Journal of Histochemistry and Cytochemistry, Vol. 49, 1421-1430, November 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Tubulo–Reticular Clusters of Peroxisomes in Living COS-7 Cells: Dynamic Behavior and Association with Lipid Droplets1

Michael Schradera
a Department of Cell Biology and Cell Pathology, Philipps University, Marburg, Germany

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


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

We characterized more complex peroxisomal structures, i.e., tubulo–reticular 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 49:1421–1429, 2001)

Key Words: peroxisome, motility, peroxisomal reticulum, microtubule, lipid droplet


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 van den Bosch et al. 1992 ; Reddy and Mannaerts 1994 ). Their importance for human health is best exemplified by the discovery of a group of severe inherited metabolic diseases in which peroxisome function is perturbed (Goldfischer et al. 1973 ; Lazarow and Moser 1989 ; Wiemer and Subramani 1994 ).

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 (Hicks and Fahimi 1977 ; Gorgas 1984 ; Yamamoto and Fahimi 1987 ; Roels et al. 1991 ) and recently at the light microscopic level (Schrader et al. 1994 , Schrader et al. 1995 ; Litwin and Bilinska 1995 ). A heterogeneous and more complex peroxisomal compartment has been observed under conditions of rapid growth, e.g., after hepatectomy (Yamamoto and Fahimi 1987 ) or after stimulation of cultured cells with defined growth factors (e.g., nerve growth factor), polyunsaturated fatty acids, or free radicals (Schrader et al. 1998a , Schrader et al. 1999 ), and in tissues actively engaged in the synthesis of special kinds of lipids, e.g., ether glycerolipids and waxes (Gorgas 1987 ). At present, however, little information is available on the exact function of complex peroxisomal structures, the mechanism of their formation, and their dynamic behavior. Recently, we reported on the tubulation and fission of elongated peroxisomes after overexpression of Pex11ßp (Schrader et al. 1998b ). These processes are believed to contribute to peroxisome proliferation in HepG2 cells (Schrader et al. 1996 , Schrader et al. 1998b ). Reticular peroxisomes, which were first proposed by Lazarow et al. 1980 , have been described by serial section reconstructions in lipid-synthezising epithelia and in regenerating liver (Gorgas 1987 ; Yamamoto and Fahimi 1987 ). However, a reticular network has not been studied in cultured cells or under in vivo conditions, and it still remains unclear if it fulfills an important metabolic function or if it plays a role in the formation of new peroxisomes by budding or fission out of a preexisting compartment (Wilcke et al. 1995 ).

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 tubulo–reticular peroxisomal clusters and we report here on their association with lipid droplets.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Schrader et al. 1998b ) or by incubation with polyethylenimine (25 kD PEI; Sigma Aldrich, Deisenhofen, Germany). For PEI transfection (Fischer et al. 1999 ; Bieber and Elsasser 2001 ), 2.4 x 104 cells/cm2 were seeded on coverslips (in 6-cm dishes) and after 24 hr the medium was changed to a volume of 2.5 ml. For three dishes, 10 µg plasmid and 100 µl of PEI stock solution (0.9 mg/ml, pH 7.4) were diluted to 750 µl each with 150 mM NaCl. After 10 minutes at room temperature the PEI solution was pipetted to the DNA and after 10 more min 500 µl of the PEI/DNA solution was added to the cells. After 6 hr the medium was changed and cells were processed for indirect immunofluorescence or live cell imaging 24–48 hr after transfection.

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) (Kalish et al. 1996 ). Rabbit anti-PMP70 (Luers et al. 1993 ), rabbit anti-acyl-CoA oxidase and, rabbit anti-catalase polyclonal antibodies (Beier et al. 1988 ) were a gift from Dr. A. Völkl (University of Heidelberg; Heidelberg, Germany). Species-specific anti-IgG antibodies conjugated to TRITC were obtained from Dianova (Hamburg, Germany).

