The Morphology of Free and Bound Polysomes in Cells and Subcellular Fractions of Suspension Cultures ofAcer pseudoplatanus

Abstract

In the course of evaluating the subcellular fractions described by Celia, Sala, and Street (1976), certain observations were made regarding the morphology of the free and bound polysomes and are described below. Suspension culture cells of Acer pseudoplatanus were fixed in glutaraldehyde and osmium tetroxide, stained with alcoholic uranyl acetate, embedded in methacrylate styrene, and, after thin sectioning, post-stained with lead citrate (Withers and Cocking, 1972). The polysome fractions were prepared for electron microscopal examination by thin sectioning and negative staining. For thin sectioning, the pelleted bound polysome (microsome) fraction was mixed with a warm solution of 2-5% (w/v) agar, then rapidly cooled. The solidified mixture was cut into small (2 mm3) blocks and treated as above. Negative staining of both polysome fractions was carried out using the method of Nonomura, Blobel, and Sabatini (1971), modified by the addition of bovine serum albumin (0-4 mg ml-1) prior to depositing on a carbon/formvar-coated grid, washing with sucrose-free buffer (Buffer 1 of Celia et al., 1976), and staining with 3% (w/v) aqueous uranyl acetate. Material was examined in an AEI EM802 electron microscope, where necessary using a ±60° tilting stage. The bound and free polysomes differed in morphology as seen in sections of the cells. Bound polysomes occurred in the familiar form of flat spirals on the ER membranes (Nanninga, 1973), double spirals of up to 30 ribosomes being frequently observed (Plate 1a, arrow). The morphology of the free polysomes was less easy to determine due to their random orientation in the cytoplasmic matrix. However, apparent helices were frequently observed. These measured some 40 nm in diameter with four or five units, each 14 nm in diameter, per turn at a pitch of 60 to 70° (Plate 1b). Occasionally there was some indication of a terminal connexion between the polysome and ER membranes (Plate 1b). The polysomes frequently appeared to form extended irregular associations in the cytoplasm (Plate lc). In negatively stained preparations of the free polysome fraction, irregular clumps and linear arrays of ribosomes were common, but up to 30 per cent of them