Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

The auxin response of actin is altered in the rice mutantYin-Yang

Summary The rice mutantYin-Yang has been selected during a screen for resistance to cytoskeletal drugs and is characterized by alterations in epidermal cell length and a precocious onset of gravitropism. The elongation response of coleoptile segments to auxin does not reveal changes of auxin sensitivity inYin-Yang. However, in contrast to the wild type, cell elongation inYin-Yang is highly sensitive to the actin-polymerisation blocker cytochalasin D. This increased sensitivity to cytochalasin D requires optimal concentrations of auxin to become manifest. The auxin response of actin microfilaments in epidermal cells differs between wild type and mutant. In the wild type, the longitudinal microfilament bundles become loosened in response to auxin. In the mutant, these bundles disintegrate partially and are replaced by a network of short filaments surrounding the nucleus. Several aspects of the mutant phenotype can be mimicked in the wild type by treatment with cytochalasin D. The mutant phenotype is discussed in terms of signal-dependent changes of actin dynamics and the putative role of actin during cell elongation.Abbreviations CD cytochalasin D - EPC ethyl-N-phenylcarbamate     

Regulated interactions of the norepineprhine transporter by the actin and microtubule cytoskeletons

One role of the actin cytoskeleton is to maintain the structural morphology and activity of the pre-synaptic terminal. We sought to determine if the actin cytoskeleton plays a role in regulating interactions between the norepinephrine transporter (NET) and alpha-Synuclein (α-Syn), two proteins expressed in the pre-synaptic terminal. In cells transfected with either 0.5 μg/mL or 3 μg/mL of α-Syn and 1 μg/mL of NET DNA, treatment with cytochalasin D, an actin depolymerizing agent, caused a dose-dependent decrease and increase, respectively, in [³H]-NE uptake. Protein interactions between NET, β-actin, and α-Syn were modified, along with levels of surface transporters. Treatment of primary brainstem neurons and frontal cortex synaptosomes with cytochalasin D caused a 115% and 28% increase, respectively, in NET activity. Depolymerization of both actin and microtubules did not alter NET activity in cells with 0.5 μg/mL α-Syn, but caused an increase in [³H]-NE uptake in cells transfected with 3 μg/mL of α-Syn and primary neurons. This is the first direct demonstration of NET activity being regulated via actin and modulated by interactions with α-Syn.     

Photocontrol of in vitro bud regeneration: A comparative study of the interaction between light and IAA in a wild type and an aurea mutant of Lycopersicon esculentum

In the presence of 0.2 μ M IAA both the wild type and the aurea mutant of Lycopersicon esculentum Mill, showed a low but significant percentage of bud formation in the dark, whereas no bud formation occurred in the dark when 20 μ M IAA was present in the medium. In both systems blue light always showed a strong promoting effect on bud regeneration, both as final percentage of regeneration and by shortening the initial lag period, suggesting the action of a specific blue light photoreceptor. Red and far-red light increased the percentage of bud differentiation in wild type explants, with both the IAA concentrations. In the aurea mutant only red at the lowest IAA concentration had such an effect. The final percentage of bud regeneration under red light was greater or equal to that found under blue light in the wild type as well as in the aurea mutant explants cultured in the presence of the lowest IAA concentration.     

Importance of actin cytoskeleton behaviour during preservation of carrot cell suspensions in liquid nitrogen

Summary Conventional methods for preservation of suspended, highly vacuolated, plant cells in liquid nitrogen (LN) usually involve equilibration in molar concentrations of cryoprotective additives, followed by slow cooling to an intermediate subzero temperature (–40 °C), before quenching in LN. Cryomicroscopy was used to monitor the reversible protoplasmic shrinkage of cryoprotected carrot cells, caused by freeze-induced dehydration. Behaviour of actin filaments was analyzed by fluorescence microscopy after labelling with rhodarnine-conjugated phalloidin, in relation to the type of pretreatment and to survival and regrowth ability after preservation at — 196 °C. Loading with dimethylsulphoxide (Me₂SO, 5%) resulted in high survival rates (70%) and regrowth. After thawing, the actin filament (MF) abundance was reduced, but the structure and distribution of the remaining MFs seemed undisturbed. Higher Me₂SO concentrations caused further reduction of MFs, which appeared fragmented after thawing. MFs were maintained by pretreatment with 0.5 M sorbitol alone but carrot cells did not survive at — 196 °C. The same pretreatment, followed by incubation with cytochalasin D (10 M), which greatly reduced MFs, enabled plasmolyzed carrot cells to survive preservation in liquid nitrogen. Thus, after both Me₂SO and sorbitol plus cytochalasin D pretreatments, partial disruption of actin filaments seemed to accompany (Me₂SO) or promote (sorbitol plus cytochalasin D) freezing tolerance at extremely low temperatures.Abbreviations CD cytochalasin D - FDA fluorescein diacetate - LN liquid nitrogen - MF actin filament - Me₂SO dimethylsulphoxide     

