Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3′-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.