COLOCALIZATION OF SINGLE RIBOSOMES WITH INTERMEDIATE FILAMENTS IN PUROMYCIN-TREATED AND SERUM-STARVED MOUSE EMBRYO FIBROBLASTS

Previous experiments have revealed a relatively weak electrostatic bin ding capacity of in vitro reconstituted intermediate filaments (IFs) a s 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 a nd these are to a great extent associated with microfilaments, in vitr o cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confo cal laser scanning microscopy, the ribosomes were detected in colocali zation with vimentin Ifs. Disassembly of polyribosomes was also achiev ed by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vi mentin Ifs being directly associated with the cell nuclei, radiating i nto the peripheral areas of the cells or showing a stress fiber-like d istribution. In both cases, considerable quantities of ribosomal mater ial were seen in close neighborhood to vimentin Ifs. Frequently, these ribosome-IF associations were coaligned with microtubules and they al so surrounded myosin I-decorated stress fibers. Double labeling with t he vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pa ttern largely superimposable on that of ribosomal protein S17. Treatme nt of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and t he 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 o f ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the ofte n seen coalignment of Ifs with microtubules and microfilaments might s erve facilitated and directional transport of ribonucleoprotein partic les from the nucleus to peripheral areas of the cell. ((C) Elsevier, P aris).