14 research outputs found

    Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

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    Background: Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages 6150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl2,0.31), while the other two types of channels are slightl

    Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

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    Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes

    Study of the molecular mechanism involved in recycling of matrix protein receptor, PEX5, during glycosome biogenesis in Trypanosoma brucei

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    Trypanosoma brucei is the parasitic protist that is responsible for human sleeping sickness in Africa, a disease for which no adequate, affordable and harmless treatment is available. The parasite lives as a so-called procyclic-form trypanosome in the midgut of the tsetse fly, the vector that transmits the trypanosomes between people. It has been previously established that glycolysis is essential for the bloodstream form of the parasite, its life-cycle stage in humans, hence representing a promising drug target. Glycolysis in trypanosomatids is compartmentalized in peroxisome-like organelles called glycosomes, a unique feature not found in cells of other eukaryotes. Biogenesis of peroxisomes, organelles found in most eukaryotes, and of glycosomes in trypanosomatids are mediated by homologous proteins called peroxins (acronym PEX). The peroxins of trypanosomes are considered potentially good drug targets not only because glycolysis is essential for the parasites, but also because trypanosomes die when proper compartmentalization of glycolytic enzymes within glycosomes is disrupted. The import of proteins into the glycosomal matrix involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS-1) present at the C-terminus of the majority of these proteins. In yeasts and mammalian cells it has previously been shown that the cargo-loaded PEX5 associates with the peroxisomal membrane, delivers its cargo and is then ubiquitinated, a modification that serves as a signal for retrieval of PEX5 from the organelle to be used for further cycles of import (monoubiquitination) or, when recycling is impaired, for its proteasome-dependent degradation (polyubiquitination). We have found stable monoubiquitinated PEX5 in cytosolic fractions of wild-type bloodstream- and procyclic-form T. brucei. This modification appeared to be resistant to DTT, suggesting the conjugation of an ubiquitin moiety to a lysine residue of PEX5. We reason that this modified PEX5 species represents recycled molecules that have been efficiently exported by the recycling peroxin complex and that are in transit to be deubiquitinated, as a physiological step in the receptor cycle. We have identified the T. brucei orthologue of PEX4, the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast. This protein is expressed in both bloodstream and procyclic forms and is associated with the cytosolic face of the glycosomal membrane, probably via its interaction with the putative TbPEX22 that we also identified. Creation of a ∆PEX4 procyclic cell line by deletion of both alleles of the TbPEX4 gene enabled us to demonstrate that this peroxin is involved in TbPEX5 monoubiquitination. Surprisingly, after transfection of this mutant with a construct for expression of the Green Fluorescent Protein with a PTS-1 followed by subcellular localization studies by live cell imaging and fluorescence microscopy, only a minor defect in glycosomal matrix protein import was observed. Analysis of the ∆PEX4 mutant by qPCR showed that other enzymes of the putative UBC repertoire were upregulated. We assume that another trypanosome-specific UBC protein has taken over the function of PEX4 in the monoubiquitination of PEX5, but with less efficiency. Interestingly, important defects in morphology and motility were found in the majority of the ∆PEX4 cells. We hypothesize that these seemingly glycosome-biogenesis unrelated defects resulting from the knockout of the TbPEX4 gene could be explained by a disregulation of the expression of UBCs that are orthologous to yeast and human UBCs involved in cell-cycle control. We have also observed that PEX5 