ARTICLE |
Correspondence to: Michael Schrader, Dept. of Cell Biology and Cell Pathology, University of Marburg, Robert-Koch Str. 5, 35037 Marburg, Germany. E-mail: schrader@mailer.uni-marburg.de
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Summary |
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We characterized more complex peroxisomal structures, i.e., tubuloreticular peroxisomal clusters, in greater detail under in vivo conditions in COS-7 cells that were transfected with a GFP-PTS1 fusion protein. Live cell imaging revealed the dynamic nature of peroxisomal clusters and allowed a detailed analysis of the motile properties of a heterogeneous peroxisome population. Furthermore, peroxisomal clusters were found to be associated with lipid droplets. The frequency of peroxisomal clusters correlated with an increase in cell density and in the size of lipid droplets. These data provide further evidence for the dynamic nature of the peroxisomal compartment and indicate that peroxisomal clusters have a function in lipid metabolism. (J Histochem Cytochem 49:14211429, 2001)
Key Words: peroxisome, motility, peroxisomal reticulum, microtubule, lipid droplet
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Introduction |
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Peroxisomes are ubiquitous subcellular organelles that are involved in a variety of important metabolic pathways, particularly those linked to the lipid metabolism, such as the biosynthesis of ether phospholipids and bile acids, and in the catabolism of very long-chain fatty acids, prostaglandins, and leukotriens (reviewed in
Microscopic observations reveal most mammalian peroxisomes to be spherical. However, the peroxisomal compartment is highly plastic and complex. Several morphologically distinct types of peroxisomes, including elongated, tubular organelles, have been described in various mammalian tissues and cell lines, first by electron microscopic studies (
In this study, we used GFP-PTS1 labeling of COS-7 and HepG2 cells to characterize more complex peroxisomal structures in greater detail under in vivo conditions. We were able to document the existence and the dynamic nature of tubuloreticular peroxisomal clusters and we report here on their association with lipid droplets.
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Materials and Methods |
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Cell Culture
HepG2 and COS-7 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 g/liter sodium bicarbonate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS) (all from Gibco BRL; Gaithersburg, MD) at 37C in a humidified atmosphere containing 5% CO2.
Transfection Experiments
HepG2 and COS-7 cells were either transfected by electroporation as described (
cDNAs and Antibodies
pGFP-PTS1, encoding the S65T mutant from the green fluorescent protein (GFP) and the PTS1 under the control of the CMV promoter, was a gift from Dr. S. J. Gould (Johns Hopkins School of Medicine; Baltimore, MD) (
Indirect Immunofluorescence and Deconvolution
Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS, pH 7.4, permeabilized with 0.2% Triton X-100, and incubated with primary and secondary antibodies as described (
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Staining of Lipid Droplets
For staining of lipid droplets, cells were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min and incubated with a 1:1000 dilution of a Nile red (Molecular Probes; Leiden, The Netherlands) stock solution (100 µg/ml in acetone) for 5 min (
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Nocodazole Treatment
Cells transfected with the GFP-PTS1 expression construct were treated with 15 µM nocodazole by adding the microtubule-destabilizing agent directly to the live cell assay (see below) and incubating for 1530 min.
Live Cell Imaging
Cells transfected with GFP-PTS1 cDNA were grown in culture dishes with a glass bottom. For time-lapse studies they were placed in a temperature- and CO2-controlled chamber (Carl Zeiss; CTI Controller 3700, TRZ 3700) on a heating stage of a Zeiss LSM 410 inverted microscope equipped with a x63/1.4 objective. As light source, an argon ion laser with wavelength of 488 nm and appropriate filter combinations were used. Images were collected at intervals of 5 sec up to 5 min, and the cells were monitored at various time intervals for a total of 14 hr. Real-time imaging studies were performed using high-magnification video-enhanced fluorescence microscopy as described (
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Results |
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COS-7 Cells Contain Tubuloreticular Clusters of Peroxisomes
For morphological characterization of their peroxisomal compartment, COS-7 cells were processed for indirect immunofluorescence and stained with antibodies directed to the peroxisomal membrane protein PMP70 (Fig 1B). In addition to a heterogeneous mixture of spherical and tubular peroxisomal structures similar to the ones described in HepG2 cells (
Some of the clusters resembled accumulations of tubulo-reticular peroxisomes (Fig 1B, Fig 1D, and Fig 1E), whereas others had a more compact, grape-like structure (Fig 1A, Fig 1C, and Fig 3). By conventional imaging methods, the immunofluorescence signal of these clusters appeared mostly as a bright focus of material (Fig 1A and Fig 1B). More detail was provided using previously developed deconvolution software (Delta Vision) (Fig 1F and Fig 1G). After deconvolution, interconnections between tubular peroxisomes were visible, forming tubuloreticular structures (Fig 1F and Fig 1G). A ring-like structure with tubular extensions is shown in Fig 1F. Furthermore, peroxisomal clusters were found to be dynamic and transient in nature. In time course experiments, an increase of the number of cells with clusters could be observed after 34 days in culture, with a maximum after 56 days, which was then followed by a slight decrease (Fig 5B). Accumulations of spherical peroxisomes were observed after prolonged time in culture and are suggested to result from the breakdown of peroxisomal clusters (Fig 2A and Fig 2B). The breakdown of elongated peroxisomes into spherical organelles could be demonstrated by live cell imaging of COS-7 cells expressing the GFP-PTS1 fusion protein (Fig 2C). The fission of elongated peroxisomes and the separation/distribution of spherical peroxisomes took about 2030 min. Although the complete breakdown of peroxisomal clusters could not yet be demonstrated in live cells, a similar mechanism is suggested.
