A]3STRACTThe uptake of radioactively labeled albumin, inulin, leucine, and glucose by Acanthamoeba castdlanii (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°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 celIs 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 /A,/hr per 106 cells. The route of uptake was examined m 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.

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the ␣-and ␤-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ϳ0.4 M or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ϳ200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer 7 dimer equilibrium with an association constant of 1.0 ؋ 10 6 M ؊1 . Together, these results indicate that CARMIL selfassociates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (ϳ1 and 2 M for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.In 1995, Zot (1) and colleagues identified a ϳ125-kDa protein from Acanthamoeba on the basis of its ability to bind to the isolated Src homology 3 (SH3) 1 domain of Acanthamoeba myosin IC (1). This protein, which they called Acan 125, coimmunoprecipitated with myosin IC and appeared to colocalize with the myosin in cellular surface projections involved in pinocytosis. The subsequent cloning of the Acanthamoeba gene for Acan 125 (2) revealed a multidomain protein dominated by a central, ϳ460-residue leucine-rich repeat (LRR) domain. LRRs are ϳ29-residue sequences that contain a loose consensus dominated by leucines, form amphipathic ␣-␤-structural units and mediate protein-protein interactions, either by serving as the ligand binding sites themselves or by increasing the affinity and/or specificity of binding at a separate site (3). The second most striking structural feature of Acan 125 is its ϳ200-residue, proline-rich COOH-terminal domain. Consistent with the fact that SH3 domains mediate protein-protein interactions by binding to proline-rich target sequences containing the core element PXXP (4), Xu et al. (2) showed that a fusion protein containing the COOH-terminal 344 residues of Acan 125 bound to the isolated SH3 domain of myosin IC and that this interaction was abrogated by an 18-residue deletion spanning two PXXP motifs fitting the consensus for SH3 domain tar...

The capacity of Acanthamoeba to distinguish nutritive yeast particles from nonnutritive 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.Phagocytic uptake of panicles depends on a specific interaction at the cell surface which results in a localized stimulus for the cell to engulf the particle. In many cells this interaction can occur with a number of indigestible substances. For example, amebas will ingest silica, carbon particles, plastic beads, and other materials of no nutritive value (1, 2). Plastic beads, in fact, are valuable tools for studying the kinetics and mechanisms of endocytosis in a wide variety of cell types because they are indiscriminately engulfed by most cells. To understand better how cells control particle turnover rates and the related membrane recycling, we have studied how Acanthamoeba handles a nutritive particle, yeast, as compared with a nonnutritive particle, plastic bead.Acanthamoeba contains a vacuolar compartment that serves as its digestive system. This compartment is maintained by the cell at virtually constant volume and surface area under standard culture conditions (3). Nevertheless, considerable membrane flow occurs through the compartment because of endocytosis (3, 4). Membrane flow, as inferred from the rate of membrane ingestion by endocytosis, is rapid during pinocytosis (~9 surface equivalents/h) and close to zero in cells that have taken up particles to the limit of their capacity (that is, are saturated). When the cell is saturated with a digestible particle such as yeast, space for particle uptake gradually becomes available as digestion progresses and undigested residues are incorporated into defecation vacuoles to be expelled from the cell. In contrast, indigestible particles like plastic beads are ingested readily by the amebas, but cannot be reduced in volume. In the experiments reported here, in addition to testing turnover rates of different types of particles, we examined whether Acanthamoeba discriminates between inert and nutritive particles and whether they do so externally or internally. MATERIALS AND METHODSCulture Methods: Acanthamoeba castellanii (Neff strain) was cultured axenically as fol...