ubiquitination was affected in procyclic cell lines in which TbPEX12 and TbPEX6 expression was decreased by RNA interference. These data thus indicate the involvement of these proteins in TbPEX5 cycling, comparable to what has previously been demonstrated for their counterparts in yeasts and mammalian cells, but nonetheless with some differences as suggested by our studies. TbPEX12 and TbPEX6 were shown to be necessary for correct glycosomal matrix protein import and for cell viability, proving that the TbPEX5 recycling process is also essential. In addition, we have investigated the presence of channel-forming activities in glycosomal membrane preparations of T. brucei bloodstream forms. We found that glycosomal membrane proteins, when reconstituted in planar lipid bilayers, gave rise to the formation of three main kinds of channels. These were revealed by electrophysiological techniques. Currents were measured with amplitudes of 70-80, 20-25 and 8-11 pA (at a holding potential of +10 mV and with 3.0 M KCl as an electrolyte). The channels were in a fully open state over the membrane potential range +150 to -150 mV and showed no sub-conductance transitions. The channel with current amplitude of 20-25 pA is anion-selective while the other two types are slightly selective for cations. The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel’s pore. We discuss the relevance of these findings for the transport of glycolytic intermediates and other metabolites between the glycosomal lumen and cytosol. We propose different explanations for the apparent paradox of on one hand the presence of non-selective channels in the glycosomal membrane and on the other hand the experimentally proved low exchange of metabolites between the glycosomal matrix and cytosol. Importantly, the discovery of these non-selective channels in the glycosomal membrane of T. brucei may set new criteria for the size and physicochemical properties of inhibitors targeted to glycolytic and other enzymes inside glycosomes that will be designed as potential drugs for sleeping sickness.Trypanosoma brucei est le protiste parasite responsable de la maladie du sommeil en Afrique ; maladie pour laquelle il n’existe aucun traitement adéquat, abordable et inoffensif. Le parasite vit sous la forme procyclique dans l’intestin de la mouche tsé-tsé, le vecteur qui transmet les trypanosomes entre les hommes. Il a été établi précédemment que la glycolyse est essentielle pour la forme sanguicole du parasite, stade du cycle de vie chez l’homme, et représente donc un cible thérapeutique prometteuse. La glycolyse des trypanosomatidés est compartimentée dans des organites de la famille des peroxisomes appelés glycosomes, une caractéristique unique que l’on ne trouve pas dans les cellules des autres eucaryotes. La biogenèse des peroxisomes, organites trouvés chez la plupart des eucaryotes, et celle des glycosomes des trypanosomatidés sont médiées par des protéines homologues appelées peroxines (PEX en abrégé). Les peroxines des trypanosomes sont considérées comme de bonnes cibles thérapeutiques potentielles, non seulement car la glycolyse est essentielle pour les parasites, mais également parce que les parasites meurent quand la compartimentation des enzymes glycolytiques dans les glycosomes est perturbée. L’importation des protéines dans la matrice glycosomale implique un récepteur cytosolique, PEX5, qui reconnaît le signal de ciblage vers les peroxisomes de type 1 (PTS1). Dans les levures et les cellules mammifères, il a déjà été démontré que le récepteur chargé de sa protéine s’associe avec la membrane peroxisomale, livre son chargement et est ensuite ubiquitylé, une modification qui sert de signal pour la sortie de PEX5 de l’organite afin d’être réutilisé dans d’autres cycles d’importation (monoubiquitination) ou quand le recyclage n’est pas possible, pour sa dégradation dépendante du protéasome (polyubiquitination). Nous avons trouvé une PEX5 monoubiquitinée stable dans les fractions cytosoliques des formes sanguicol et procyclique sauvages de T. brucei. Cette modification semble être résistante au DTT, suggérant la liaison du groupement ubiquitine à une lysine de PEX5. Nous estimons que cette forme de PEX5 modifiée représente des molécules recyclées qui ont été exportées efficacement pour le recyclage du