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 (Schrader et al. 1995 ). Transfected cells were processed for immunofluorescence 24–48 hr after transfection. For double-labeling experiments, transfected cells were incubated with antibodies to peroxisomal marker proteins and subsequently with goat anti-rabbit IgG conjugated to TRITC. 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 Kodak TMY film or digitalized. Deconvolution studies were performed with a Delta Vision system (Applied Precision; Issaquah, WA) using an Axiovert S100 2T microscope (Carl Zeiss) for optical sectioning. The deconvolved images in Fig 1F and Fig 1G were taken at 200-nm intervals with a x100 Plan Apo objective with a 1.4 numerical aperture through focus.



View larger version (90K):
[in this window]
[in a new window]
 
Figure 1. Tubulo–reticular peroxisomal clusters can be visualized in COS-7 cells by GFP-PTS1. Fluorescence localization of GFP-PTS1 in living (A) and fixed (C–G) COS-7 cells. (B) Localization of PMP70, a peroxisomal membrane protein. (C–E) Higher-magnification view of tubulo–reticular clusters of peroxisomes seen by GFP-PTS1 fluorescence. (F,G) Deconvolved images of peroxisomal clusters (see Materials and Methods for details). (F) A ring-like peroxisome (arrow) with tubular extensions is shown. Arrows point to peroxisomal clusters (A,B) or interconnections of peroxisomes (F,G). N, nucleus. Bars: A–E,G = 5 µm; F = 2.5 µm.

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 (Greenspan et al. 1985 ). Alternatively, lipid droplets in fixed cells were stained with Sudan black (Serva; Heidelberg, Germany) (0.1% in 70% ethanol) for 20 min. Excess dye was removed by washing in PBS. GFP and Nile red were localized simultanously using a FITC filter set. For quantitative evaluations, 100–200 cells per coverslip (with three to five coverslips per preparation) were examined at the fluorescence microscope and categorized according to the size of lipid droplets. The data in Fig 5 are from three or four independent experiments. Measurements of the size of lipid droplets were performed on photographs at x1000–2000.



View larger version (29K):
[in this window]
[in a new window]
 
Figure 2. Tubulo–reticular peroxisomes in COS-7 cells are transient structures. (A,B) Accumulations of spherical peroxisomes after breakdown of peroxisomal clusters in COS-7 cells labeled with GFP-PTS1. (C) Breakdown and distribution of elongated peroxisomes in COS-7 cells. Peroxisomes were labeled by GFP-PTS1 and observed in vivo by time-lapse microscopy. Elapsed time is in minutes. Bars = 5 µm.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 3. Time-lapse imaging of GFP-PTS1 labeled peroxisomal clusters in living COS-7 cells. The dynamic nature of the clusters and the changes in morphology are shown. Arrowheads point to regions where interconnections are detached, whereas arrows point to regions where connections are formed. Elapsed time is in seconds. Bar = 5 µm.



View larger version (38K):
[in this window]
[in a new window]
 
Figure 4. Co-localization of GFP-PTS1-labeled peroxisomal clusters and lipid droplets in COS-7 cells stained with Nile red. (A) Single GFP-PTS1-labeled peroxisomes (green, arrows) can be observed in association with or close to lipid droplets (yellow) in COS-7 cells stained with Nile red. (B–E) Higher-magnification view showing peroxisomal clusters (green, arrowheads) which are in close association with lipid droplets (yellow). Small lipid droplets can be observed associated with the tips of elongated peroxisomes (D,E, arrows). Bars = 5 µm.



