Uroid Region Research Articles

Transcriptional Gene Silencing Reveals Two Distinct Groups of Entamoeba histolytica Gal/GalNAc-Lectin Light Subunits

The Entamoeba histolytica cell surface Gal/GalNAc-inhibitable lectin is a heterodimer between a heavy (170 kDa) subunit linked via a disulfide bond to a light (31 to 35 kDa) subunit. Five light subunit genes with high homology have been identified (Ehlgl1 to -5). We have previously shown that silencing of the expression of Ehlgl1, in the G3 trophozoites which had already been silenced in the amoebapore gene (Ehap-a), also suppressed the transcription of Ehlgl2 and -3 (strain RBV). The total absence of the lgl1 to -3 subunits in the RBV trophozoites affected their ability to cap the surface Gal-lectin molecules to the uroid region. We have now found that in the RBV trophozoites, the lgl4 and -5 subunits (31 kDa) are overexpressed and appear to compensate for the missing lgl1 to -3 in the heterodimer complex. Transcriptional silencing of Ehlgl5 was achieved by transfection of G3 trophozoites with a plasmid containing the open reading frame of Ehlgl5 ligated to the 5' promoter region of the Ehap-a gene. The transfected trophozoites (strain L5) were silenced in Ehlgl5 and the closely related Ehlgl4, while the expression of the larger lgl1 to -3 subunits was upregulated. L5 trophozoites retained their ability to cap the Gal-lectin molecules. Attempts to simultaneously silence all of the Ehlgl genes have failed so far, possibly due to their crucial importance to the Gal-lectin functions. Our ability to silence part of the genes belonging to the same family can serve as a tool to study the relationships and functions of the members of other gene families.

Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica

In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Ionic currents and cytoplasmic streaming in Amoeba proteus

Amoeba proteus may drive a current through themselves, and this current may determine the direction of cytoplasmic streaming. Cytoplasm streams from the more contracted tail or uroid region to the extending pseudopods. The positions of the uroid and pseudopods change as the amoeba changes direction. The membrane potential of the amoeba may change with location in the cell. This voltage gradient may regulate the magnitude and direction of cytoplasmic streaming. Bingley and Thompson (1962), recorded from amoebae using intracellular microelectrodes and reported that the membrane potential of the uroid region was more negative than that of the pseudopods. In contrast, Nuccitelli et al. (1977), using a vibrating probe, observed that current entered the uroid region and exited through the pseudopods. They concluded that the uroid region was more depolarized than the pseudopods. We revisited this issue by recording membrane potential simultaneously from two regions of the amoeba and by recording ionic currents using the two‐electrode voltage clamp. Our data support the findings of Nuccitelli et al. Cytoplasm may stream from more depolarized to more hyperpolarized regions. Moreover, overshooting depolarizations of the cell may produce global cytoplasmic contractions and rounding of the amoeba.

Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica.

Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand-receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.

Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica.

Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica. The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum, alpha-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica.

Distribution and function of myosin II as a main constituent of the microfilament system in Physarum polycephalum

Summary Morphological and physiological investigations on different plasmodial stages of Physarum polycephalum demonstrate a manifold functional significance of the cytoskeletal protein myosin II. During morphogenesis of the complex microfilament system myosin is first incorporated into the submembraneous cell cortex and then into nascent cytoplasmic fibrils with a gradual duplication after 6–8 min. Hence, myosin exhibits a varying topographical distribution in that fibrils of the plasmodial front contain 35% less myosin than fibrils of the uroid region; finally, mature fibrils show a sarcomeric-like arrangement of the motor protein with a periodicity of 0.7 μm. The contraction behavior of comparable specimens after detergent extraction correlates with the myosin content and distribution. Young stages attain only 60% of the contractile efficiency as measured for older ones, and uroid regions contract with a 4 to 6 times higher efficiency than front regions. However, a fibrillar system is not implicitly essential for motive force generation since plasmodial stages that only contain a filament cortex also perform distinct contractions. Altogether, results of the present paper point to the outstanding significance of myosin II in Physarum as a motor producing cytoplasmic streaming, an element stabilizing cytoskeletal organization and a factor probably controlling plasmodial polarity and aging.

Functional analysis of actin fibrils in Physarum polycephalum

Fluorochromed heavy meromyosin (TRITC-HMM) was microinjected as a molecular probe into small sandwich-plasmodia of Physarum polycephalum with the aim to demonstrate the spatial morphology and to analyze the dynamic activity of the fibrillar actin system in the living state. The plasmodia display different fibrillar organizations with a polygonal arrangement in the front region (FR) and a parallel or helical arrangement along protoplasmic veins in the intermediate (IR) and uroid region (UR). Quantitative evaluations by measuring the total length, lifetime, dynamic activity, long-term stability and optical density of fibrils reveal distinct differences between the three plasmodial regions: The total length (FR = 27.1 ± 18.5 μm, IR = 24.8 ± 12.9 μm, UR= 12.3 ± 4.7 μm), the lifetime (FR = 12.2 ± 3.4 min, IR=10.5 ± 3.7 min, UR = 6.0 ± 3.4 min), and the dynamic activity as measured in length changes per min (FR = 17.9 ± 11.3 μm, IR = 13.1 ± 3.9 μm, UR = 8.3 ± 3.9 μm) distinctly decrease from the front to the uroid region. On the other hand, the greatest stability as determined by lifetime changes in length (FR = -2.4 ± 16.2 μm, IR = 0.3 ± 10.1 μm, UR = -6.6 ± 8.9 μm) and the highest optical density as expressed in grey-values (FR = 57.0 ± 14.1 gv, IR = 115.6 ± 26.1 gv, UR 62.5 ± 8.1 gv) were found for actomyosin fibrils of the intermediate region. The morphological and physiological data of the present paper are discussed with respect to the biological significance of the fibrillar microfilament system in Physarum polycephalum.

