnm1
(Saccharomyces cerevisiae), DRP-1 (Caenorhabditis
elegans), and mammalian
DLP11 are homologues involved
in the control of mitochondrial morphology and division (21, 27-30).
The mammalian DLP1 is suggested to function in the maintenance of
mitochondrial morphology (31-34). It forms a homotetrameric complex
similar to dynamin (35) and has been localized to mitochondria but also
to other cellular organelles (34, 36, 37). Recently, it has been
demonstrated that DLP1 is able to form rings and tubulate membranes in
a nucleotide-dependent manner both in living cells and
in vitro (38).
In the present study, we have investigated whether DLP1 has an
influence on peroxisomal morphology and division. Using Pex11p
expression to induce tubular peroxisomes as well as peroxisome proliferation, we were able to localize DLP1 in spots on elongated peroxisomes. Expression of a dominant-negative DLP1 mutant deficient in
GTP hydrolysis or silencing of DLP1 by siRNA inhibited peroxisomal fission and caused tubulation of peroxisomes. These findings provide the first evidence suggesting that a dynamin-like protein, DLP1, is
involved in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.
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EXPERIMENTAL PROCEDURES |
cDNAs and Antibodies--
Wild-type DLP1 (DLP1-WT), DLP1
fused to GFP (DLP1-WT-GFP), and the point mutants GFP-DLP1-K38A and
GFP-Dyn2(aa)-K44A were described previously (34, 39). The C-terminally
tagged version of Pex11p
myc in pcDNA3 was described in Ref. 13.
Rabbit anti-PMP70 (40) and rabbit anti-acyl-CoA oxidase polyclonal
antibodies (41) were a gift from Dr. A. Völkl (University of
Heidelberg). The sheep anti-catalase antibody was obtained from The
Binding Site (Birmingham, UK). The anti-Myc epitope monoclonal antibody was described in Ref. 13. The rabbit polyclonal anti-DLP1 (DLP1-MID) and anti-Dyn2 (MC63) antibodies were described previously (36). The
monoclonal anti-mangan superoxide dismutase antibody (MnS-1) was
obtained from Alexis Corp. (San Diego, CA). The monoclonal anti-tubulin
antibody (DM1
) was obtained from Sigma. Species-specific anti-IgG
antibodies conjugated to TRITC or fluorescein isothiocyanate were
obtained from Dianova (Hamburg, Germany).
Cell Culture, RNA Interference, and Transfection
Experiments--
HepG2 and COS-7 cells were obtained from the American
Type Culture Collection (Manassas, VA). They were cultured in
Dulbecco's modified Eagle's medium supplemented with 2 g/liter sodium
bicarbonate, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (all from PAA
Laboratories GmbH) at 37 °C in a humidified atmosphere containing
5% CO2. Cells were transfected with DNA constructs by
incubation with polyethyleneimine (25 kDa; Sigma) as described (42) and
processed for immunofluorescence 24-48 h after transfection. The level
of Pex11p
myc overexpression was 1.5-4-fold. 21-Nucleotide RNAs were
obtained from Dharmacon (Lafayette, LA) and annealed according to the
manufacturer's instructions. The siRNA sequence targeting human DLP1
(accession number NM012062) corresponded to the coding region 785-803
relative to the first nucleotide of the start codon. Transfection of
siRNA was carried out using Oligofectamine (Invitrogen) according to
the manufacturer's instructions. Cells were assayed for silencing and
peroxisome morphology 48 h after transfection by
immunofluorescence and immunoblotting with anti-DLP1 and anti-tubulin antibodies.
Immunofluorescence and Confocal Microscopy--
Cells grown on
glass coverslips were fixed with 4% paraformaldehyde in
phosphate-buffered saline, pH 7.4, permeabilized with 0.2% Triton
X-100 or 25 µg/ml digitonin, and incubated with primary and secondary
antibodies as described (13). Transfected cells were processed for
immunofluorescence 24-48 h after transfection. For double-labeling
experiments, cells transfected with GFP constructs were incubated with
rabbit anti-PMP70 (or other peroxisomal marker proteins) and
subsequently with goat anti-rabbit IgG conjugated to TRITC. For
visualization of Pex11p
myc, cells were labeled with anti-Myc (TRITC)
antibodies. Samples were examined using a Leitz Diaplan (Leica,
Wetzlar, Germany) or an Axiovert 100 microscope (Carl Zeiss, Jena,
Germany) equipped with the appropriate filter combinations and
photographed on Eastman Kodak Co. TMY film or digitalized. Confocal
images (150-nm sections) were captured with a Leica TCS MP
confocal microscope (Leica Microsystems, Bensheim, Germany) with
appropriate spectrometer settings for each fluorophor. Digital images
were optimized for contrast and brightness using Micrograph Picture
Publisher software.
