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Richard E Triemer - One of the best experts on this subject based on the ideXlab platform.
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ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1
Journal of Phycology, 2002Co-Authors: Woongghi Shin, Stacy Brosnan, Richard E TriemerAbstract:In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid Cytostomes, colorless euglenoid Cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/ pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.
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ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1
Journal of Phycology, 2002Co-Authors: Woongghi Shin, Stacy Brosnan, Richard E TriemerAbstract:In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid Cytostomes, colorless euglenoid Cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/ pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.
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RECONSTRUCTION OF THE FEEDING APPARATUS IN PLOEOTIA COSTATA (EUGLENOPHYTA) AND ITS RELATIONSHIP TO OTHER EUGLENOID FEEDING APPARATUSES
Journal of Phycology, 1999Co-Authors: Eric W. Linton, Richard E TriemerAbstract:The ultrastructure of the feeding apparatus in Ploeotia costata Farmer and Triemer was determined and compared to other euglenoid feeding apparatuses. The feeding apparatus opened subapically onto the ventral surface and extended nearly the entire length of the cell. It consisted of four parts at the anterior surface: a comb, Cytostome/pocket, vanes, and supporting rods. The comb was a multilayered structure of three horizontal microtubular rows encased in cement and formed the dorsal lip of the apparatus. The Cytostome/pocket was located between the comb and the supporting rods, tapered into the cell as the cytopharynx and was surrounded by five vanes. The electron-opaque vanes extended the entire length of the feeding apparatus and were lined with microtubules for most of their length. Finally, two cement supporting rods that were joined by a crosspiece at the anterior end formed the ventral lip. The rods separated briefly before merging with the vanes. As the merged rods and vanes descended into the cell, they gradually narrowed and terminated. Comparisons of the feeding apparatus with Ploeotia vitrea, Diplonema ambulator, Lentomonas applanatum, and other euglenoids have led to the conclusion that the Type II feeding apparatus is found only in Ploeotia species.
Wanderley De Souza - One of the best experts on this subject based on the ideXlab platform.
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Loss of the Cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis.
Journal of structural biology, 2016Co-Authors: Juliana C. Vidal, Wanderley De Souza, Carolina De L. Alcantara, Narcisa L. Cunha-e-silvaAbstract:Trypanosoma cruzi epimastigotes uptake nutrients by endocytosis via the Cytostome-cytopharynx complex - an anterior opening (Cytostome) continuous with a funnel-shaped invagination (cytopharynx) that extends to the posterior of the cell, accompanied by microtubules. During metacyclogenesis - the transformation of epimastigotes into human-infective metacyclic trypomastigotes - the Cytostome-cytopharynx complex disappears, as trypomastigotes lose endocytic ability. To date, no studies have examined Cytostome-cytopharynx complex disappearance in detail, or determined if endocytic activity persists during metacyclogenesis. Here, we produced 3D reconstructions of metacyclogenesis intermediates (Ia, Ib, Ic) using electron microscopy tomography and focused ion beam-scanning electron microscopy (FIB-SEM), concentrating on the Cytostome-cytopharynx complex and adjacent structures, including the preoral ridge (POR). Parasite endocytic potential was examined by incubation of intermediate forms with the endocytic tracer transferrin (Tf)-Au. Ia, Ib and Ic cells were capable of internalizing Tf-Au, and had a shorter cytopharynx than that of epimastigotes, with the Cytostome/POR progressively displaced towards the posterior, following the movement of the kinetoplast/flagellar pocket. While some Ic cells had a short cytopharynx with an enlarged proximal end (∼300nm in diameter, larger than that of the Cytostome), other Ic cells had no cytopharynx invagination, but retained the cytopharynx microtubules, which were also present in metacyclics. We conclude that Cytostome-cytopharynx disappearance and loss of endocytic ability are late events in metacyclogenesis, during which the Cytostome is displaced towards the posterior, probably due to a link to the kinetoplast/flagellar pocket. Retention of the cytopharynx microtubules by metacyclics may allow prompt Cytostome-cytopharynx reassembly in amastigotes, upon host cell infection.