View larger version (24K):
[in this window]
[in a new window]
 
Figure 5. Correlation between the number of peroxisomal clusters, the size of lipid droplets, and cell density. (A) Influence of cell density on the size of lipid droplets in COS-7 cells. COS-7 cells were seeded at the cell density indicated and were processed after 48 hr. Lipid droplets were stained with Nile red or Sudan black and cells were analyzed as described in Materials and Methods. At a density of 1x cells appeared well separated and single, whereas at a density of 12x they were highly confluent. (B) Time course experiment showing the number of cells with peroxisomal clusters and with enlarged lipid droplets after prolonged time in culture. Peroxisomes were labeled with GFP-PTS1 or with antibodies to the peroxisomal membrane protein PMP70. Lipid droplets were stained with Nile red or Sudan Black. Cells were seeded at a density of 0.5x (x = 3 x 103 cells/3.5 cm2) at Day 0 and reached a density of 10–12x after 5–6 days. Data are presented as mean ± SD from three or four independent experiments.

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 15–30 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 1–4 hr. Real-time imaging studies were performed using high-magnification video-enhanced fluorescence microscopy as described (Valetti et al. 1999 ; Schrader et al. 2000 ) and the motile events recorded on video tape. Two or three coverslips were observed per experiment and between five and 10 different fields were viewed on each coverslip. For analysis of real-time imaging data, the tracks of individual GFP-labeled peroxisomes were traced from the video monitor onto transparency sheets and the distance of each translocation measured manually. The start and stop frames corresponding to each movement were used to determine the duration and to calculate velocities. A movement was defined as a saltation (at a single velocity) of 0.5 µm or longer. For peroxisomes undergoing multiple movements, each individual saltation was scored separately.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

COS-7 Cells Contain Tubulo–reticular 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 (Schrader et al. 1994 , Schrader et al. 1995 ), some COS-7 cells contained clusters of peroxisomes (Fig 1B). To exclude fixation artifacts, COS-7 cells were transfected with a cDNA construct encoding a GFP-PTS1 fusion protein. When live cells were observed, peroxisomal clusters similar to the ones in fixed cells could be seen (Fig 1A). The GFP-PTS1 fusion protein was targeted exclusively to peroxisomes, as indicated by its co-localization with different peroxisomal marker proteins in fixed cells (not shown). Usually, one to seven clusters of peroxisomes varying in size from 5 to 15 µm were found in approximately 20% of the cells under routine culture conditions (Fig 5B). The clusters were also positive for other peroxisomal marker proteins (e.g., catalase, acyl CoA oxidase; not shown).

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 tubulo–reticular 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 3–4 days in culture, with a maximum after 5–6 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 20–30 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, tubulo–reticular 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 (Rapp et al. 1996 ; Huber et al. 1997 ; Wiemer et al. 1997 ).

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 (Rapp et al. 1996 ; Huber et al. 1997 ; Wiemer et al. 1997 ). The dynamic properties of the microtubule-dependent movements of morphologically different individual peroxisomes in COS-7 cells were analyzed in detail and compared to those of HepG2 cells, which were also transfected with the GFP-SKL construct and observed in vivo (Table 1). The morphological and biochemical properties of the peroxisomes in HepG2 cells have been well characterized in recent studies (Schrader et al. 1994 , Schrader et al. 1996 ). Our analysis revealed that the frequency of microtubule-dependent movements was higher in COS-7 than in HepG2 cells. This was also true for the microtubule-dependent movements of other subcellular organelles, e.g., endosomes and lysosomes, which were labeled with Cy3–{alpha}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 (Rapp et al. 1996 ) and CV-1 cells (Wiemer et al. 1997 ) (Table 1) and might be a general feature of microtubule-based peroxisomal motility.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Analysis of microtubule-dependent peroxisomal movementsa

Peroxisomal Clusters Are Associated with Lipid Droplets
Because it is known that peroxisomes play important functions in lipid metabolism (van den Bosch et al. 1992 ; Reddy and Mannaerts 1994 ) and can be found associated with lipid droplets in adipocytes (Blanchette 1966 ; Novikoff et al. 1980 ; Blanchette-Mackie et al. 1995 ), we stained lipid droplets in GFP-PTS1-transfected COS-7 cells with Nile red and performed co-localization studies (Fig 4). Single peroxisomes were occasionally observed to be associated with lipid droplets (Fig 4A) as described for adipocytes (Blanchette 1966 ; Novikoff et al. 1980 ; Blanchette-Mackie et al. 1995 ). Surprisingly, about 60% of the peroxisomal clusters were associated with lipid droplets (Fig 4B–4E). Small lipid droplets were frequently observed at the tips of elongated peroxisomes.

