HIGH-RESOLUTION VIEW OF THE TRUE CYTOSOLIC MEMBRANE-SURFACE OF PHAGOSOMES OF KNOWN AGES PURIFIED FROM PARAMECIUM

Techniques were used for viewing the true cytosolic surfaces of the me mbranes of intracellular organelles by field emission scanning electro n microscopy (SEM). Cells of Paramecium multimicronucleatum were fed b riefly with magnetic beads followed by a chase which advanced the newl y formed digestive vacuoles (DVs) to predetermined ages. These bead-co ntaining phagosomes were isolated from the whole cell homogenates with a magnet and were determined to be intact by fluorescence microscopy. Antigenically; these DVs were similar to those in situ. The DVs prepa red for transmission electron microscopy or SEM showed extensive adher ence of cellular debris. The use of 0.2 M KCI in the wash buffer elimi nated much of this debris and exposed the true vacuolar surfaces, Thre e populations of tightly bound vesicles and numerous globular particle s of 10 to 20 nm became visible on the DV surfaces. The attached vesic les, hating diameters of similar to 300 and similar to 200 nm each, co rresponded to the acidosomes and lysosomes that are known to be associ ated with the DV-I and DV-II, respectively. High resolution SEM also r evealed a third set of small vesicles (50-150 nm), which were previous ly not known to be associated with DVs. The 10 to 20 nm globular parti cles were judged to be the cytosolic extensions of transmembrane prote in complexes as their patterns of distribution on DVs of various ages corresponded to the transmembrane particles previously seen in these m embranes in freeze-fracture studies.