Isolation of Peroxisomes--
Peroxisomes were isolated from rat
liver according to a protocol described previously (43). For some
experiments, peroxisomes were isolated from the livers of adult male
Wistar rats (Charles River, Sulzfeld, Germany), which were fed with the
potent peroxisome proliferator bezafibrate (41) (Roche Molecular
Biochemicals). Briefly, one liver was homogenized (1 stroke, 2 min,
1000 rpm) using a Potter S homogenizer (Braun, Melsungen, Germany) in
ice-cold homogenization buffer (5 mM MOPS, pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.1% ethanol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM
-aminocaproic acid, and 0.2 mM dithiothreitol). After
subcellular fractionation, a crude peroxisomal fraction (light
mitochondrial fraction D) was sedimented into exponentially shaped
OptiPrep (Axis-Shield, Oslo, Norway) gradients (40). The highly
purified peroxisomal fraction with a density of 1.24 g/ml (usually
>95% pure) was used for further experiments. For determination of
marker enzyme activities, the following standard procedures were
employed: catalase (44), esterase (45), and cytochrome c
oxidase (46). The assay for
-glucuronidase was performed at 56 °C
(47). Protein was determined using standard procedures. All assays were
run with a recording spectrophotometer (Uvikon 810 (Kontron,
Munich, Germany) or Beckman model 24).
Gel Electrophoresis and Immunoblotting--
Protein samples were
separated by SDS-PAGE, transferred to nitrocellulose (Schleicher and
Schüll) using a semidry apparatus and analyzed by immunoblotting.
Immunoblots were processed using horseradish peroxidase-conjugated
secondary antibodies and enhanced chemiluminescence reagents (Amersham
Biosciences). For quantification, immunoblots were scanned and
processed using Pcbas software.
Quantitation and Statistical Analysis of Data--
For
quantitative evaluation of peroxisome morphology, 100-200 cells per
coverslip were examined and categorized as cells with tubular (2-5
µm in length; Figs. 1B and 4D), rod-shaped
(0.5-1 µm; Figs. 1A and 4E), or spherical
peroxisomes (0.1-0.3 µm; Fig. 9A) as described (15-17).
Cells co-expressing Pex11p
myc and GFP-DLP1-K38A were usually filled
with extremely elongated (up to 15 µm) peroxisomes (hypertubulation).
Usually, 3-5 coverslips per preparation were analyzed, and 4-8
independent experiments were performed. Significant differences between
experimental groups were detected by analysis of variance for unpaired
variables using Microsoft Excel. Data are presented as means ± S.D., with an unpaired t test used to determine statistical
differences. p values <0.05 are considered as significant,
and p values <0.01 are considered as highly significant.
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RESULTS |
DLP1 Localizes to Tubular Peroxisomes--
To investigate the role
of DLP1 in peroxisome fission and maintenance of morphology, we took
advantage of the human hepatoblastoma cell line HepG2, which has been
recently used to propose a general model for peroxisome morphogenesis
(16, 48). The peroxisomal compartment in HepG2 cells exhibits marked
morphological heterogeneity (15). Elongated and tubular peroxisomes,
which are frequently detected in cultured cells, undergo dynamic
changes and are observed to divide into
spherical organelles (Fig. 1A). Overexpression of the
human PEX11
gene alone was sufficient to induce a
pronounced proliferation of peroxisomes through a multistep process
involving peroxisome elongation (Fig. 1, B and D)
and segregation of Pex11p
from other peroxisomal membrane proteins
(Fig. 1B), followed by peroxisome division (Fig. 1,
C and D) (16). Only 6 h after transfection, the peroxisomes in 90% of the Pex11p
myc-expressing cells displayed an elongated, tubular morphology. Peroxisome tubules declined rapidly
in abundance over the following 24-48 h and were replaced by numerous,
small spherical peroxisomes (Fig. 1, C and D). In controls (untransfected or vector alone), 30-40% of the cells exhibited tubular peroxisomes after ~20 h, which is dependent on
culture conditions (15, 16). However, elongated peroxisomes in controls
were also observed to divide and declined with time in culture, giving
rise to spherical organelles (Fig. 1, C and D).
These observations indicate that HepG2 cells, and especially those
expressing Pex11p
myc provide an excellent model system to study
peroxisomal elongation and fission in more detail.

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Fig. 1.
Pex11p expression
induces proliferation of peroxisomes in HepG2 cells. A,
HepG2 cells were processed for indirect imunofluorescence using
an antibody specific for PMP70, a peroxisomal membrane protein. Note
the elongated peroxisomes in the cell on the left, in
contrast to their segmented appearance in the cell on the
right. The arrows point to peroxisomes that are
supposed to divide. B-D, overexpression of Pex11p myc
induces peroxisome elongation prior to fission and proliferation.
B, HepG2 cells were transfected with Pex11p myc, a
Myc-tagged version of a peroxisomal membrane protein, and labeled with
antibodies specific for PMP70 (red) and the Myc epitope tag
(green). Peroxisome proliferation coincides with the
elongation of peroxisomes and the segregation of Pex11p myc from
PMP70 (or peroxisomal matrix proteins). The arrow points to
an elongated peroxisome with multiple, alternating bands of
Pex11p myc and PMP70. C, expression of Pex11p myc
finally results in the formation of numerous spherical peroxisomes.
Note the smaller size of peroxisomes after expression of Pex11p myc
(asterisks) compared with controls (cell on the
left). D, peroxisome morphology at different time
points after transfection. time (h), time after
transfection; squares, control cells (vector alone or
untransfected); triangles, Pex11p myc-expressing cells;
black, tubular peroxisomes; white, rodlike and
spherical peroxisomes; N, nucleus; bars, 10 µm
(A and C) or 2.5 µm (B).
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As described previously (36, 34), DLP1 has been localized to
mitochondria, endoplasmic reticulum tubules and other, yet unidentified
cellular organelles in mammalian cells. As shown in Fig.