Induced pinocytosis and cytoskeletal organization in Amoeba proteus — a combined fluorescence and electron microscopic study

Induced pinocytosis was studied in Amoeba proteus by combining fluorescence microscopy, low-light-level-TV intensification, image processing and electron microscopy. After external incubation of living cells with fluorochromed bovine serum albumin (BSA-TRITC) or a complex of colloidal gold and fluorochromed bovine serum albumin (Au(12)-BSA-TRITC) the fate of internalized material was followed for more than 2 h. Endosomes containing the ingested marker/membrane receptor-complex gather rapidly (15-30 min) within the uroid region and fuse successively to one large secondary endosome which either segregates its content into the external medium by exocytosis or is pinched off with some surrounding cytoplasm from the cell body. In cases where endosome-fusion does not occur, the distinct vacuoles release their content exocytotically in a progressive fashion. The fluorescence and electron microscopic results are in agreement with the in vivo investigations and support the suggestion that the microfilament system is responsible for the generation of motive force necessary to promote endocytosis, endosome gathering, fusion, sequestration and exocytosis.

Antibody-induced caps in Entamoeba histolytica: isolation and electrophoretic analysis.

Polyspecific antibodies bound to the cell surface of Entamoeba histolytica are polarized toward the uroid region and spontaneously released by the amoeba as supramolecular aggregate or cap. We purified these exfoliated structures and analyzed them by electron microscopy and sodium dodecyl sulfate electrophoresis. Isolated caps were mainly composed of membranes and contained five major [35S]methionine-labeled bands; many more bands appeared (together with immunoglobulins) after silver staining. Surface immunoprecipitates contained about twelve major [35S]methionine-labeled polypeptides, five of which had molecular weights similar to those of radiolabeled polypeptides in the cap. Some proteins in the caps had the same electrophoretic migration pattern as that from iodinated cell-surface proteins and polypeptides from isolated plasma membranes. Results suggest that only half of the surface-immunoprecipitated antigens are enriched in the cap.

Spatial organization and fine structure of the cortical filament layer in normal locomoting Amoeba proteus.

The fine structural organization of a cortical filament layer in normal locomoting Amoeba proteus was demonstrated using improved fixation and embedding techniques. Best results were obtained after application of PIPES-buffered glutaraldehyde in connection with substances known to prevent the depolymerization of F-actin, followed by careful dehydration and freeze-substitution. The filament layer is continuous along the entire surface; it exhibits a varying thickness depending on the cell polarity, measuring several nm in advancing regions and 0.5-1 micron in retracting ones. Two different types of filaments are responsible for the construction of the layer: randomly distributed thin (actin) filaments forming an unordered meshwork beneath the plasma membrane, and thick (myosin) filaments mostly restricted to the uroid region in close association with F-actin. The cortical filament layer generates the motive force for amoeboid movement by contraction at posterior cell regions and induces a pressure flow that continues between the uroid with a high hydrostatic pressure and advancing pseudopodia with low one. The local destabilization of the cell surface as a precondition for the formation of pseudopodia is enabled by the detachment of the cortical filament layer from the plasma membrane. This results in morphological changes by the active separation of peripheral hyaloplasmic and central granuloplasmic regions.

Surface redistribution and release of antibody-induced caps in entamoebae.

Polyspecific antibodies bound to Entamoeba induced surface redistribution of membrane components toward the uroid region. Capping of surface antigens was obtained with a single layer of antibodies in E. histolytica and E. invadens. This surface segregation progressed to a large accumulation of folded plasma membrane that extruded as a defined vesicular cap. A spontaneous release of the cap at the end of the capping process took place. These released caps contained most of the antibodies that originally bound to the whole cell surface. Two-thirds of radiolabeled antibodies bound to the surface of E. histolytica were released into the medium in 2 h. Successive capping induced by repeated exposure of E. invadens to antibodies produced conglomerates of folded surface membrane, visualized as stacked caps, in proportion to the number of antibody exposures. These results indicate the remarkable ability of Entamoeba to rapidly regenerate substantial amounts of plasma membbrane. The properties of surface redistribution, liberation of caps, and plasma membrane regeneration, may contribute to the survival of the parasite in the host during infection.

Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate.

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.

Adelphamoeba galeacystis n. g., n. sp., an Amoeboflagellate Isolated from Soil*

SYNOPSIS. An amoeboflagellate isolated from common soil is described. The amoeboid stage is typically limax and contains a well differentiated uroid region. The flagellate has 2 flagella, which emerge anteriorly and are equal in length. It has a ventral cytostome near the anterior border. The cyst is helmet‐shaped and without opercula. Polar masses are present during nuclear division.