Entamoeba histolytica:Identification of Functional Gsand GiProteins as Possible Signal Transduction Elements in the Interaction of Trophozoites with Fibronectin

Soid-Raggi, L. G., Torres-Maárquez, M. E., and Meza, I. 1998.Entamoeba histolytica:Identification of functional G_sand G_iproteins as possible signal transduction elements in the interaction of trophozoites with fibronectin.Experimental Parasitology90, 262–269. Trophozoites ofEntamoeba histolyticaadhere to several components of the extracellular matrix. Binding is mediated by specific receptors identified in the parasite surface. Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation. The process requires activation of signaling pathways in which PLC, IP₃, Ca²⁻, and PKC participate. These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins. We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated byVibrio choleraeandBordetella pertussistoxins. Three of them are also recognized by antibodies prepared against the α-subunit of G_s-and G_i-proteins. Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins. Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity. Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin. The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment. Our present data show the presence and the functionality of G_s- and G_i-like proteins and their apparent activation duringin vitrointeraction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms inE. histolytica.     

Entamoeba histolytica: role of amebic proteinases and polymorphonuclear leukocytes in acute experimental amebiasis in the rat

The injection of 1 x 10(6) trophozoites of axenically grown Entamoeba histolytica strain HM-1 in the subcutaneous tissue of the rat results in an acute and self-limited inflammatory process, characterized by the early onset of conspicuous tissue necrosis and focal hemorrhage in the vicinity of the parasites, followed by infiltration with polymorphonuclear leukocytes. The process develops for 5-10 hr but during that period amebic trophozoites progressively disappear, leukocytes undergo degenerative changes, and the lesion tends to heal in 72-96 hr. In leukopenic animals (less than 1000 white blood cells/ml) tissue necrosis and hemorrhage are equally conspicuous in the neighborhood of amebas. Inhibition of amebic proteinase activity prior to injection by heat denaturation, p-hydroxy-mercuri-benzoate (PHMB), soybean trypsin inhibitor (STI), and human alpha-2-macroglobulin (alpha 2M), alone or in various combinations, results in absence or notorious decrease in tissue necrosis as well as in clearly diminished inflammatory reaction. This effect is particularly evident when cysteine proteinases are either specifically or generally inhibited. On the other hand, amebic proteinase inhibition with alpha 2M and STI does not interfere with the cell-killing capacity of trophozoites co-incubated in vitro for 2 hr with rat peritoneal cells enriched for macrophages. We conclude that in acute experimental amebiasis produced in the subcutaneous tissue of the rat, amebic cysteine (and perhaps other) proteinases are primarily responsible for necrosis and are also important, but not essential, for inflammation. We also suggest that in this model polymorphonuclear leukocytes are not required for tissue necrosis. Finally, in an in vitro model, the cell-killing capacity of amebas is not influenced by the proteinase activity of the parasite.     

Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.     

Further studies on the distribution of cytochromes in methanogenic bacteria

Abstract Growth of Methanobacterium thermoautotrophicum strain Marburg was strongly inhibited by 2 μg/ml of pseudomonic acid. This inhibition was relieved by 10 mM l -isoleucine. The susceptibility of a pseudomonic acid-resistant mutant was 50 times lower than in the wild type. Isoleucyl-tRNA synthetase activity in cell extracts from the wild type was strongly inhibited by pseudomonic acid whereas the corresponding enzyme in mutant extracts was only marginally inhibited by the antibiotic.     

