SUBMICROSCOPIC CYTOCHEMICAL ORGANIZATION OF INTERPHASE NUCLEI REVEALED BY PROTEIN REAGENTS AND GALLOCYANIN-CHROMALUM. A STUDY OF EHRLICH ASCITES CELLS.

Frozen-dried Ehrlich ascites cells have been studied with the aid of acylating, bifunctional arylating, mercaptide- and pyrrole-body forming protein reagents. These were employed as post-fixatives or electron dense stains, or as both. Specimens were treated with anhydrous vapors or non-aqueous solutions of these reagents immediately after freezing-drying, and sections observed in the electron microscope directly. Others, similarly treated, were observed after superimposition of the electron-dense dye-lake, gallocyanin-chromalum, to localize the basophilia of nucleoprotein. All of the treatments, including enzyme digestion controls, were carried out on specimen pellets rather than on sections and required no special water-soluble embedding media. The salient disclosures are: 1. When used alone, certain protein reagents reveal both fibrillar elements (60–100 A wide) and particulate elements (about 125 A) in the nucleolus. The fibrils are rich in protein-bound sulphydryl groups and constitute an anastomotic system. Cytoplasmic particles stained by these reagents are of roughly the same size as those in the nucleolus. 2. Gallocyanin-chromalum stained specimens exhibit image density differences which correspond closely to the familiar, light microscopical images of chromatin, nucleoli, interchromatinic space and cytoplasm yielded by independent cytochemical methods. The nucleoli show a clear differentiation of a dense band of perinucleolar chromatin and less dense ribosomal particles (about 250 A). The latter consist of integral, 3 or more closely clustered unit-particles each measuring 40 A or less. The nucleolar ribosomes together with innumerable, free, 40 A particles seem to occupy spaces corresponding to the interstices of the anastomotic protein fibrils. 3. Ribonuclease abolishes the density of both nucleolar and cytoplasmic particles as revealed subsequently by gallocyanin-chromalum. However, in specimens pretreated with an electron-dense protein reagent prior to enzyme digestion, a low but appreciable density attributable to residual protein persists in cytoplasmic ribosomes. But the 250 A ribosomes and smaller particles of the nucleolus are completely extracted; only the anastomotic pattern of protein fibrils in the nucleolus and the chromatinic structures are left behind. 4. Deoxyribonuclease abolishes the basophilia of the perinucleolar and other chromatinic structure without greatly affecting the ribonuclease-sensitive, nucleolar particles. Extranucleolar particles within the nucleus, on the other hand, were more susceptible to deoxyribonuclease. 5. There is morphologically visualizable suggestive evidence for the view that quantitative, compositional or configurational differences occur between ribosomal particles of the nucleolus and the cytoplasm. The differences involve image density of basophilia and protein. 6. Electron micrographs of favorable sectioning planes disclose nuclear ribosomes (messenger-RNA ?) seemingly in movement from the immediate vicinity of the nucleolus towards the nuclear boundary and cytoplasm. 7. The interchromatinic space is occupied, almost as a continuum, by extremely fine fibrils which can be visualized by means of the denser protein reagents but not gallocyanin-chromalum.