Live Cell Imaging of Peroxisomal Clusters and Single Peroxisomes
To analyze the dynamic properties of the peroxisomal clusters, live cell imaging of COS-7 cells expressing the GFP-PTS1 fusion protein was performed (Fig 3). Interestingly, tubuloreticular clusters exhibited highly dynamic movements which were accompanied by drastic changes in shape (Fig 3). Remodeling of these structures could be observed, involving the formation and/or detachment of tubular processes that appeared to interconnect adjacent peroxisomes (Fig 3). These movements were not inhibited by nocodazole treatment, suggesting that they were microtubule-independent. Such short-range, non-directed oscillatory movements were seen for the majority of the peroxisome population in a given cell (90%). All morphologically distinct peroxisomal structures exhibited random, vibration-like, microtubule-independent movements similar to the ones described recently in CHO and CV-1 cells (
A small subset (10%) of the peroxisomes in COS-7 cells underwent fast directional movement that could be blocked completely by nocodazole, indicating that it was microtubule-dependent. Similar observations have been made using CHO and CV-1 cells transfected with a GFP-PTS1 construct (
2-macroglobulin (not shown), indicating cell-specific differences in organelle motility. Nevertheless, peroxisomes in HepG2 cells exhibited similar velocities (mean 0.58 µm/sec) and moved over comparable distances (up to 10 µm) as in COS-7 cells (Table 1). It should be noted that spherical as well as elongated peroxisomes moved in a microtubule-dependent manner, although the movements of spherical organelles were more frequent. However, extremely elongated tubules (>3 µm), reticular structures, or peroxisomal clusters in COS-7 cells were not observed to translocate along microtubules. Elongated peroxisomes moved with slightly lower velocities than spherical ones, but over longer distances (Table 1). Furthermore, peroxisome velocities and distances traveled in COS-7 and HepG2 cells were comparable to the motile properties of GFP-labeled peroxisomes reported for CHO (
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Peroxisomal Clusters Are Associated with Lipid Droplets
Because it is known that peroxisomes play important functions in lipid metabolism (
The size of the lipid droplets was highly dependent on the cell density in culture (Fig 5A). COS-7 cells with small lipid droplets (<0.9 µm in diameter) were more frequent at lower density (3540% of the cells at 3 x 103 cells/3.5 cm2) and decreased in frequency with higher cell densities. In contrast, cells with larger lipid droplets (>2.0 µm in diameter) were more frequent at higher cell densities. Interestingly, peroxisomal clusters were also observed to be more frequent at higher cell densities (Fig 5B). In a dynamic culture situation, the number of cells with peroxisomal clusters increased with time and increasing cell density. The increase in peroxisomal clusters was accompanied by an increase in the number of cells with enlarged lipid droplets. After a maximum at Days 5 and 6, a slight decrease was observed. This decrease is presumably caused by an unefficient supply of the cells with nutrition due to the high cell density. At any time, about 60% of the peroxisomal clusters were found to be associated with lipid droplets, mostly with the larger ones. However, the size of the peroxisomal clusters was increased after prolonged time in culture. These findings support a special role for tubuloreticular peroxisomal clusters in lipid metabolism in conjunction with lipid droplets.
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Discussion |
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In this study, we used GFP-PTS1 labeling of cultured cells to characterize more complex peroxisomal structures in more detail under in vivo conditions. We analyzed the microtubule-dependent movements of a heterogeneous peroxisome population in greater detail and provide insight into the dynamic behavior of peroxisomal clusters using live cell imaging. Furthermore, our data indicate that tubuloreticular peroxisomal clusters in COS-7 cells are associated with lipid droplets and correlate with their size and with cell density.
Peroxisomal TubuloReticular Clusters in COS-7 Cells
As indicated, GFP-PTS1 targeting to peroxisomes provides a useful tool for the in vivo labeling of a variety of morphologically distinct and complex peroxisomal structures, including tubules as well as reticula. GFP-PTS1 labeling of COS-7 cells revealed the existence and the dynamic nature of tubuloreticular networks of peroxisomes in living cells that are reminiscent of the "peroxisomal reticulum" postulated by
Like the tubular peroxisomes described recently in HepG2 cells, the tubuloreticular clusters were transient in nature and were found to divide into spherical peroxisomes. However, the formation and breakdown of tubular peroxisomes in HepG2 cells was most prominent at low cell densities after 2448 hr in culture (
Microtubule-based Motility of Peroxisomes
Our detailed live cell imaging analysis of microtubule-based peroxisomal movement revealed some general features of peroxisomal motility: (a) microtubule-based peroxisome movement might be a low-frequency event; (b) peroxisomes do not show a high degree of bidirectional movements, which is a characteristic of particles involved in trafficking of proteins and membranes (unpublished observations); (c) peroxisomal movements occur in all areas of the cytoplasm and show no direction preference, and organelles move towards and away from the cell center in a random fashion; and (d) peroxisomes can move over very long distances. The motile properties of peroxisomes in both HepG2 and COS-7 cells were comparable to data obtained in other cell lines by time-lapse microscopy (Table 1;
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Footnotes |
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1 Presented at the Symposium on Peroxisomes, ICHC 2000, XIth International Congress of Histochemistry and Cytochemistry, York, UK, 2000.
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Acknowledgments |
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Supported by a fellowship of the Deutsche Forschungsgemeinschaft (DFG) to MS.
I am grateful to Drs S.J. Gould and A. Völkl for providing antibodies and cDNA constructs. I thank Dr H.-P. Elsässer for assistance with image acquisition. The technical assistance of Katharina Elsässer and Waltraud Sperling is gratefully acknowledged.
Received for publication November 27, 2000; accepted June 18, 2001.
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