Scanning Electron Microscopy of Attached Trophozoites of Pathogenic Entamoeba histolytica1

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2–8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

Localization of actin in Dictyostelium amebas by immunofluorescence

Antibody prepared against avian smooth muscle actin has been used to localize actin in the slime mold, Dictyostelium discoideum. The distribution of actin in migrating cells is different from that in feeding cells. Migrating amebas display fluorescence primarily in advancing regions whereas feeding amebas show uniform fluorescence throughout. The reaction is specific for actin since the fluorescence observed is blocked when the antibody is absorbed by actin purified from avian skeletal muscle, human platelets, and Dictyostelium. These results, in addition to describing the distribution of actin in D. discoideum, demonstrate that actins from these diverse sources share at least one common antigenic determinant.     

Scanning electron microscopy of attached trophozoites of pathogenic Entamoeba histolytica

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2-8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

Localization and regulation of phospholipase D2 by ARF6

Phospholipase D (PLD) and ADP-ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co-localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP-tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans-Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co-expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans-Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative ARF6 selectively inhibited PLD2. In contrast, dominant active ARF1 or Rac1 stimulated both PLD isozymes but the ARF1 effect on PLD2 was very small. Cytochalasin D did not affect the activation of PLD by phorbol ester. The localizations of PLD and ARF6 were also analyzed by fractionation after methyl-beta-cyclodextrin extraction to deplete cholesterol. The results showed that all PLD isoforms and ARF6 mutants existed in the membrane fraction, but only wild type ARF6 was dependent on the presence of cholesterol. These experiments showed that wild type ARF6 had a similar location with PLD isoforms on cell staining, but it did not colocalize with PLD isoforms in fractionation experiments. It is proposed that activated ARF6 translocates to the cholesterol independent microdomain and then activates PLD2 there. It is further concluded that PLD2 is selectively activated by ARF6 in vivo and that disruption of the actin cytoskeleton does not affect this activation.     

Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0·30 ± 0·03 (mean ± SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0·49 ± 0·03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0·5 ± 0·07 (n = 4), indicating a 1·7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10^?6 and 10^?5 M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10^?4 M ), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted. We concluded that the G/total actin ratio was significantly increased in malignant keratinocytes. This seems to be correlated with altered microfilament morphology and resistance to cytochalasin B treatment. Our results suggest that the process of malignant transformation may be characterized by changes in the state of the polymerization of actin and in the stability of the microfilament network indicating that both features could potentially serve as markers determine the transformed state of keratinocytes.     

Microtubule disassembly enhances reversible cytochalasin-dependent disruption of actin bundles in characean internodes

Summary Parallel bundles of actin filaments at the cortex-endoplasm interface provide tracks for myosin-generated cytoplasmic streaming in characean internodes. These bundles resist disassembly or structural modification when exposed to 10 μM cytochalasin D (CD) even though this concentration of CD rapidly (within minutes) but reversibly arrests streaming. Unexpectedly, we discovered that prolonged treatment with lower concentrations of CD could partially disassemble the subcortical actin bundles. Actin bundles became discontinuous following one- to several-day treatment with concentrations (6 μM) that reduced but did not arrest streaming, and the residual fragments mostly remained parallel to the chloroplast files. When microtubules were concurrently disassembled with tubulin-specific drugs, however, low CD concentrations (2.5–3 μM) completely arrested bulk streaming, disrupted the largely 2-dimensional actin bundle array and caused the formation of a coarse, thick-meshed actin network that extended from the cortex to the endoplasm. Despite such massive reconstruction, drug removal enabled cells to recover continuous parallel bundles and streaming. Recovery was possible if both or just one of the drugs were removed. In recovered cells, the streaming pattern frequently redeveloped in new directions that did not follow the chloroplast files, and later, chloroplast files readjusted to the new polarity established by the actin bundles. This first report on the complete and reversible disassembly of characean actin bundles provides new insights into the mechanism of actin bundle assembly and organization and supports the idea of indirect interactions between actin filaments and microtubules.     

Actin mRNA Levels and Actin Synthesis During the Encystation of Entamoeba invadens

ABSTRACT. Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate that encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeleton occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.     

Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function

The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3 L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.