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The Cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes disassembles during cell division
Journal of cell science, 2016Co-Authors: Carolina De L. Alcantara, Wanderley De Souza, Juliana C. Vidal, Narcisa L. Cunha-e-silvaAbstract:The Cytostome-cytopharynx complex is the main site for endocytosis in epimastigotes of Trypanosoma cruzi It consists of an opening at the plasma membrane surface - the Cytostome - followed by a membrane invagination - the cytopharynx. In G1/S cells, this structure is associated with two specific sets of microtubules, a quartet and a triplet. Here, we used electron microscopy and electron tomography to build 3D models of the complex at different stages of the cell cycle. The Cytostome-cytopharynx is absent in late G2 and M phase cells, whereas early G2 cells have either a short cytopharynx or no visible complex, with numerous vesicles aligned to the Cytostome-cytopharynx microtubules. The microtubule quartet remains visible throughout cell division (albeit in a shorter form), and is duplicated during G2/M. In contrast, the microtubule triplet is absent during late G2/M. Cells in cytokinesis have an invagination of the flagellar pocket membrane likely to represent early stages in Cytostome-cytopharynx assembly. Cells in late cytokinesis have two fully developed Cytostome-cytopharynx complexes. Our data suggest that the microtubule quartet serves as a guide for new Cytostome-cytopharynx assembly.
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The three-dimensional structure of the Cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes
Journal of Cell Science, 2014Co-Authors: Carolina De L. Alcantara, Wanderley De Souza, Juliana C. Vidal, Narcisa L. Cunha-e-silvaAbstract:The Cytostome-cytopharynx complex is the main site of endocytosis of Trypanosoma cruzi epimastigotes. Little is known about the detailed morphology of this remarkable structure. We used serial electron tomography and focused-ion-beam scanning electron microscopy to reconstruct the entire complex, including the surrounding cytoskeleton and vesicles. Focusing on cells that had taken up gold-labeled tracers, we produced three-dimensional snapshots of the process of endocytosis. The Cytostome cytoskeleton was composed of two microtubule sets--a triplet that started underneath the Cytostome membrane, and a quartet that originated underneath the flagellar-pocket membrane and followed the preoral ridge before reaching the cytopharynx. The two sets accompanying the cytopharynx formed a 'gutter' and left a microtubule-free side, where vesicles were found to be associated. Cargo was unevenly distributed along the lumen of the cytopharynx, forming clusters. The cytopharynx was slightly longer during the G2 phase of the cell cycle, although it did not reach the postnuclear region owing to a bend in its path. Therefore, the cytopharynx is a dynamic structure, undergoing remodeling that is likely associated with endocytic activity and the preparation for cell division.
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The endocytic pathway in the epimastigote form of
2013Co-Authors: Dirceu E. Teixeira, Marlene Benchimol, Paulo Henrique Crepaldi, Wanderley De SouzaAbstract:(A) Endocytosis occurs in two sites of macromolecular ingestion: the Cytostome-cytopharinx complex and the flagellar pocket. (B) In Cytostome-cytopharinx complex the macromolecules migrate through the cytopharynx and are internalized via small vesicles, which are formed in the final portion of the cytopharynx. (C) Subsequently, the macromolecules cross through the early tubular endosomal network and are delivered to a reservosome (D). (E) The macromolecules are also internalized via vesicles that form in the flagellar pocket. (F) The endocytic pathway continues through a network of long tubules and vesicles extending to the posterior end of the cell body, returning to the opposite direction and eventually merging with the reservosome. (G) Our model also suggests that cruzipain molecules, as well as other proteases, are processed and leave the Golgi complex. (H) Vesicles containing these molecules also interact with the endocytic pathway and are transported to reservosomes. These images were made based on micrographs of transmission electron microscopy.
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Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi.
Parasitology international, 2010Co-Authors: Gustavo Miranda Rocha, Kildare Miranda, Narcisa L. Cunha-e-silva, Sergio Henrique Seabra, Tecia Maria Ulisses De Carvalho, Wanderley De SouzaAbstract:The flagellar pocket and the Cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The Cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between Cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.
Woongghi Shin - One of the best experts on this subject based on the ideXlab platform.
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ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1
Journal of Phycology, 2002Co-Authors: Woongghi Shin, Stacy Brosnan, Richard E TriemerAbstract:In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid Cytostomes, colorless euglenoid Cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/ pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.