2, affinity-purified antibodies to DLP1
stain punctate vesicular structures in HepG2 cells that are
concentrated at the perinuclear region and appear to form linear arrays
(Fig. 2, A and B). The intensity of the fine
punctate staining pattern was increased by transfection of HepG2 cells
with either a wild type DLP1 construct (Fig. 2B) or
expression of a wild type DLP1-GFP fusion protein (Fig. 2C).
The expression of the constructs did not influence the normal
intracellular distribution of DLP1. Similar observations have been made
in other mammalian cells (36, 34). To determine whether DLP1 associates
with multiplying peroxisomes, HepG2 cells expressing Pex11p
myc were
fixed, permeabilized, and double-stained with antibodies to DLP1 and to
the Myc tag of Pex11p
myc. In addition, HepG2 cells were
co-transfected with Pex11p
myc and DLP1-WT-GFP. Interestingly,
DLP1-positive structures were found to distribute along the length of
elongated peroxisomes (Fig. 2, D-H). DLP1 was found to
align along some tubular peroxisomes in spots and was associated with
the tips of some tubules (Fig. 2, D and E). In
addition, other nonperoxisomal intracellular structures, presumably
mitochondria, were positive for DLP1. Co-localization with
tubulo-reticular peroxisomes was confirmed by confocal laser-scanning microscopy. DLP1 (Fig. 2F) or DLP1-WT-GFP (Fig.
2H) were found to form small patches along the tubular
structures and at the tips. Similar observations were made in
untransfected controls; however, co-localization was less frequent
(Fig. 2G). In contrast, colocalization with peroxisomes was
not observed when an antibody to dynamin II, an ubiquitously expressed
dynamin isoform, was used (not shown).

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Fig. 2.
DLP1 localizes to elongated peroxisomes in
Pex11p -expressing HepG2 cells.
A-C, DLP1 is associated with numerous punctate vesicular
structures in HepG2 cells. Immunofluorescence microscopy of control
cells (A) or HepG2 cells transfected with DLP1-WT
(B), stained with affinity-purified antibodies to DLP1.
C, HepG2 cells transfected with DLP1-WT-GFP.
D-H, HepG2 cells expressing Pex11p myc only
(D-F) or Pex11p myc in combination with DLP1-WT-GFP
(H) and untransfected controls (Con)
(G) were processed for indirect immunofluorescence using
antibodies to the Myc epitope tag (D, F,
H), to PMP70 (G), and to DLP1 (E-G).
F, G, and H, confocal images showing
localization of DLP1 (red) (F and G)
or DLP1-WT-GFP (green) (H) along elongated
peroxisomes in Pex11p myc-expressing cells (F and
H) and untransfected controls (G). The
arrows in A point to linear arrays of DLP1
staining; arrows in D-H point to regions of
co-localization. Bars, 10 µm (A-C) or 2.5 µm
(D-H).
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DLP1 Associates with Highly Purified Peroxisomes and Is Induced
during Peroxisome Proliferation--
To determine whether DLP1 was
associated with isolated peroxisomes, we performed subcellular
fractionation experiments with rat liver. A crude peroxisomal fraction,
which was obtained by differential centrifugation, was further purified
on exponentially shaped OptiPrep gradients (Fig.
3A). Peroxisomes were mainly
recovered in fractions 2-4 with the mean equilibrium density of 1.24 g/ml. Mitochondria mostly band in a density range of about 1.15 g/ml (fractions 12 and 13), and microsomes (density of about 1.11 g/ml) were
predominantly found in fractions 14 and 15. Some activity of the
peroxisomal marker enzyme catalase was also found in the uppermost
gradient fractions, reflecting the fragmentation of the fragile
peroxisomes during centrifugation (Fig. 3A). The
determination of marker enzyme activities in the main organelle
fractions revealed no contamination of the peroxisomal fraction with
mitochondria, microsomes, or lysosomes (Table
I). These data demonstrate that the
isolated peroxisomal fraction is well separated from other cell
organelles and highly pure (43). The resulting fractions were analyzed
by immunoblotting with an anti-DLP1 antibody, which has been
characterized previously on rat liver subcellular fractions (36). The
DLP1-specific antibody detected a single band of the expected size (80 kDa) in rat liver cytosol and in a rat brain homogenate (Fig.
3B). Brain DLP1 ran slightly higher on SDS-PAGE than DLP1
from the cytosolic fraction or from other rat tissues (36). Whereas
DLP1 was prominent in the cytosolic fraction, there was a small but
reproducible amount of DLP1 in the highly purified peroxisomal
fraction. In addition, a small amount was detected in the mitochondrial
and the microsomal fractions under our experimental conditions (Fig.
3B). In contrast, DLP1 was absent from a lysosomal fraction,
which has been suggested previously (36).

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Fig. 3.
DLP1 is associated with highly purified
peroxisomes and is induced during peroxisome proliferation.
A, highly purified peroxisomes were isolated from rat liver.
Distribution of marker enzyme activities following OptiPrep gradient
centrifugation of a crude peroxisomal fraction is shown. Relative
concentration = (U ×  V)( U × V)-1, where V represents the
volume of a single fraction, U its corresponding enzyme
activity,  V the total gradient volume, and
U the total units recovered from all gradient fractions.
CytcOx, cytochrome c oxidase. B,
localization of DLP1 in the gradient fractions. Equal amounts of
protein (30 µg/lane) were run on 12.5% acrylamide gels, blotted onto
nitrocellulose membranes, and incubated with antibodies to DLP1.