complexe de peroxines et qui sont en transit vers une désubiquitination, comme une étape physiologique dans le cycle du récepteur. Nous avons identifié chez T. brucei, l’orthologue de PEX4, l’enzyme de liaison de l’ubiquitine (UBC) responsable de la monoubiquitination de PEX5 chez la levure. Cette protéine est exprimée dans les formes procyclique et sanguicole et est associée à la face cytosolique de la membrane glycosomale, probablement via une interaction avec la probable TbPEX22 que nous avons également identifiée. La création d’une lignée cellulaire procyclique ∆PEX4 par délétion des deux allèles du gène TbPEX4 nous a permis de démontrer que cette peroxine est impliquée dans la monoubiquitination de TbPEX5. Etonnament, après la transfection de ce mutant avec une construction exprimant la « Green Fluorescent Protein » munie d’un PTS-1 et des études de localisation subcellulaire par des images de cellules vivantes et de la microscopie à fluorescence, seule une petite diminution de l’importation des protéines matricielles dans les glycosomes est observée. L’analyse du mutant ∆PEX4 par qPCR a montré que d’autres enzymes du répertoire probable des protéines UBC sont sur-régulées. Nous supposons qu’une autre protéine UBC a repris la fonction de PEX4 dans la monoubiquitination de PEX5, mais de manière moins efficace. Il est intéressant de noter que des défauts importants dans la morphologie et la mobilité ont été trouvés dans la majorité des cellules ∆PEX4. Nous supposons que ces défectuosités, qui résultent de l’élimination du gène TbPEX4 et qui ne semblent pas être liées à la biogenèse des glycosomes, peuvent être expliquées par une dérégulation de l’expression d’UBCs qui sont des orthologues des UBCs humaines et de levures impliquées dans le contrôle du cycle cellulaire. Nous avons également observé que l’ubiquitination de PEX5 était affectée dans des lignées cellulaires procycliques dont l’expression de TbPEX12 et TbPEX6 avaient été réduites par interférence à l’ARN. Ces résultats indiquent donc un rôle de ces protéines dans le cycle de TbPEX5, comparable à ce qui avait déjà été démontré pour les protéines équivalentes dans la levure et les cellules mammifères, mais cependant avec quelques différences comme le suggèrent nos études. Il a été montré que TbPEX12 et TbPEX6 sont nécessaires pour l’importation correcte des protéines de la matrice glycosomale et pour la viabilité cellulaire, prouvant que le recyclage de TbPEX5 est également essentiel. De plus, nous avons étudié la présence d’activités de formation de canaux dans des préparations de membranes glycosomales des formes sanguicoles de T. brucei. Nous avons trouvé que les protéines de la membrane glycosomale, quand elles sont reconstituées dans des doubles couches lipidiques planes, permettaient la formation de trois types de canaux. Ils ont été révélés par des techniques d’électrophysiologie. Des courants d’amplitudes de 70-80, 20-25 et 8-11 pA (à un potentiel fixe de +10 mV et avec 3.0 M KCl comme électrolyte) ont été mesurés. Les canaux étaient dans un état complètement ouvert dans une gamme de potentiels de membrane de +150 à –150 mV et ne montraient pas de transition de sous-conductance. Le canal, avec un courant d’amplitude de 20-25 pA, est sélectif pour les anions, tandis que les deux autres sont légèrement sélectifs pour les cations. Le canal sélectif pour les anions a montré un courant de rectification intrinsèque qui pourrait suggérer une asymétrie fonctionnelle du pore du canal. Nous discutons la pertinence de ces découvertes pour le transport des intermédiaires glycolytiques et d’autres métabolites entre la lumière du glycosome et le cytosol. Nous proposons différentes explications pour le paradoxe apparent où d’un côté on a la présence de canaux non sélectifs dans la membrane glycosomale et de l’autre côté de faibles échanges de métabolites entre la matrice du glycosome et le cytosol prouvés expérimentalement. Il est important de noter que la découverte de ces canaux non sélectifs dans la membrane glycosomale de T. brucei met en place de nouveaux critères pour la taille et les propriétés physicochimiques des inhibiteurs dirigés contre la glycolyse et d’autres enzymes à l’intérieur des glycosomes qui seront modélisés comme médicaments potentiels pour la maladie du sommeil.(SBIM 3) -- UCL, 201

