Endocytosis in Entamoeba histolytica. Evidence for a unique non-acidified compartment.

Abstract

Endocytosis by mammalian cells leads to the rapid uptake of exogenous solutes within plasma-membrane-derived vesicles. In general, fusion of these vesicles with primary or secondary iysosomes occurs and most biological macromolecules are degraded to small molecular weight components (1). Extensive bidirectional flow of membrane takes place between the plasma membrane and the lysosomal apparatus during endocytosis (7). Recent evidence has suggested that solutes pass through at least one intracellular compartment before reaching the lysosome (2-6) and representative portions of endocytic markers (2,4,6,8) are returned to the cell surface.We now report the existence of a distinct, intermediate vacuolar compartment in the parasitic amoeba, Entamoeba histolytica. This is the primary pinocytic route in this organism. It is equilibrated with the pH of the ambient milieu and allows for the flow of solutes into and out of the cytoplasm. Only after many hours of intraceilular residence are the solutes detected in a separate, acidified lysosomelike compartment. Materials and MethodsCulture ofE. histolytica. E. histolytica trophozoites, strain HM 1 :IMSS, were generously provided by Dr. L. Diamond, the National Institute for Allergy and Infectious Diseases, and were maintained in axenic culture in TYI-S medium (9). For studies on internalization of fluid phase markers, fluorescein-labeled dextran (fluorescein isothiocyanate [FITC] ~-dextran, average molecular weight 70,000, Sigma Chemical Co., St. Louis, MO) or horseradish peroxidase (HRP, Sigma) was added directly to the culture medium at concentrations ranging between 1 and 10 mg/ml. After incubation for the indicated times, amoebae were harvested by chilling followed by centrifugation at 500 g for 5 min. Trophozoites were then washed by resuspension in cold 19 mM phosphate buffer, pH 7.2, containing 0.27 mM NaCI (PD), or in TYI-S medium and centrifugation. For studies on reincubation of amoebae, trophozoites were harvested and washed with TYI-S medium, resuspended in TYI-S medium, and gently spun (50 g) onto the sides of culture tubes. The cultures were then carefully transferred to a 37 °C water bath and incubated on their sides. These manipulations were necessary to insure rapid warming of amoebae on reincubation.

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…In a way reminiscent of macropinosomes, these post-Iysosomes are again surrounded by actin and sequentially acquire coronin and vacuolin, which targets them for exocytosis (Jenne et al 1998). Similar large non-acidic endosomal vacuoles exist in other protists such as E. histolytica (Aley et al 1984 ). D. discoideum, like other lower eukaryotes, partly secretes its lysosomal enzymes (FlorinChristensen et al 1989;Hohman and Bowers 1984;1986), which is likely due to incomplete retrieval from the post-lysosomal compartment.…”

Zanvil Alexander Cohn 1926-1993.

“…Aley also applied the methods that were being used to study the macrophage cell surface to several microbes that were of great interest to the lab (below): Trypanosoma cruzi (190,205), Leishmania donovani (224), and Entamoeba histolytica (151,152,239). With graduate fellow Margaret Kielian, Zan monitored phagosome-lysosome fusion in macrophages (154,184,185).…”

Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases

“…Trafficking of fluid phase to and from this compartment accounts for most of the pinocytosis carried out by E. histolytica. The second (late) compartment consists of smaller vesicles with acidified contents [6]. The physiological relevance of these two compartments has not been discerned.…”

“…In addition, this organism exhibits robust fluid phase pinocytosis, turning over 30% of its volume per hour [6,7]. There are two pinocytic compartments in E. histolytica [6]. The first (early) compartment consists of non-acidic, large vesicles (> 2 μm in diameter) that rapidly exchange their contents with the external milieu.…”

“…It is known, however, that these amoebae can actively phagocytose large particles such as bacteria and erythrocytes, and the ability to phagocytose bacteria appears to be correlated with virulence [4,5]. In addition, this organism exhibits robust fluid phase pinocytosis, turning over 30% of its volume per hour [6,7]. There are two pinocytic compartments in E. histolytica [6].…”