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ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1
Journal of Phycology, 2002Co-Authors: Woongghi Shin, Stacy Brosnan, Richard E TriemerAbstract:In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid Cytostomes, colorless euglenoid Cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/ pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.
Maurilio J. Soares - One of the best experts on this subject based on the ideXlab platform.
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Clathrin expression in Trypanosoma cruzi
BMC cell biology, 2014Co-Authors: Ligia Cristina Kalb, Cassiano Martin Batista, Iriane Eger, Yohana Camila Antunes Frederico, Stenio Perdigão Fragoso, Maurilio J. SoaresAbstract:Clathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins. Analysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the Cytostome/cytopharynx complex. Clathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the Cytostome/cytopharynx complex.
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Transferrin uptake in Trypanosoma cruzi is impaired by interference on Cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis
Experimental parasitology, 2007Co-Authors: Jose R Correa, Georgia Correa Atella, Marcelo M. Batista, Maurilio J. SoaresAbstract:Transferrin uptake by Trypanosoma cruzi epimastigotes ocurrs mainly through the Cytostome/cytopharynx. Here, we present evidences for the association of sterol-rich membrane domains with the transferrin endocytic site. Assays using pharmacological treatments to disrupt clathrin-coated pits and hinder caveolae formation showed no association between transferrin uptake and clathrin-dependent endocytosis, but indicated that cholesterol stability in membrane domains is essential for the endocytosis of transferrin. Furthermore, it was observed a connection between the integrity of cytoskeleton elements at the cytopharynx and the function of the Cytostome. Our data show that T. cruzi epimastigotes depend on a specialized pathway for transferrin uptake, which is cholesterol-dependent, clathrin-independent, and closely associated with the structural stability of the Cytostome/cytopharynx cytoskeleton.
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transferrin uptake may occur through detergent resistant membrane domains at the cytopharynx of trypanosoma cruzi epimastigote forms
Memorias Do Instituto Oswaldo Cruz, 2007Co-Authors: Jose R Correa, Camila Vargas, Georgia Correa Atella, Maurilio J. SoaresAbstract:Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a Cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise Cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the Cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.
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low temperature blocks fluid phase pinocytosis and receptor mediated endocytosis in trypanosoma cruzi epimastigotes
Parasitology Research, 2000Co-Authors: R C B Q De Figueiredo, Maurilio J. SoaresAbstract:Gold-labeled albumin and transferrin were used to follow at the ultrastructural level the early events and the effect of low temperature on protein uptake by Trypanosoma cruzi epimastigotes. In parasites incubated for 5 min at 28 °C with protein-gold complexes, extracellular markers were found only at the Cytostome and/or the flagellar pocket regions, whereas intracellular gold particles were detected inside small uncoated vesicles located nearby. Within 10 min, labeling was also observed in uncoated vesicles close to the nucleus. Only after 30 min could the tracers be detected in the reservosomes. Weak labeling in the Cytostome and flagellar pocket of parasites incubated at 4 °C with the albumin-gold solution indicated that albumin uptake occurred by fluid-phase pinocytosis. On the other hand, intense labeling at the Cytostome was observed in parasites incubated at 4 °C with gold-labeled transferrin, showing that receptor-mediated endocytosis occurs mainly at this site. Both proteins were absent from the cells at 4 °C and 12 °C. Raising the temperature from 12 °C to 28 °C led to transferrin labeling in intracellular vesicles dispersed throughout the cytoplasm, but not in reservosomes. Our results suggest that low temperatures affect the transport and pinching of endocytic vesicles as well as the rate of delivery of transferrin to reservosomes.
Stacy Brosnan - One of the best experts on this subject based on the ideXlab platform.
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ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1
Journal of Phycology, 2002Co-Authors: Woongghi Shin, Stacy Brosnan, Richard E TriemerAbstract:In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid Cytostomes, colorless euglenoid Cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/ pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.
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ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1
Journal of Phycology, 2002Co-Authors: Woongghi Shin, Stacy Brosnan, Richard E TriemerAbstract:In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid Cytostomes, colorless euglenoid Cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/ pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.
