The Wnt/Beta-Catenin Pathway and Cytoskeletal Filaments Are Involved in the Positioning, Size, and Function of Lysosomes during Chick Myogenesis

Lysosomes are highly dynamic organelles involved in the breakdown and recycling of macromolecules, cell cycle, cell differentiation, and cell death, among many other functions in eukaryotic cells. Recently, lysosomes have been identified as cellular hubs for the modulation of intracellular signaling pathways, such as the Wnt/beta-catenin pathway. Here we analyzed morphological and functional characteristics of lysosomes in muscle and non-muscle cells during chick myogenesis, as well as their modulation by the Wnt/beta-catenin pathway. Our results show that (i) muscle and non-muscle cells show differences in lysosomal size and its distribution, (ii) lysosomes are found in spherical structures in myoblasts and fibroblasts and tubular structures in myotubes, (iii) lysosomes are found close to the plasma membrane in fibroblasts and close to the nucleus in myoblasts and myotubes, (iv) lysosomal distribution and size are dependent on the integrity of microtubules and microfilaments in myogenic cells, (v) alterations in lysosomal function, in the expression of LAMP2, and in Wnt/beta-catenin pathway affect the distribution and size of lysosomes in myogenic cells, (vi) the effects of the knockdown of LAMP2 on myogenesis can be rescued by the activation of the Wnt/beta-catenin pathway, and (vii) the chloroquine Lys05 is a potent inhibitor of both the Wnt/beta-catenin pathway and lysosomal function. Our data highlight the involvement of the Wnt/beta-catenin pathway in the regulation of the positioning, size, and function of lysosomes during chick myogenesis

Endocytosis in Trypanosoma cruzi

4 p. : il.Endocytic activity is particularly intense in Trypanosoma cruzi epimastigotes, while in amastigotes and trypomastigotes it is untraceable. Cargo molecules enters through the cytostome or flagellar pocket at the parasite anterior region, goes along a branched early endosomal network of tubules and vesicles spread from nuclear periphery to the posterior pole, until delivery to reservosomes, the final compartment. Reservosomes are acid compartments that store protein and lipid cargo and also accumulate lysosomal hydrolases, modulating digestive activity. Although T. cruzi infective forms are unable to uptake molecules, its lysosome related organelles represent a potential targets for anti-parasitic chemotherapy

New insights into the morphology of Trypanosoma cruzi reservosome

7 p. : il.Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions

Subcellular proteomics of Trypanosoma cruzi reservosomes

13 p. : il.Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles

ACAT-related activity assay.

<p>The ability of <i>T</i>. <i>cruzi</i> epimastigotes to esterify cholesterol was tested using Sandoz 58-035-58-035 for 16–18 h (A) or Avasimibe for 24 h (B) in different concentrations, followed by addition of BSA-³H-palmitate (800,000 DPM; 1 mg/mL) for 24 h. Lipid analysis was performed by TLC; CHOE spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of two independent experiments in duplicate, and analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p

Phthalimido-thiazoles as building blocks and their effects on the growth and morphology of Trypanosoma cruzi.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2016-05-13T16:12:59Z No. of bitstreams: 1 Gomes PATM Phthalimido....pdf: 1703584 bytes, checksum: 52e53fea525abd4a9ca1d384144f5738 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2016-05-13T17:31:23Z (GMT) No. of bitstreams: 1 Gomes PATM Phthalimido....pdf: 1703584 bytes, checksum: 52e53fea525abd4a9ca1d384144f5738 (MD5)Made available in DSpace on 2016-05-13T17:31:23Z (GMT). No. of bitstreams: 1 Gomes PATM Phthalimido....pdf: 1703584 bytes, checksum: 52e53fea525abd4a9ca1d384144f5738 (MD5) Previous issue date: 2016Universidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilUniversidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilUniversidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilUniversidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilUniversidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilUniversidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilLaboratório de Imunologia Keizo Asami-LIKA/UFPE. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilUniversidade Federal de Pernambuco. Departamento de Ciências Farmacêuticas. Centro de Ciências da Saúde. Recife, PE, BrasilChagas disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affects approximately 6e7 million people worldwide. Benznidazole is the only drug approved for treatment during the acute and asymptomatic chronic phases; however, its efficacy during the symptomatic chronic phase is controversial. The present work reports the synthesis and anti-T. cruzi activities of a novel series of phthalimido-thiazoles. Some of these compounds showed potent inhibition of the trypomastigote form of the parasite at low cytotoxicity concentrations in spleen cells, and the resulting structure-activity relationships are discussed. We also showed that phthalimido-thiazoles induced ultrastructural alterations on morphology, flagellum shortening, chromatin condensation, mitochondria swelling, reservosomes alterations and endoplasmic reticulum dilation. Together, these data revealed, for the first time, a novel series of phthalimido-thiazoles-structure-based compounds with potential effects against T. cruzi and lead-like characteristics against Chagas disease

Morphometric analysis of the number and the area occupied by lipid bodies in the cytoplasm of epimastigotes cultivated in LIT medium supplemented with 10% dFCS.

<p>¹ n = 30 parasite profiles</p><p>*<i>P</i> > 0.05. The results are expressed as the mean ± S.E.M using one-way ANOVA.</p><p>Morphometric analysis of the number and the area occupied by lipid bodies in the cytoplasm of epimastigotes cultivated in LIT medium supplemented with 10% dFCS.</p

<i>Trypanosoma cruzi</i> Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions

<div><p><i>Trypanosoma cruzi</i> epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.</p></div

Free and total (after saponification) cholesterol and ergosterol contents in lipid starved <i>T</i>. <i>cruzi</i> epimastigotes, kept in LIT medium supplemented with 10% dFCS for 0, 24, 48 and 72h.

<p>All samples correspond to 5 μg/mL of protein.</p><p>Free and total (after saponification) cholesterol and ergosterol contents in lipid starved <i>T</i>. <i>cruzi</i> epimastigotes, kept in LIT medium supplemented with 10% dFCS for 0, 24, 48 and 72h.</p

Incorporation of ³H-cholesterol into cholesteryl esters in epimastigotes.

<p>Parasites were incubated in LIT medium with 10% FCS (control) or 10% dFCS (starved) in the presence of LDL-³H-cholesterol (1,000,000 DPM; 2 mg/mL) for 3 days at 28°C. Parasites were washed and their lipids were extracted and analysed by TLC. The cholesterol (CHO) and cholesteryl-ester (CHOE) spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of three independent experiments analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p