Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology.

The uptake of radioactively labeled albumin, inulin, leucine, and glucose by Acanthamoeba castellanii (Neff strain) was measured. The uptake is linear with time and appears to be continuous under the conditions of these experiments. Uptake is abolished at 0 degrees C. No evidence for saturation of the uptake mechanism was obtained with either albumin or leucine. Each of the four tracer molecules enters the ameba at a similar rate when the uptake is calculated as volume of fluid ingested per unit time. The data suggest that each of these molecules enters the cell by pinocytosis. The highest rate of uptake was obtained with cells in their usual culture medium containing proteose peptone, glucose, and salts but pinocytosis also continued at a reduced rate in a simple salt solution. The calculated volume of fluid taken in during pinocytosis in culture medium was about 2 microl/hr per 10(6) cells. The route of uptake was examined in the electron microscope using horseradish peroxidase (HRP) as a tracer. HRP activity was found exclusively within membrane profiles within the cytoplasm, confirming the pinocytotic mode of uptake. An estimate of the rate of surface membrane turnover due to pinocytosis was made using the biochemical and morphological data obtained. This estimate suggests that the plasma membrane turnover of one cell is on the order of several times an hour.

Acanthamoeba discriminates internally between digestible and indigestible particles.

The capacity of Acanthamoeba to distinguish nutritive yeast particles from non-nutritive plastic beads during phagocytosis was investigated. When cells were allowed to phagocytose yeast to capacity, endocytosis stopped and subsequent presentation of particles (either yeast or beads) did not result in further uptake. By contrast, when cells were allowed to phagocytose plastic beads to capacity and a second dose of particles was presented (either yeast or beads), the cells exocytosed the internal particles and took up new ones. Yeast rendered indigestible by extensive chemical cross-linking were taken up at rates similar to those of untreated yeast, but, like beads, they were exocytosed when a second dose of particles was presented. The results show that an internal distinction is made between vacuoles containing yeast and vacuoles containing plastic beads, and they are consistent with the hypothesis that the presence within the vacuoles of material capable of being digested prevents exocytosis.

Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba.

Stereologic analysis was made of cell surface membrane (PM) and two interrelated cytoplasmic membrane systems, the vacuole membranes (VM) and small vesicle membranes (SVM). Volumes and surface areas of the three membrane compartments were measured during steady-state pinocytosis, when membrane recycling is rapid, and during phagocytosis, when a shift to a lower rate of membrane uptake by endocytosis occurs (B. Bowers, 1977, Exp. Cell Res. 110:409). Total membrane area in the three compartments was 3.2 micrometers 2/micrometers 3 of protoplasmic volume and was constant throughout the experiments. In pinocytosing cells, 32% of the membrane was in the PM, 25% in the vM, and 43% in the SVM. The vacuole compartment occupies approximately 20% of the total cell volume, and the small vesicle, approximately 3%. As the endocytic uptake of membrane from the surface decreased, there was an increase in PM area and a marked decrease in SVM area. The VM area remained constant even though "empty" vacuoles were almost completely replaced by newly formed phagosomes within 45 min. This demonstrates directly a rapid flux of membrane though this compartment. A model, taking into consideration these and other data on Acanthamoeba, is proposed to account for the observed membrane shifts. The data suggest that the vacuolar (digestive) system of Acanthamoeba is central to cellular control of endocytosis and membrane recycling.