Br, rat brain homogenate (5 µg/lane); Cyt,
cytosolic fraction (10 µg/lane); Po, peroxisomes;
Mit, mitochondria; Mic, microsomes;
Lys, lysosomes. C, highly purified peroxisomes
from controls ( ) (Co) and rats treated with the peroxisome
proliferator bezafibrate (+) (Beza) were isolated, separated
on 12.5% acrylamide gels, blotted onto nitrocellulose, and incubated
with antibodies to peroxisomal acyl-CoA oxidase (AOX),
catalase (CAT), or DLP1. Equal amounts of protein (acyl-CoA
oxidase and catalase, 4 µg/lane; DLP1, 40 µg/lane) were loaded onto
the gels. For DLP1, immunoblots loaded with different amounts of
protein (30-50 µg/lane) were quantitated and are expressed as
means ± S.D. Proteins were detected by ECL using
streptavidin-peroxidase. The positions of molecular mass markers (in
kDa) are indicated on the right. A-C, three
subunits of acyl-CoA oxidase with molecular masses of 72, 52, and 20.5 kDa. Similar results were obtained in three independent
experiments.
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Table I
Properties of the purified organelle fractions
The relative specific activity (RSA) was determined with respect to the
crude peroxisomal fraction. Values given are means ± S. D. The data are from three independent experiments. CytcOx, cytochrome
c oxidase; -Gluc, -glucuronidase.
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To investigate whether the amount of DLP1 in the peroxisomal fraction
was increased after the induction of peroxisome proliferation by
pharmacological compounds, rats were treated with the potent peroxisome
proliferator bezafibrate (41). Rodents are known to respond to a
variety of xenobiotics with a massive peroxisome proliferation, whereas
primates are "low responders." Peroxisome proliferation is usually
accompanied by an induction of peroxisomal
-oxidation enzymes and an
increase in peroxisome size and number. After treatment of rats with
bezafibrate, an induction of acyl-CoA oxidase, a key enzyme of
peroxisomal
-oxidation, was observed in a highly purified
peroxisomal fraction when compared with untreated controls (Fig.
3C). In contrast, catalase is not induced or is only
slightly induced (Fig. 3C). The determination of marker
enzyme activities in the peroxisomal fractions isolated from
bezafibrate-treated rats revealed a purity similar to the controls (not
shown). In electron microscopic studies, an increase in the size and
number of peroxisomes was also observed (41). After immunoblotting, DLP1 was found to be increased about 2-fold in the peroxisome fractions
obtained from bezafibrate-treated animals when compared with controls
(Fig. 3C). Similar results were obtained when rat Fao cells
were treated with the potent peroxisome proliferator ETYA (not shown).
DLP1 Mutants Cause Morphological Changes of Peroxisomes--
To
examine whether a mutated DLP1 affects peroxisome morphology, we
transfected HepG2 cells with a DLP1-GFP construct harboring a
lysine-to-alanine (K38A) mutation in GTP binding element 1 (34). It has
been shown recently that recombinant DLP1-K38A protein is capable of
binding GTP but is deficient in its hydrolysis due to reduced GTPase
activity (38). We transiently co-transfected HepG2 cells with
Pex11p
myc and the mutated GFP-DLP1-K38A. Cells were immunostained
24 h after transfection with antibodies to Pex11p
myc (Fig.
4). In contrast to the normal
distribution of DLP1, GFP-DLP1-K38A assembled into large cytoplasmic
aggregates in addition to associating with punctate vesicular
structures (Fig. 4; see also Ref. 34). Recent ultrastructural studies
revealed that the large cytoplasmic aggregates are composed of tubular membrane clusters that are coated with DLP1 in a periodic manner (38).
In cells co-expressing Pex11p
myc and GFP-DLP1-K38A, a pronounced
elongation of peroxisomes ("hypertubulation") was observed (Figs.
4, A-C, and 6). The cytoplasm of these cells was filled with numerous, largely elongated peroxisomes. They usually ranged in
length from 2 to 8 µm, but tubular forms measuring 10-15 µm were
also found. Interestingly, extended tubulo-reticular networks of
peroxisomes were frequently observed when confocal laser-scanning microscopy was applied, suggesting that fusion of elongated peroxisomes was promoted (Fig. 5A).
Furthermore, some of the DLP1-positive cytoplasmic aggregates were
found to localize to the tips of elongated peroxisomes (Fig.
5B). In cells expressing Pex11p
myc only (or in
combination with DLP1-WT or DLP1-WT-GFP), tubular peroxisomes were less
frequent and usually less elongated (up to 5 µm) (Figs. 4D
and 6). "Hypertubulation" of
peroxisomes was also not observed in cells expressing GFP-DLP1-K38A
only. However, peroxisomes were significantly more elongated and
rod-like than in control cells (Figs. 4, E and F,
and 7).

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Fig. 4.
Inhibition of DLP1 function induces
"hypertubulation" of peroxisomes in
Pex11 p-expressing cells. HepG2 cells were
co-transfected with Pex11p myc/GFP-DLP1-K38A (A-D), with
Pex11p myc/GFP-Dyn2-K44A (G and H), or with
GFP-DLP1-K38A only (E and F) and immunostained
with antibodies to the Myc tag of Pex11p (A,
C, D, and G) or the peroxisomal
membrane protein PMP70 (E). The corresponding GFP
fluorescence of DLP1-K38A (B and F) and Dyn2-K44A
(H) is shown on the right. Higher magnification
images of boxed regions in A are shown
in C and D. Note the pronounced elongation of
peroxisomes in DLP1-K38A co-expressing cells. Asterisks,
co-expressing (A, B, G, and
H) or transfected cells (E and F).
