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The Importance of Microtubules in Determination of Shape and Intracellular

Distribution of Peroxisomes" MICHAEL SCHRADER, JANIS K. BURKHARDT,b EVELINE BAUMGART,

GEORG LUERS, ALFRED VOLKL, AND H. DARIUSH FAHIMI'

Institute for Anatomy and Cell Biology (I4 University of Heidelberg, and

bEuropean Molecular Biology Laboratory Heidelberg, Germany

The shape, intracellular transport, and distribution of most membrane-bound or- ganelles is determined by their interaction with the cytoskeleton.' Whereas the spe- cific interaction of cytoskeletal elements-especially the microtubular network- with the membranes of the Golgi complex,2 endosomal ~ompartment,~ and mito- chondria4 has been well documented, little information is available on peroxisomes.

In this study we investigated the role of the microtubules in the determination of shape and intracellular distribution of peroxisomes in the well-differentiated human hepatoblastoma cell line, HepG2. We have recently shown4 that peroxisomes in HepG2 cells exhibit several morphologically distinct forms: elongated tubular (up to 5 pm), short rod-shaped (0.5 pm), and small spherical (0.1-0.3 Fm) ones, which are present in neighboring cells in the same culture. Quantitative immunofluorescence studies suggest that the spherical peroxisomes arise by fission from the tubular or rod-shaped ones. Moreover, the transformation of the different peroxisomal forms seems to be dependent on cell density, but is not influenced by cell proliferation.

In normal untreated HepG2 cells peroxisomes, as revealed by immunofluores- cence with an antibody to catalase, are uniformly distributed in the cytoplasm (FIG. Ib). In double immunofluorescence preparations with antibodies to tubulin and cata- lase, a matching pattern for tubulin and peroxisomes is observed (FIG. l a and b). The association of peroxisomes with microtubules in such preparations is best visualized by confocal laser scanning microscopy (not shown here).

Treatment of cells with microtubule-depolymerizing agents (FIG. Ic) (colcemid, nocodazole, vinblastine) induced first a significant increase in the frequency of tubu- lar peroxisomes and later on interfered with the uniform distribution of peroxisomes leading to cluster formation (FIG. l c and d). Taxol, a microtubule-stabilizing drug, had no effect on peroxisomes. The observed effects were completely reversible after withdrawal of the microtubule-depolymerizing drugs and thus were not due to side effects of those substances. These data show that the intactness of the microtubular

"This work was supported by SFB 352 (project C 7) and LFSP of the State of Baden-Wurttem- berg, Germany.

"Address correspondence to Prof. H. D. Fahimi, Department of Anatomy and Cell Biology 11, University of Heidelberg, Im Neuenheimer Feld 307, D-69 120 Heidelberg, Germany.

669

670 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. (a and b) The same HepG2 cell showing the matching distribution of micro- tubules and peroxisomes as revealed by double inimunofluorescence. (e and d) Double ini- munofluorescence. The same cell treated with nocodazole. Note the depolymerization of micro- tubules and the clustering of peroxisomes. (e and f ) In vitvo binding of isolated peroxisomes to microtubules (e). Protease treatment of peroxisomes abolished the binding (0. Magnification: ~640.

SCHRADER et al.: MICROTUBULES AND PEROXISOMES 671

network is of great importance for the distribution of peroxisomes after the fission process. Furthermore, an in vitro binding assay was established using highly purified rat liver peroxisomes isolated by Metrizamide density gradient centrifbgation and Taxol-stabilized microtubules from bovine brain. Video-enhanced microscopy re- vealed strong binding of purified peroxisomes to microtubules in the presence or ab- sence of cytosol (FIG. le). Binding was abolished by pretreatment of peroxisomes with proteases or KCI (FIG. lf), but could be restored after treatment with KCl by the addition of cytosol.

These data show for the first time the specific binding of peroxisomes to micro- tubules, suggesting that microtubules play an important role in the determination of shape and intracellular distribution of peroxisomes.

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