Helices and aggregates of helices (chromatoid bodies) composed of ribosomelike particles appear in cysts and slow-growing trophozoites of Entamoeba invadens. We found that similar helix aggregates were formed abundantly in actively growing E. invadens trophozoites treated with a variety of direct or indirect inhibitors of protein synthesis. The inhibitor-induced helices appeared cytochemically and ultrastructurally identical to those seen in cysts.Numerous single helices and small arrays occurred randomly distributed throughout the trophozoite cytoplasm within 15 rain after treatment with NaF, which rapidly and completely stopped all nucleic acid and protein synthesis. Cycloheximide (CH), which inhibited protein synthesis as effectively as NaF, stimulated aggregate formation more slowly, and only after a delay of 30-60 rain. CH temporarily blocked NaF-stimulated aggregate formation. Aggregation was slowest with actinomycin-D, which strongly inhibited RNA synthesis but depressed protein synthesis only slowly. These results suggested that release of ribosomes from mRNA was required for aggregation.Inhibition by CH was reversible, and aggregates disappeared from CH-treated amebas shortly after they were transferred to inhibitor-free growth medium. There was no evidence that helices assembled about a structural organizer within the cell or that the process involved metabolic activity. It was concluded that the inhibitor-induced helices were composed of mature, normally functional ribosomes and that helix formation was a spontaneous and reversible consequence of the accumulation within the cell of free monosomes (or subunits) which were prevented from binding to mRNA.Considerable evidence has accumulated which indicates that the aggregated ribonucleoprotein helices (chromatoid bodies) found in cysts of Entamoeba are composed of ribosomal particles (18,14,15). There are, however, uncertainties about the precise structural and functional nature of the unit particles within the helices and about the total composition of the arrays of helices.

Some structural and functional properties of ribosomes from polysomes and from helix aggregates of Entamoeba invadens have been compared by sucrose gradient analysis and assays of in vitro protein synthesis.Actively growing trophozoites, lacking helices, presented normal polysome profiles in sucrose gradients. The single large ribosomal helix aggregate (chromatoid body) of cysts disappeared as the cells were disrupted. Gradient profiles of cyst extracts contained predominantly large and small ribosome subunit peaks and no evidence of remaining helix fragments or mRNA-bound polysomes. Sequential profiles of trophozoites incubated with NaF or cycloheximide (which both stimulate ribosome aggregation, but at different rates) showed that polysome breakdown occurred before aggregates appeared and, again, that helices broke down to subunits in vitro. Radioactive ribosomes synthesized during vegetative growth were collected into helices during encystation. Subunits of these ribosomes cosedimented with comparable particles isolated from trophozoites. Ribosomes from both trophozoites and cysts were active in cell-free protein synthesis, although activity in cyst extracts required the addition of trophozoite-soluble fraction. It was concluded that ribosomes from polysomes and helices in E. invadens were probably identical and that the ability to form helices was an intrinsic property of mature mRNA-free ribosomes of this organism.During cellular differentiation of Entamoeba from vegetative trophozoites to cysts, a large symmetrical array of ribosome-containing helices is formed. This so-called chromatoid body appears to contain virtually all of the cyst ribonucleoprotein (1). Helices and smaller helix aggregates have also been observed at times in Entamoeba trophozoites, t Dr. Kamolwat Vinijchaikul is now deceased. and these are ultrastructurally similar to the cyst arrays (2).In the preceding paper (4) we suggested that there was a correlation between the natural appearance of ribosome aggregates in amebas and periods of reduced metabolic activity. We demonstrated that massive aggregation could be induced by treatment of actively growing trophozoites with inhibitors of protein synthesis. These aggregates 540