Bars, 10 µm.
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Fig. 5.
Co-expression of
Pex11p myc and GFP-DLP1-K38A promotes the
formation of reticular peroxisomal networks. A and
B, confocal images of HepG2 cells co-expressing Pex11p myc
(red) and GFP-DLP1-K38A (green). Cells were
immunostained with an anti-Myc antibody. A, higher
magnification view of a tubulo-reticular peroxisomal network induced by
co-expression. B, note the association of
GFP-DLP1-K38A-positive cytoplasmic aggregates with the tips of
elongated peroxisomes. The arrows in B point to
regions of co-localization. Bars, 2.5 µm.
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Fig. 6.
Inhibition of DLP1 function blocks fission of
peroxisomes in HepG2 and COS-7 cells. HepG2 (A) and
COS-7 cells (B) expressing Pex11p myc/GFP-DLP1-K38A,
Pex11p myc/GFP-Dyn2-K44A, or Pex11p myc only were immunostained 24 and 48 h after transfection as described in the legend to Fig. 4.
Note the accumulation of peroxisome tubules and the complete absence of
spherical peroxisomes in Pex11p myc/GFP-DLP1-K38A co-expressing
cells. C, control experiments with HepG2 cells expressing
Pex11p myc only, Pex11p myc/DLP1-WT, or Pex11p myc/DLP1-WT-GFP.
Similar results were obtained in control experiments with COS-7 cells
(not shown). For quantitative evaluation of peroxisome morphology,
cells were examined under the fluorescence microscope and categorized
as cells with spherical (s), rod-shaped (r), or
tubular (t) peroxisomes (see "Experimental Procedures").
The data are from 4-8 independent experiments and are expressed as
means ± S.D. (*, p < 0.01; #, p < 0.05 when compared with Pex11p myc only expressors).
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Fig. 7.
Expression of GFP-DLP1-K38A is sufficient to
cause elongation of peroxisomes in HepG2 and COS-7 cells. HepG2
(A) and COS-7 cells (B) expressing GFP-DLP1-K38A
only were immunostained 24 and 48 h after transfection with
antibodies to PMP70, a peroxisomal membrane protein, and quantitated as
described in Fig. 6. Note the accumulation of elongated peroxisomes
(rods and tubules) in cells expressing GFP-DLP1-K38A.
C, control experiments with HepG2 cells expressing DLP1-WT
or DLP1-WT-GFP or with untransfected cells. Similar results were
obtained in control experiments with COS-7 cells (not shown).
Con, control cells (untransfected, DLP1-WT, or DLP-WT-GFP).
The data are from six independent experiments and are expressed as
means ± S.D. (*, p < 0.01; #, p < 0.05). For quantitative evaluation, cells were categorized as cells
with spherical (s), rod-shaped (r), or tubular
(t) peroxisomes.
|
|
To investigate whether the effect on peroxisome morphology was
restricted to DLP1 (K38A) expression, we co-transfected cells with
Pex11p
myc and a mutated form of dynamin II (GFP-Dyn2(aa)-K44A) (39).
Dyn2, which is ubiquitously expressed, has been recently identified as
one of three isoforms of conventional brain dynamin I (49-51).
Expression of GFP-Dyn2(aa)-K44A did not result in "hypertubulation" of peroxisomes or the formation of reticular structures (Fig. 4,
G and H). The dramatic effect on peroxisome
morphology appeared to be specific for the dynamin-like protein DLP1 (K38A).
Perturbance of DLP1 Function Blocks Peroxisomal Fission--
The
striking induction of elongated tubulo-reticular peroxisomes in cells
expressing GFP-DLP1-K38A prompted us to investigate the influence on
peroxisomal fission more carefully in a time course experiment. Besides
HepG2 cells, COS-7 cells were used, which possess an elaborate
peroxisomal compartment (18). Cells co-expressing Pex11p
myc and
GFP-DLP1-K38A were immunostained 24 and 48 h after transfection
with antibodies to Pex11p
myc, and the cells were quantitated
according to their different peroxisomal forms (Fig. 6). In cells
expressing Pex11p
myc only or Pex11p
myc in combination with either
DLP1-WT or DLP1-WT-GFP, a typical decrease in tubular peroxisomes was
noted after 48 h, whereas a prominent increase in cells containing
spherical peroxisomes was observed (Figs. 1, C and
D, and 6). Furthermore, segmented tubules, presumably in
division, were detected (see Fig. 1B). However, in cells
expressing both Pex11p
myc and GFP-DLP1-K38A, tubular peroxisomes
were found to accumulate. After 48 h, nearly all co-expressors
contained largely elongated peroxisome tubules, whereas cells with
spherical peroxisomes were missing completely (Fig. 6). Segmented
tubules were not observed any more, but reticular networks of
peroxisomes were detectable (Fig. 5A). In cells
co-expressing GFP-Dyn2(aa)-K44A and Pex11p
myc, tubular peroxisomes
were slightly increased after 48 h compared with Pex11p
myc
alone (Fig. 6, A and B). The accumulation of
elongated peroxisomes was more pronounced in COS-7 than in HepG2 cells.
