EXPRESSION OF FIBRONECTIN, LAMININ AND RIBOSOMES IN NORMAL AND NOCODAZOLE-TREATED NEONATAL HEART-CELLS IN CULTURE - A STUDY BY LASER-SCANNING CONFOCAL MICROSCOPY AND IMMUNOCYTOCHEMISTRY

Polyclonal and monoclonal antibodies were used to examine the effects of the synthetic microtubule disruptive drug nocodazole on the subcell ular expression of fibronectin, laminin, and ribosomes in primary cult ures of neonatal cardiac ventricular cells. Non-invasive serial optica l sectioning was carried out by immunolaser scanning confocal microsco py. In addition, fibronectin and laminin were immunolabelled with pero xidase or gold conjugates for electron-microscopic examination. Immuno labelling for the large 60S ribosome subunit in fibroblast-like non-my ocytes showed that punctate ribosome structures with a multi-subunit c omposition were present in perinuclear region. Double immunostaining w ith antibodies directed against ribosomes and cellular fibronectin ind icated that the punctate structures were cisternae of the rough endopl asmic reticulum. No clear effects of nocodazole treatment were detecte d on the distribution of cytoskeleton-bound ribosomes. Following immun olabelling for both glycoproteins and double immunolabelling for cellu lar fibronectin and the 60 S ribosome subunit, fibronectin and laminin were found in the perinuclear cisternae of the rough endoplasmic reti culum and in pleomorphic secretory vesicles. The cisternal stacks of t he Golgi complex appeared either unstained or were only weakly labelle d. When these cells were exposed to nocodazole, fibronectin and lamini n accumulated in peripheral parts of the cytoplasm, including cellular processes. These peripheral accumulations of immunostaining for fibro nectin and laminin did not reflect Golgi staining, as shown by double labeling experiments versus wheat-germ-agglutinin staining, and, by ex posing cultures to a high dose of brefeldin A.