Colocalization of single ribosomes with intermediate filaments in puromycin-treated and serum-starved mouse embryo fibroblasts

Abstract

Previous experiments have revealed a relatively weak electrostatic binding capacity of in vitro reconstituted intermediate filaments (IFs) as well as of natural IFs of whole cell mount preparations for purified ribosomal particles of mammalian origin. In order to demonstrate that such associations also occur in vivo, intact cells were subjected to double immunofluorescence microscopy using antibodies directed against vimentin and ribosomal protein S17. Since in proliferating cells the majority of the ribosomal particles are assembled into polyribosomes and these are to a great extent associated with microfilaments, in vitro cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confocal laser scanning microscopy, the ribosomes were detected in colocalization with vimentin IFs. Disassembly of polyribosomes was also achieved by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vimentin IFs being directly associated with the cell nuclei, radiating into the peripheral areas of the cells or showing a stress fiber-like distribution. In both cases, considerable quantities of ribosomal material were seen in close neighborhood to vimentin IFs. Frequently, these ribosome-IF associations were coaligned with microtubules and they also surrounded myosin I-decorated stress fibers. Double labeling with the vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pattern largely super-imposable on that of ribosomal protein S17. Treatment of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and the ribosomes stayed in contact with the vimentin IFs. On the basis of these results, it is conceivable that IFs play a role in the storage of ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the often seen coalignment of IFs with microtubules and microfilaments might serve facilitated and directional transport of ribonucleoprotein particles from the nucleus to peripheral areas of the cell.