However, tubular peroxisomes in these cells were usually less elongated
and less frequent than in DLP1-K38A/Pex11p
myc expressors. Although
less obvious in COS-7 cells, the HepG2 cells still kept their ability
to generate spherical peroxisomes by fission of elongated ones (Fig.
6A). We therefore assume that the enrichment of elongated
peroxisomes in Dyn2-K44A/Pex11p
myc expressors may be due to an
additional proliferative stimulus exerted by the inhibition of Dyn2
function and not to a direct role in peroxisome division.
Next, we addressed whether the expression of GFP-DLP1-K38A alone was
able to influence tubulation and fission of peroxisomes in HepG2 and
COS-7 cells (Fig. 7). Cells were immunostained 24 and 48 h after
transfection with antibodies to PMP70, a peroxisomal membrane protein.
In contrast to co-expression with Pex11p
myc, a pronounced
"hypertubulation" was not observed (see Fig. 4, E and
F). However, a significant shift toward more elongated
peroxisomal forms (rods and tubules) was detected 24 h after
transfection, which was more pronounced after 48 h, when compared
with control cells (untransfected, DLP1-WT, or DLP1-WT-GFP) (Fig.
8). Furthermore, an accumulation of
elongated peroxisomal forms was observed, whereas the majority of the
control cells contained spherical peroxisomes after 48 h (Fig. 7).
Dyn2 (K44A)-expressing cells were indistinguishable from control cells
when peroxisomal forms were compared (not shown). These findings
indicate that mutated DLP1 (K38A) has the capacity to cause the
appearance of tubular peroxisomal membranes even in the absence of a
proliferative stimulus, although elongation appears to be more
pronounced in the presence of a stimulus. We conclude from these data
that DLP1 is involved in the fission process of peroxisomes.

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Fig. 8.
Inhibition of DLP1 function does not
influence the intracellular distribution of peroxisomes. HepG2
cells were transfected with GFP-DLP1-K38A (B and
C) and immunostained with antibodies to mitochondria
(anti-mangan superoxide dismutase) (A and B) or
peroxisomes (anti-PMP70) (C). Note the collapse of
mitochondria toward the cell center (B), in contrast to the
uniform intracellular distribution of elongated peroxisomes.
Con, untransfected control cell. N, nucleus.
Bars, 10 µm.
|
|
Inhibition of DLP1 Function Does Not Change Peroxisomal
Distribution--
It has been reported that the inhibition of DLP1
function results in a change of the normal distribution of mitochondria
(31, 32, 34, 52). Similar observations were made in DLP1
mutant-expressing HepG2 and COS-7 cells, where mitochondria were often
found to be clustered and wrapped around the nucleus when compared with controls (Fig. 8, A and B). In contrast, the
uniform intracellular distribution of peroxisomes was not affected by
the expression of GFP-DLP1-K38A, either alone (Fig. 8C) or
in combination with Pex11p
myc (Fig. 4). The tubular peroxisomes
observed in cells expressing GFP-DLP1-K38A only or in combination with
Pex11p
myc are not stained by antibodies to mitochondrial or
endoplasmic reticulum marker proteins but are positive for other
peroxisomal marker proteins (e.g. PMP70) (13). Although
similar in morphology to elongated mitochondria, tubular peroxisomes
are thinner and less elongated and do not exhibit bulbous structures
(Fig. 8, compare A and C). Furthermore, their intracellular
distribution remains unchanged after GFP-DLP1-K38A expression, which is
in contrast to tubular mitochondria.
Silencing of DLP1 by siRNA Causes Tubulation and Aggregate
Formation of Peroxisomes--
To verify the results obtained with the
mutated, nonfunctional DLP1 (K38A), we conducted RNA interference
experiments to "knock down" the expression of DLP1. Silencing of
DLP1 in HepG2 cells was mediated by transfecting the cells with
21-nucleotide siRNA duplexes that targeted human DLP1. Efficient
siRNA-mediated gene silencing in mammalian cells has recently been
reported by Tuschl and co-workers (53). HepG2 cells were processed for
immunofluorescence 48 h after transfection using antibodies
directed to DLP1 and catalase. As shown in Fig.
9, the expression of DLP1 was
specifically reduced by the cognate siRNA duplex. However, a fine
punctate and diffuse cytoplasmic staining was still visible (Fig.
9D). A reduction of DLP1 after transfection was also
observed in immunoblots of cell homogenates (Fig. 9F). In
controls treated with buffer or a noneffective siRNA, the expression of
DLP1 was not reduced (Fig. 9, B and F). We also
obtained an siRNA for DLP1, which was not effective in silencing DLP1.
The peroxisomes in these controls were overwhelmingly spherical, and
tubular peroxisomes were usually completely absent after 3 days in
culture (Fig. 9A; see also Fig. 1). Interestingly, the
peroxisomes in transfected cells became highly elongated (40-60% of
the cell population) and formed tubulo-reticular aggregates. The
elongated peroxisomes induced by siRNA often had a segmented appearance
but were not observed to separate into spherical organelles. Elongation
of mitochondria and clustering around the nucleus was also visible
after siRNA-mediated DLP1 silencing (not shown). These observations
further confirm a role for DLP1 in peroxisomal fission and in the
maintenance of peroxisomal morphology in mammalian cells.

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Fig. 9.