    Extracellular Vesicles in Trypanosoma cruzi Infection: Immunomodulatory Effects and Future Perspectives as Potential Control Tools against Chagas Disease.

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    Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is a neglected tropical disease and a major public health problem affecting more than 6 million people worldwide. Many challenges remain in the quest to control Chagas disease: the diagnosis presents several limitations and the two available treatments cause several side effects, presenting limited efficacy during the chronic phase of the disease. In addition, there are no preventive vaccines or biomarkers of therapeutic response or disease outcome. Trypomastigote form and T. cruzi-infected cells release extracellular vesicles (EVs), which are involved in cell-to-cell communication and can modulate the host immune response. Importantly, EVs have been described as promising tools for the development of new therapeutic strategies, such as vaccines, and for the discovery of new biomarkers. Here, we review and discuss the role of EVs secreted during T. cruzi infection and their immunomodulatory properties. Finally, we briefly describe their potential for biomarker discovery and future perspectives as vaccine development tools for Chagas Disease

    Autophagy in parasitic protists: unique features and drug targets

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    Eukaryotic cells can degrade their own components, cytosolic proteins and organelles, using dedicated hydrolases contained within the acidic interior of their lysosomes. This degradative process, called autophagy, is used under starvation conditions to recycle redundant or less important macromolecules, facilitates metabolic re-modeling in response to environmental cues, and is also often important during cell differentiation. In this review, we discuss the role played by autophagy during the life cycles of the major parasitic protists. To provide context, we also provide an overview of the different forms of autophagy and the successive steps in the autophagic processes, including the proteins involved, as revealed in recent decades by studies using the model organism Saccharomyces cerevisiae, methylotrophic yeasts and mammalian cells. We describe for trypanosomatid parasites how autophagy plays a role in the differentiation from one life cycle stage to the next one and, in the case of the intracellular parasites, for virulence. For malarial parasites, although only a limited repertoire of canonical autophagy-related proteins can be detected, autophagy seems to play a role in the removal of redundant organelles important for cell invasion, when sporozoites develop into intracellular trophozoites inside the hepatocytes. The complete absence of a canonical autophagy pathway from the microaerophile Giardia lamblia is also discussed. Finally, the essential role of autophagy for differentiation and pathogenicity of some pathogenic protists suggests that the proteins involved in this process may represent new targets for drug development. Opportunities and strategies for drug design targeting autophagy proteins are discussed

    Detection of channel-forming activities in subcellular fractions.

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    <p>Fractions 2–4 (glycosomes), 8–11 (fragments of flagella), and 15–18 (mitochondria) from Optiprep density gradients (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>) were combined and treated with Genapol X-080 to solubilize membrane proteins (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). After sedimentation of insoluble material, aliquots of the resulting supernatants were used for MCR (<b>A</b>–<b>C</b>) or SCA (<b>D</b>). (<b>A</b>) Traces of the current monitoring in the presence of glycosomal (upper panel) or mitochondrial (lower panel) preparations. The middle trace represents a timescale-expanded current recording of the upper trace. The bath solution contained 3 M KCl and the applied voltage was +10 mV. (<b>B</b>) Histograms of insertion events registered in subcellular fractions (see panel <b>A</b>). Bin size is 4.0 pA. The total number of insertion events (I.e.) is indicated. Here and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3</a> C (upper panel) all insertion events with current increments over 180 pA (for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3C</a>, lower panel −90 pA) are combined in one bin (180 pA or 90 pA, respectively). Note that the amount of insertion events in the flagella fraction (see <b>B</b>, middle panel) is lower than that observed in other fractions. This is mainly due to low channel-forming activity (per protein content) in the preparations of this fraction. For the sake of compatibility we used the same amounts of protein for measurements in different fractions. (<b>C</b>) Histograms of insertion events detected for glycosomal preparations using NH<sub>4</sub>Cl as the electrolyte. Bin size: 4 pA (upper panel) or 2 pA (lower panel). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figures 3A and 3B</a> for other details. (<b>D</b>) Trace of the current monitoring using the glycosomal fraction (initial holding potential +10 mV) indicating the insertion (marked by one asterisk) of a large-conductance channel that spontaneously closed (marked by two asterisks) after stepwise (each step is +10 mV) increase in the holding potential up to 50 mV.</p

    SCA of a very-low-conductance channel.

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    <p>(<b>A</b>) Current recording of a single very-low-conductance channel. The bath solution (panels <b>A</b>, <b>B</b>, and <b>C</b>) contained 3 M KCl. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. (<b>B</b>) Current trace of the channel in response to the shown voltage-ramp protocol. Dotted line indicates the current level at zero holding potential. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Current traces of a single channel in response to the indicated voltage-step protocol. (<b>D</b>) Ion-selectivity of the channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>D</b></a> for details.</p

    SCA of a high-conductance channel.

