Dynamics of the orphan myosin MyoF over Trypanosoma cruzi life cycle and along the endocytic pathway.

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.

Evolutionary conservation of actin-binding proteins in Trypanosoma cruzi and unusual subcellular localization of the actin homologue.

The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cell-substrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi.

Isolation and biochemical characterization of the costa of Tritrichomonas foetus.

The costae are cytoskeletal structures found in Trichomonadidae. Both the structural organization and composition of this organelle are still unknown. In the present work we have introduced a new methodology for the costa isolation. Using sucrose density-gradient centrifugation an enriched costa fraction was obtained. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the costa contains several proteins, with major bands corresponding to apparent molecular masses of 122, 115, 112, 93, 87, 82, 59, 52, 44, 41, 32, and 26 kDa. No significant amount of carbohydrates was detected in the costa fraction. The fractionation methodology described here has the advantage of using normal centrifugation methods and is being applied to trichomonas in the size range of Tritrichomonas foetus and Trichomonas vaginalis.

Leishmania-induced tyrosine phosphorylation in the host macrophage and its implication to infection.

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.

Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids.

The flagellar membrane of trypanosomatids has certain ultrastructural and antigenic characteristics that make its biochemical analysis very interesting. We have obtained a highly purified flagellar membrane fraction from epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas samuelpessoai. The fractions consisted of regular spherical vesicles. Membrane fractions fixed in a glutaraldehyde solution containing filipin and freeze-fractured showed few intramembranous particles, and many protuberances indicative of filipin-sterol complexes. The protein composition of all the subflagellar fractions was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The flagellar membrane fraction showed more than twenty bands. We wish particularly to point out the presence of six bands corresponding to proteins of 160, 135, 41, 31, 26 and 18 kDa which were not present in the axoneme-paraxial structure fraction. The blot of flagellar membrane proteins successively incubated with concanavalin A and horseradish peroxidase showed intense binding of the lectin to proteins of 117 and 87 kDa, and less strong binding to several other minor bands. Our results showed that although the membrane of the flagellum of trypanosomatids had few proteins, it seemed rich in glycosylated elements.

Purification of the paraflagellar rod of the trypanosomatid Herpetomonas megaseliae and identification of some of its minor components.

The paraflagellar rod (PFR) is a component of the flagellar cytoskeleton of trypanosomatid protozoa, representing a filamentous structure that runs alongside the common 9 + 2 microtubular axoneme. The high degree of ultrastructural complexity and organization of the PFR suggests that it might be formed by numerous biochemical components. However, biochemical analysis of the PFR has revealed, to date, a modest degree of complexity in what concerns both major and minor PFR proteins. In this paper the preparation of purified PFR fractions by a combination of conventional cell-fractionation procedures, non-ionic detergent treatment and limited proteolysis is described. Comparative SDS-PAGE analysis of the different purification steps indicates that the purified PFR fractions possess high amounts of the well-known major PFR proteins (77 and 83 kDa). Also, bands of 147, 139, 129 and 122 kDa are clearly enriched in such fractions and may correspond to minor PFR components. A slight enrichment in a specific fraction of a doublet of bands of 181/188 kDa suggest the participation of these proteins in the composition of the bridges between the PFR and the axoneme.

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus.

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.

Acetylated alpha-tubulin in Trypanosoma cruzi: immunocytochemical localization.

We have used monoclonal antibodies specific for acetylated and non-acetylated alpha-tubulin to localize microtubules containing acetylated alpha-tubulin in all developmental forms of the life cycle of Trypanosoma cruzi. This was demonstrated using immunofluorescence and by transmission electron microscopy of thin sections, negative stained cells, and replicas of whole Triton X-100 extracted cells immunolabeled with antibody-gold complex. The antibody specific for acetylated alpha-tubulin (6-11B-1) binds to the flagellar, as well as to the sub-pellicular microtubules. The extent of labeling of the sub-pellicular microtubules with the monoclonal antibody recognized alpha-acetylated tubulin was smaller than that observed with the antibody which recognizes all tubulin isoforms. In relation to the developmental forms, the extent of labeling of the microtubules with antibody 6-11B-1 was larger in epimastigote and trypomastigote than in amastigote forms. Incubation of the parasites for 1 h at 0 degrees C or in the presence of either colchicine or vinblastine did not interfere with the sub-pellicular microtubules. These observations, in agreement with those reported for Trypanosoma brucei brucei (Schneider et al., 1987; Schulze et al., 1987; Sasse & Gull, 1988) indicate that the sub-pellicular microtubules of trypanosomatids represent stable microtubules containing acetylated alpha-tubulin (or the alpha 3-tubulin isotype).

Localization of acetylated alpha-tubulin in Tritrichomonas foetus and Trichomonas vaginalis.

We used monoclonal antibodies specific for acetylated and nonacetylated alpha-tubulin to detect and to localize microtubules containing acetylated alpha-tubulin (stable microtubules) in the pathogenic protozoa Tritrichomonas foetus and Trichomonas vaginalis. SDS-PAGE analysis showed that tubulin is a major protein of both parasites, being enriched in cytoskeletal preparations of whole cells extracted with Triton X-100. The monoclonal antibodies, which recognize all isoforms of alpha-tubulin (B-5-1-2) and only acetylated alpha-tubulin (6-11B-1), bind to the tubulin of T. foetus and T. vaginalis as seen by immunoblotting. Tubulin-containing structures were localized using immunofluorescence microscopy and transmission electron microscopy of the whole cytoskeleton previously incubated in the presence of the anti-tubulin antibodies and a second antibody-gold complex, and then processed using the negative staining or replica techniques. The results obtained indicate that, in addition to the flagellar microtubules, those which form the peltar-axostyle system represent stable microtubules containing acetylated alpha-tubulin.

Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis.

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis.

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.

Further studies on the structural analysis of the cuticle of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

In order to obtain further information on the structural organization of the cuticle of nematodes, this structure was isolated from adult forms of the filariid Litomosoides chagasfilhoi. The purity of the fraction was determined by light and transmission electron microscopy, deep-etching, high resolution scanning electron microscopy, atomic force microscopy, immunocytochemistry, gel electrophoresis (SDS-PAGE) and Western blot. The epicuticle presented a rugous surface with parallel rows and several globular particles that could be involved in the absorption of nutrients and secretion of products. Analysis by SDS-PAGE of purified cuticles revealed five major polypeptides corresponding to 151, 41, 28, 13 and 11 kDa. A polyclonal antibody against a synthetic 18 amino-acid peptide that corresponds to the sequence of domain E of the Haemonchus contortus3A3 collagen gene recognized several protein bands on the Western blot of purified cuticle, and labeled all cuticular layers, as shown by immunocytochemistry.