Silencing of DLP1 induces tubulation and
aggregate formation of peroxisomes. siRNA-mediated DLP1 silencing
in HepG2 cells. Cells were assayed for silencing and peroxisome
morphology 48 h after transfection by immunofluorescence
(A-E) and immunoblotting (F) with anti-DLP1,
anti-catalase (Cat), and anti-tubulin (Tub)
antibodies. A and B, control cells
(Con) (treated with buffer or noneffective siRNA).
C-E, HepG2 cells transfected with DLP1 siRNA duplex. The
arrows point to peroxisomal aggregates. Nuclei are marked
with asterisks. In E, a high magnification view
of elongated reticular peroxisomes is shown. F, immunoblots
of homogenates prepared from controls and transfected cells (siRNA)
using anti-DLP1 and anti-tubulin antibodies. Equal amounts of protein
(DLP1, 75 µg/lane; tubulin, 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 (A-D) or
5 µm (E).
|
|
 |
DISCUSSION |
In this study, we have combined two experimental approaches,
induction of peroxisome proliferation by Pex11p
and expression of
dominant negative mutants, to examine whether DLP1 plays an important
role in peroxisomal fission and in the maintenance of peroxisomal
morphology in mammalian cells.
A Direct Role for Pex11p
in Peroxisomal Fission?--
The
growing recognition of the dynamic nature of the peroxisomal
compartment in eukaryotic cells has inspired the query into the
investigation of the cellular machinery that mediates such a complex
behavior. At present, however, little information is available on the
molecular components involved in peroxisome growth and division. The
characterization of Pex11p in both yeast and mammalian cells and in
trypanosomes has led to the proposal that it is directly involved in
the regulation of peroxisome growth in size and number (8, 9, 11, 13,
54). Since cells that lack Pex11p accumulate a few large peroxisomes in
contrast to Pex11p overexpressors, which contain numerous small
peroxisomes, it has been assumed that Pex11p is involved in the fission
process. In support of a more direct role of Pex11p in peroxisome
division, it has been shown that rat Pex11p
binds coatomer in
vitro by its cytoplasmically exposed C-terminal dilysine motif.
Consequently, recruitment of coatomer by Pex11p
has been proposed to
initiate vesiculation or budding of peroxisomes (11). However, the
dilysine motif is not conserved in other Pex11p homologues, and
coatomer does not bind to yeast Pex11p or human Pex11p
. Furthermore,
mutations of the C terminus of Pex11p were not found to affect its
function in peroxisome division (55), and studies on PEX3-mediated
peroxisome biogenesis suggest that it can proceed independently of both
COPI and COPII (56). Here we have demonstrated that peroxisomal fission requires a functional DLP1, indicating that Pex11p
, which is supposed to control constitutive peroxisome abundance in mammals (13),
is not directly mediating the vesiculation process of the organelle. In
support of a secondary, indirect role of Pex11p in peroxisome division,
van Roermund et al. (57) proposed a function for yeast
ScPex11p in medium-chain fatty acid
-oxidation rather than in
peroxisomal fission. They favor the idea that ScPex11p is involved in
the transport of fatty acids or cofactors across the peroxisome
membrane and suggest that it is part of a signaling event that
modulates peroxisome proliferation. In contrast, Li and Gould
(58) showed that Pex11p promotes peroxisome division in the absence of
peroxisomal metabolic activity. They suggest that Pex11 proteins play a
direct role in peroxisome division and that their loss inhibits
peroxisome metabolism indirectly. If Pex11p
is more directly
involved in the fission process, it must act upstream of DLP1. But what
could be its function in peroxisome division? The rapid tubulation of
the peroxisomal membrane induced by Pex11p
overexpression (alone or
in combination with a DLP1 mutant) might be indicative of a change in
membrane lipid composition or a modification of peroxisome lipids
mediated by Pex11p
function. Since the association of DLP1 to
peroxisomal membranes was more pronounced after Pex11p
expression,
we speculate that a membrane-modifying activity of Pex11p
could
initiate and/or favor the binding of DLP1 and other factors of the
fission machinery to the peroxisomal membrane.
A Role for DLP1 in Peroxisomal Division--
The function of
mammalian DLP1 is still a matter of debate. DLP1 has been shown to
participate in mitochondrial morphogenesis and fission in mammalian
cells and yeast (27, 28, 31, 32, 34). However, DLP1 is also found to
occur at cytoplasmic sites other than mitochondria, including
microtubules and endoplasmic reticulum cisternae (36, 34, 38), and
evidence for the action in other cellular processes has been reported
(34, 37). The data presented in this study support an additional role
for DLP1 in peroxisomal division. A direct role of DLP1 in peroxisomal fission is substantiated by the localization of DLP1 in spots on
peroxisomal tubules. The DLP1 spots were detected by immunofluorescence of endogenous DLP1 with a DLP1-specific antibody but also with GFP-tagged DLP1. Since the segmentation of elongated peroxisomes has
been connected with peroxisomal fission (13), these morphological observations are likely to be consistent with a direct role of DLP1 in
peroxisomal division. Furthermore, DLP1 was found to be associated with
highly purified peroxisomes from rat liver. Based on our biochemical
results, it is unlikely that this association is due to contamination
of the peroxisomal fraction with mitochondria or microsomes.
Interestingly, the association of DLP1 to peroxisomes was increased
under proliferative conditions induced by the peroxisome proliferator
bezafibrate. In addition, the localization of DLP1 to tubular
peroxisomes was more pronounced after expression of Pex11p
.
