Morphological characteristics and ganglioside composition of substratum adhesion sites in a hepatoma cell line (CMH5123 cells) during different phases of growth

Abstract

This study compares the ganglioside composition of tissue culture substrate-attached material (SAM) with that of cell bodies in a line of transformed hepatocytes derived from the minimal deviation Morris hepatoma 5123c (CMH5123 cells). We examined both confluent cultures (late-phase cultures) and cells which were allowed to attach for only 3 h (early-phase cultures). We also determined to what extent ganglioside compositions of SAM and cell bodies from early- and late-phase cultures of CMH5123 cells are affected by the block of complex ganglioside biosynthesis induced by treatment with chelating agents (EGTA+EDTA). The morphological characteristics of SAM were monitored by scanning electron microscopy during the different steps of this study. In early-phase cultures, SAM was composed of fragments of filopodia and small vesicles probably representing newly formed substratum adhesion sites. In contrast, SAM of late-phase cultures was made up of large pools of membranous material resulting from the breakage of thick retraction fibers connecting the cell body with broad, mature adhesion sites. SAM of early-phase cultures yielded ganglioside profiles with a higher content of GM1 and GD1 a than those of cell bodies, while in late-phase cultures there was no difference between SAM and cell body gangliosides. When cells were grown in the presence of chelating agents, SAM of earlyphase cultures was composed of vesicles and filopodial fragments similar to those found in early-phase cultures grown in regular media; these morphological features also appeared in SAM of confluent cultures (in contrast to the membranous material characteristic of latephase cultures grown in regular media). In early-phase cultures grown in the presence of chelating agents, gangliosides of SAM were enriched in complex homologs relative to their content in cell bodies. These ganglioside characteristics were also found in SAM of confluent cultures grown in the presence of chelating agents, reflecting the presence of newly formed adhesion sites. On the basis of these results, we may conclude that the molecular assembly of newly formed adhesion sites implies the preferential distribution of several surface components involved in cell adhesion, including complex gangliosides.