INTERACTION OF MICROTUBULES WITH PEROXISOMES - TUBULAR AND SPHERICAL PEROXISOMES IN HEPG2 CELLS AND THEIR ALTERATIONS INDUCED BY MICROTUBULE-ACTIVE DRUGS

We have studied the interaction of microtubules with peroxisomes and t he influence of changes in the microtubular network on the peroxisomal compartment. From the several cell lines analyzed for this purpose, H epG2 cells proved to be the best candidate exhibiting both a well-deve loped cytoskeleton and a peroxisomal compartment with great plasticity , Three distinct types of peroxisomes: small spherical (0.1-0.3 mu m), rod-shaped (0.5 mu m) and elongated tubular (up to 5 mu m) ones were identified in this cell line. A shift of the elongated tubular forms t o spherical particles was noted by increasing the density of cells in culture, whereas no correlation between the distinct peroxisomal forms and the cellular proliferation could be observed. At time points when the elongated tubular peroxisomes were disappearing, many spherical p eroxisomes arranged like 'chains of beads on a string' were observed, suggesting that the fission of elongated tubular forms may give rise t o newly developing spherical peroxisomes, A clear association of spher ical peroxisomes with microtubules was,visualized by double immunofluo rescence in combination with confocal laser scanning microscopy (CLSM) , Treatment with a variety of microtubule-depolymerizing drugs (colcem id, nocodazole, vinblastine) induced a significant increase in the fre quency of tubular peroxisomes and led to the formation of peroxisomal clusters, These effects were reversible since already 1 to 2 h after r emoval of the drugs from the culture medium, a uniform distribution of spherical peroxisomes was reestablished, Taxol, a microtubule-stabili zing drug, on the other hand exerted no significant effects on the per oxisomal compartment. The direct interaction of microtubules with pero xisomes in vitro was demonstrated using highly purified rat liver pero xisomes and taxol-stabilized microtubules from bovine or pig brain, Th e binding of peroxisomes to microtubules was visualized by video-enhan ced contrast micoscopy (VECM) and was abolished by pretreatment of per oxisomes with 100 mM KCI ('stripping'), proteinase K or trypsin, Incub ation with cytosol restored the binding capacity of KCI-treated peroxi somes, but did not complement the protease treatment, The data present ed provide for the first time evidence for a direct interaction of mic rotubules with the peroxisomal compartment indicating that this cytosk eletal system plays an important role in the morphogenesis and intrace llular distribution of peroxisomes.