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 (35–40% 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 tubulo–reticular peroxisomal clusters in lipid metabolism in conjunction with lipid droplets.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 tubulo–reticular peroxisomal clusters in COS-7 cells are associated with lipid droplets and correlate with their size and with cell density.

Peroxisomal Tubulo–Reticular 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 tubulo–reticular networks of peroxisomes in living cells that are reminiscent of the "peroxisomal reticulum" postulated by Lazarow et al. 1980 . These authors suggested that peroxisomes may be transiently interconnected, if not permanently, by fission and fusion, into a "peroxisomal reticulum." The reticula observed in vivo in COS-7 cells exhibited highly dynamic, microtubule-independent movements and were engaged in transient contacts. Whether or not these interactions facilitate the metabolic functions of peroxisomes and represent fusion events is now under investigation.

Like the tubular peroxisomes described recently in HepG2 cells, the tubulo–reticular 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 24–48 hr in culture (Schrader et al. 1996 ), whereas peroxisomal clusters in COS-7 cells were more common at higher cell densities. The formation of peroxisomal clusters has also been described in HepG2 cells after treatment with microtubule-depolymerizing agents (Schrader et al. 1996 ) or during maturation of peroxisomes in differentiating hepatoblastoma cells after a prolonged time in culture (Stier et al. 1998 ). Furthermore, their association with lipid droplets in COS-7 cells supports a role in lipid metabolism rather than in peroxisome proliferation, as described for tubular peroxisomes in HepG2 cells (Schrader et al. 1996 , Schrader et al. 1998b ). Interestingly, reticular peroxisomes were described by serial section reconstruction in lipid-synthesizing epithelia (Gorgas 1987 ) and regenerating rat liver (Yamamoto and Fahimi 1987 ). An elaborate peroxisomal network was found in tissues actively engaged in the synthesis of special kinds of lipids (Gorgas 1987 ), whereas in regenerating rat liver the reticulum consisted of focal interconnections between three to five dilated peroxisomes (Yamamoto and Fahimi 1987 ). Previous studies have shown tubular and segmented structures to be a characteristic feature of the peroxisomal compartment in a variety of cell lines (Schrader et al. 1995 , Schrader et al. 1996 ; Litwin and Bilinska 1995 ), but a reticulum had not been observed thus far under culture conditions. This failure might be due to the transient nature and the enormous plasticity of complex peroxisomal structures, and to the technical approach selected for their detection. Their preservation requires special precautions in fixation (Schrader et al. 1995 ). In addition, reticular structures are not easily resolved by conventional fluorescence microscopy. Studies with other cell lines using deconvolution microscopy indicate that reticular structures are more spread than previously expected (unpublished observation).

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; Rapp et al. 1996 ; Wiemer et al. 1997 ). Furthermore, peroxisome velocities and distances traveled were similar to the motile properties of other intracellular organelles, e.g., endosomes and lysosomes in COS-7 cells or hepatocytes (Valetti et al. 1999 ; Schrader et al., unpublished observations). The physiological relevance of these motile events is poorly understood, but translocations might be a prerequisite for the interaction of individual peroxisomes (Schrader et al. 2000 ). Furthermore, little is known about the molecular basis of peroxisomal movement. In a recent study (Schrader et al. 2000 ; Thiemann et al. 2000 ), we obtained evidence for the involvement of the dynein/dynactin complex in peroxisomal motility. Peroxisomal movements could be inhibited by disrupting dynein function either by overexpression of the dynamitin subunit of dynactin (Echeverri et al. 1996 ), or by microinjection of a function-blocking antibody to the dynein intermediate chain. These observations support a role for the dynein/dynactin motor in peroxisome motility and indicate that besides the complex import machinery other multiprotein complexes are present on the peroxisomal membrane which are not only required for regulated tethering to and translocation along microtubules but also for fission and, presumably, even fusion events of the peroxisomal compartment. It is a great challenge for future investigations to characterize further components of these machineries and to determine how they are regulated.