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    <p>(<b>A</b>) Current trace of a single high-conductance channel. The insertion event (marked by an asterisk) was registered at +10 mV and the applied voltage was then switched to −10 mV. The dashed line indicates the current level (zero) before insertion of the channel. The data in panels <b>A</b>, <b>B</b>, and <b>C</b> were collected using 3 M KCl as the electrolyte. (<b>B</b>) Current trace of the channel in response to the indicated voltage-ramp protocol. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Single channel currents in response to the indicated voltage-step protocol. (<b>D</b>) Dependence of the single channel conductance on the KCl concentration. After detection of a single channel insertion using 3 M KCl as bath solution (holding potential +10 mV), the electrolyte was diluted and registration of the current amplitudes of the same channel was conducted at 2.0 M and 1.0 M KCl, respectively. Data points are mean±SD for at least 4 independent measurements. (<b>E</b>) Current traces of a single channel in response to a low-speed linear increase (upper trace) or decrease (lower trace) of the holding potential. The bath solution contained 1.0 M NH<sub>4</sub>Cl, 20 mM Tris-Cl, pH 7.8, and 2 mM DTT at both sides of the membrane. Note that the channel was still open even at hyperpolarizing holding potentials of ±150 mV. (<b>F</b>) Current-voltage relationship of the high-conductance channel under asymmetric salt conditions: 3.0 M KCl <i>trans</i>/1.5 M KCl <i>cis</i> compartment. The insertion of a single channel was detected at 3 M KCl at both sides of the membrane and at a voltage of +10 mV, then the electrolyte concentration in the <i>cis</i> compartment was decreased by dilution and an initial current recording was conducted at zero potential followed by stepwise (±10 mV) change of the applied voltage. Data points are mean±SD, n = 4–5. Bars in some cases are smaller than symbols.</p

    SCA of a low-conductance channel.

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    <p>(<b>A</b>) Current traces of two low-conductance channels. The bath solution (<b>A–C</b>) contained 3 M KCl at both sides of the membrane. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. Note that two types of channels were registered. Most of them showed a larger current amplitude at +10 mV than at −10 mV (upper trace). In contrast, some channels displayed an opposite trend (lower trace). (<b>B</b> and <b>C</b>) Current traces of the channels in response to the indicated voltage-ramp (<b>B</b>) and voltage-step (<b>C</b>) protocols. Most detected channels displayed a current rectification at negative holding potentials (upper panels). However, in a few cases the rectification was observed at positive holding potentials (lower panels). (<b>D</b>) Dependence of the low-conductance channel activity on the electrolyte concentration. The channel insertion was registered at a holding potential of +10 mV using 3 M KCl as a bath solution. After confirming that the channel shows current rectification at negative voltages by application of a voltage-ramp protocol, the electrolyte in the chambers was diluted to 2.0 M or 1.0 M KCl and the current amplitudes were measured at +10 mV. Data are mean±SD, n = 4–5. (<b>E</b>) Ion selectivity of the low-conductance channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>F</b></a> for details. The current-voltage relationship of channels (rectification at negative voltages) was validated using a voltage-ramp protocol.</p

    Electron microscopy of cellular organelles separated by Optiprep gradient centrifugation.

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    <p>Fractions enriched in glycosomes (fractions 2–5, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>), fragments of flagella (fractions 8–11) or mitochondria and other organelles (fractions 15–18) were combined and processed for EM examination (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). (<b>A</b> and <b>B</b>) Isolated glycosomes shown at lower (<b>A</b>) and higher (<b>B</b>) magnifications. The fraction consists mostly of glycosomes. Some contamination by fragments of flagella is also visible. Importantly, fragments of flagella (paraflagellar rods and axonemes) show no sign of attachment to the flagellar membrane. Note the presence of intact glycosomes as electron-dense vesicles surrounded by a single membrane (marked by arrows in panel <b>B</b>). (<b>C</b> and <b>D</b>) Fractions enriched in flagella at low (<b>C</b>) and high (<b>D</b>) magnifications. One can see many paraflagellar rods in longitudinal section (<b>C</b>) and recognize flagellar axonemes (marked by arrows in panel <b>D</b>). Some glycosomes are also visible in panel <b>C</b>. (<b>E</b> and <b>F</b>) Composition of the fraction from the top of the Optiprep gradient that is enriched with mitochondria. Several types of organelles – mitochondria, lysosomes, lipid droplets, clathrin-coated vesicles, and components from the flagellar apparatus – can be observed. Note the shrinking of the mitochondrial inner membrane (see panel <b>F</b>) apparently due to osmotic misbalance. Scale bars: 2 µm (<b>C</b> and <b>E</b>); 1 µm (<b>A</b>); 0.5 µm (<b>D</b> and <b>F</b>), and 0.1 µm (<b>B</b>).</p
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