Expression of Pex11p
has been shown to result in rapid elongation of
peroxisomes, followed by segregation of peroxisomal proteins and
formation of numerous small peroxisomes (13), thus representing a
strong stimulus for peroxisome proliferation. These observations make
sense, because a recruitment of DLP1 to peroxisomes would be required
during rapid growth and division of the peroxisomal compartment. It
might also explain why an association of DLP1 with peroxisomes has not
yet been noted in other studies.
A most striking observation was the complete inhibition of peroxisome
division after expression of the mutated DLP1-K38A in conjunction with
Pex11p
. The characteristic appearance of small peroxisomes was
completely inhibited by co-expression of DLP1-K38A, and further
tubulation of the organelles as well as the formation of peroxisomal
networks was observed. The peroxisomal network is likely to result from
a shift in the balance between fission and fusion of peroxisomes. A
significant increase in the frequency of elongated peroxisomes was also
observed after expression of DLP1-K38A alone independently of Pex11p
expression, although the organelles appeared to be less elongated. The
expression of a mutated dynamin II had a weak effect on peroxisomal
morphology but did not inhibit peroxisomal division in HepG2 cells.
These observations were confirmed by siRNA-mediated silencing of DLP1 and are consistent with a direct role of DLP1 in peroxisomal fission.
In support of this, Hoepfner et al. (59) have recently
demonstrated that in yeast lacking the dynamin-related protein Vps1, the number of peroxisomes was reduced, and peroxisomes appeared as
large tubular structures. The authors suggest that Vps1p may be
involved in peroxisome fission and consequently in the regulation of
peroxisome abundance in yeast. Interestingly, peroxisomes in vps1
cells were still able to divide, but peroxisome
morphology and number was unaffected in cells lacking Dnm1p. Both yeast
Vps1p and Dnm1p share about 42% homology with mammalian DLP1 (36). Based on a common function in mitochondrial division (21, 27, 28), it
has been inferred that Dnm1p is the yeast homologue to DLP1, but this
is still a matter of debate. Whether DLP1 can be considered a true
mammalian homologue of Vps1p and Dnm1p and whether the mechanism of
peroxisome division is completely comparable between yeast and
mammalian cells has to be elucidated.
How Does DLP1 Tubulate and Divide Peroxisomal Membranes?--
It
has been demonstrated recently that DLP1-K38A mutant protein was able
to bind but not hydrolyze GTP, which resulted in an increased affinity
for membranes (38). Furthermore, recombinant DLP1 was capable of
forming oligomeric protein ring structures in the presence of GTP
S
that were found to deform liposomes into tubules. These findings
demonstrated that, despite the limited homology to conventional
dynamins (35%), DLP1 tubulates and constricts cytoplasmic membranes in
a similar manner. We therefore conclude that hydrolysis of GTP by DLP1
is required for proper fission of peroxisomes. For conventional
dynamin, which is involved in receptor-mediated endocytosis, it has
been shown that it is a membrane-active molecule capable of penetrating
into the acyl chain region of membrane lipids and that local lipid
metabolism can influence dynamin-lipid interactions (60). In contrast
to dynamin, DLP1 lacks a pleckstrin homology or proline-rich domain, known to interact with acidic phospholipids, but was capable of deforming phosphatidylserine-containing liposomes into tubules (38).
Whether the specific interaction of DLP1 with the peroxisomal membrane
is mediated by lipids and/or requires other cytosolic or peroxisomal
membrane proteins has to be elucidated.
Although the exact mechanism of DLP1 action is unclear at present, it
might act as a "peroxisomal pinchase" either by constricting or
destabilizing the peroxisomal membrane, thus leading to peroxisomal fission. Whether members of the dynamin family act as mechanochemical enzymes (pinchase model) or function as a molecular switch regulating downstream effectors of membrane fission (molecular switch model) is
still a matter of debate (61). The striking similarities between
mitochondrial and peroxisomal division and morphogenesis in mammalian
cells may point to a similar, perhaps general, mechanism of
intracellular membrane fission mediated by DLP1. We therefore favor a
model of peroxisome fission similar to the one proposed for
mitochondrial division (29, 21, 30). It is likely that dynamin-related
GTPases act together with accessory proteins (e.g. cytoskeletal motor proteins, lipid-modifying enzymes) and the local
lipid composition in the membrane to mediate constriction and final
scission of peroxisomes.
 |
ACKNOWLEDGEMENTS |
We thank Drs. S. J. Gould (The Johns
Hopkins Unversity) and A. Völkl (University of Heidelberg)
for providing antibodies and cDNA constructs, G. Schneider and W. Sperling (University of Marburg) for excellent technical assistance,
and V. Kramer (University of Marburg) for help with the photographic
work and image processing.
 |
FOOTNOTES |
*
This work was supported in part by a grant of the Medizin
Stiftung (Marburg, Germany) (to A. K.).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.
To whom correspondence should be addressed: Dept. of Cell
Biology and Cell Pathology, University of Marburg, Robert-Koch Str. 5, D-35037 Marburg, Germany. Tel.: 6421-28-63857; Fax: 6421-28-66414; E-mail: schrader@mailer.uni-marburg.de.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M211761200
 |
ABBREVIATIONS |
The abbreviations used are:
DLP1, dynamin-like
protein 1;
PMP, peroxisomal membrane protein;
WT, wild type;
TRITC, tetramethylrhodamine isothiocyanate;
GFP, green fluorescent protein;
MOPS, 4-morpholinepropanesulfonic acid;
siRNA, small interfering
RNA.
 |
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