  Footnotes

1 Presented at the Symposium on Peroxisomes, ICHC 2000, XIth International Congress of Histochemistry and Cytochemistry, York, UK, 2000.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Beier K, Völkl A, Hashimoto T, Fahimi HD (1988) Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting. Eur J Cell Biol 46:383-393[Medline]

Bieber T, Elsässer HP (2001) Preparation of a low molecular weight polyethylenimine for efficient cell transfection. BioTechniques 30:74-77[Medline]

Blanchette EJ (1966) Ovarian steroid cells. II. Lutein cell. J Cell Biol 31:517-543[Abstract/Free Full Text]

Blanchette–Mackie E, Dwyer NK, Barber T, Coxey RA, Takeda T, Rondinone CM, Theodorakis JL, Greenberg AS, Londos C (1995) Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes. J Lipid Res 36:1211-1226[Abstract]

Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB (1996) Molecular characterization of the 50kD subunit of dynactin reveals a function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol 132:617-633[Abstract]

Fischer D, Bieber T, Li Y, Elsässer HP, Kissel T (1999) A novel non-viral vector for DANN delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm Res 16:1273-1279[Medline]

Goldfischer S, Moore CL, Johnson AB, Spiro AJ, Valsamis MP, Wisniewski HK, Ritch RH, Norton WT, Rapin I, Gartner LM (1973) Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science 182:62-64[Medline]

Gorgas K (1984) Peroxisomes in sebaceous glands.V. Complex peroxisomes in the mouse preputial gland: serial sectioning and three dimensional reconstruction studies. Anat Embryol 169:261-270[Medline]

Gorgas K (1987) Morphogenesis of peroxisomes in lipid-synthesizing epithelia. In Fahimi HD, Sies H, eds. Peroxisomes in Biology and Medicine. Berlin, Heidelberg, Springer-Verlag, 3-17

Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965-973[Abstract]

Hicks L, Fahimi HD (1977) Peroxisomes (microbodies) in the myocardium of rodents and primates. Cell Tissue Res 175:467-481[Medline]

Huber CM, Saffrich R, Anton M, Passreiter M, Ansorge W, Gorgas K, Just WW (1997) A heterotrimeric G protein-phospholipase A2 signaling cascade is involved in the regulation of peroxisomal motility in CHO cells. J Cell Sci 110:2955-2968[Abstract/Free Full Text]

Kalish JE, Keller GA, Morrell JC, Mihalik SJ, Smith B, Cregg JM, Gould SJ (1996) Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins. EMBO J 15:3275-3285[Abstract]

Lazarow PB, Moser HW (1989) Disorders of peroxisome biogenesis. In Beaudet AL, Scriver CR, Sly WS, Vallel D, eds. The Metabolic Basis of Inherited Disease. 6th ed New York, McGraw–Hill. 1479–1509Vol. II

Lazarow PB, Shio H, Robbi M (1980) Biosynthesis of peroxisomes and the peroxisome reticulum hypothesis. In Bucher T, Sebald W, Weiss H, eds. 31st Mosbach Colloquium. Biological Chemistry of Organelle Formation. New York, Springer-Verlag, 187-206

Litwin JA, Bilinska B (1995) Morphological heterogeneity of peroxisomes in mouse Leydig cells. Folia Histochem Cytobiol 33:255-258[Medline]

Lüers G, Hashimoto K, Fahimi HD, Völkl A (1993) Biogenesis of peroxisomes: Isolation and characterization of two distinct peroxisomal populations from normal and regenerating rat liver. J Cell Biol 121:1271-1280[Abstract]

Novikoff AB, Novikoff PM, Rosen OM, Rubin CS (1980) Organelle relationships in cultured 3T3-L1 preadipocytes. J Cell Biol 87:180-196[Abstract]

Rapp S, Saffrich R, Anton M, Jäkle U, Ansorge W, Gorgas K, Just W (1996) Microtubule-based peroxisome movement. J Cell Sci 109:837-849[Abstract/Free Full Text]

Reddy JK, Mannaerts GP (1994) Peroxisomal lipid metabolism. Annu Rev Nutr 14:343-370[Medline]

Roels FM, Espeel M, Pauwels M, De Craemer D, Egberts HJA, Van der Spek P (1991) Different types of peroxisomes in human duodenal epithelium. Gut 32:858-865[Abstract]

Schrader M, Baumgart E, Fahimi HD (1995) Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system. Histochem J 27:615-619[Medline]

Schrader M, Baumgart E, Völkl A, Fahimi HD (1994) Hetero-geneity of peroxisomes in human hepatoblastoma cell line HepG2. Evidence of distinct subpopulations. Eur J Cell Biol 64:281-294[Medline]

Schrader M, Burkhardt JK, Baumgart E, Lüers G, Spring H, Völkl A, Fahimi HD (1996) Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG 2(cells and their alterations induced by microtubule-active drugs. Eur J Cell Biol 69):24-35

Schrader M, King SJ, Stroh TA, Schroer TA (2000) Real-time imaging reveals a peroxisomal reticulum in living cells. J Cell Sci 113:3663-3671[Abstract/Free Full Text]

Schrader M, Krieglstein K, Fahimi HD (1998a) Tubular peroxisomes in HepG2 cells: selective induction by growth factors and arachidonic acid. Eur J Cell Biol 75:87-96[Medline]

Schrader M, Reuber BE, Morrell JC, Sanchez GG, Obie C, Stroh TA, Valle D, Schroer TA, Gould SJ (1998b) Expression of PEX11ß mediates peroxisome proliferation in the absence of extracellular stimuli. J Biol Chem 273:29607-29614[Abstract/Free Full Text]

Schrader M, Wodopia R, Fahimi HD (1999) Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells. J Histochem Cytochem 47:1141-1148[Abstract/Free Full Text]

Stier H, Fahimi HD, Van Veldhoven PP, Mannaerts GP, Völkl A, Baumgart E (1998) Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha). Differentiation 64:55-66[Medline]

Thiemann M, Schrader M, Völkl A, Baumgart E, Fahimi HD (2000) Interaction of peroxisomes with microtubules: in vitro studies using a novel peroxisome-microtubule binding assay. Eur J Biochem 267:6264-6275[Abstract/Free Full Text]

Valetti C, Wetzel DM, Schrader M, Hasbani MJ, Gill SR, Kreis TE, Schroer TA (1999) Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170. Mol Biol Cell 10:4107-4120[Abstract/Free Full Text]

van den Bosch H, Schutgens RBH, Wanders RJA, Tager JM (1992) Biochemistry of peroxisomes. Annu Rev Biochem 61:157-197[Medline]

Wiemer EAC, Subramani S (1994) Protein import deficiencies in human peroxisomal disorders. Mol Genet Med 4:119-152[Medline]

Wiemer EAC, Wenzel T, Deerinck TJ, Ellisman MH, Subramani S (1997) Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules. J Cell Biol 136:71-80[Abstract/Free Full Text]

Wilcke M, Hultenby K, Alexson SEH (1995) Novel peroxisomal populations in subcellular fractions from rat liver. J Biol Chem 270:6949-6958[Abstract/Free Full Text]

Yamamoto K, Fahimi HD (1987) Three-dimensional reconstruction of a peroxisomal reticulum in regenerating rat liver: evidence of interconnections between heterogeneous segments. J Cell Biol 105:713-722[Abstract]