BACKGROUND: Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis, a member of the Excavata supergroup of eukaryotes. The mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron-sulfur clusters. RESULTS: In live experiments, no fusion events were observed between the mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in mitosome dynamics. Unlike in mitochondria, division of the mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER-mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4. CONCLUSIONS: This study provides the first report on the dynamics of mitosomes. We show that together with the loss of metabolic complexity of mitochondria, mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.
- MeSH
- biologická evoluce MeSH
- dynaminy metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- Giardia lamblia cytologie metabolismus MeSH
- interfáze MeSH
- koenzym A-ligasy metabolismus MeSH
- mitochondriální dynamika * MeSH
- mitochondrie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Faecal samples from 162 wild animals were collected from 32 distinct sites of Łęczyńsko-Włodawskie Lakeland (eastern Poland). The presence of Giardia duodenalis (Stiles, 1902) was assessed by a Direct Fluorescence Assay (DFA) and by Polymerase Chain Reaction (PCR) and sequencing of a fragment of the beta-giardin gene. DFA showed the presence of cysts of G. duodenalis in 12 of 162 faecal samples (7%), namely in four wild boars (15%), four foxes (19%), two roe deer (4%), and two wolves (29%). PCR identified 34 of the 162 (21%) samples as positive, including 11 wild boars (41%), five red deer (18%), 11 roe deer (23%), four moose (17%), two wolves (29%) and a single sample from the European badger. Thus, PCR detected a significantly higher number of infection than DFA (P = 0.0005). However, 14 of 34 PCR products could not be sequenced because of their insufficient amount; the low number of cysts, poor conservation of the faeces or presence of PCR inhibitors may have contributed to weak DNA amplification. Sequence analysis of the remaining 20 products showed the presence of assemblage B in wild boars, red deer and roe deer, whereas samples from wolves were identified as assemblage D. This is the first detection of assemblage B in wild boars and deer. As assemblage B has zoonotic potential, wild animals from eastern Poland may act as reservoirs of cysts of G. duodenalis infectious for humans.
- MeSH
- divoká zvířata klasifikace parazitologie MeSH
- feces parazitologie MeSH
- fluorescenční protilátková technika přímá statistika a číselné údaje MeSH
- Giardia lamblia * cytologie patogenita MeSH
- giardiáza epidemiologie etiologie MeSH
- polymerázová řetězová reakce statistika a číselné údaje MeSH
- sekvenční analýza statistika a číselné údaje MeSH
- statistika jako téma MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Polsko MeSH
Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.
- MeSH
- biotinylace MeSH
- Escherichia coli enzymologie MeSH
- frakcionace buněk MeSH
- Giardia lamblia chemie cytologie metabolismus MeSH
- giardiáza parazitologie MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- ligasy tvořící vazby C-N metabolismus MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- organely chemie metabolismus MeSH
- proteiny z Escherichia coli metabolismus MeSH
- protozoální proteiny analýza izolace a purifikace metabolismus MeSH
- represorové proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- anaerobióza MeSH
- genetické vektory genetika MeSH
- Giardia lamblia cytologie genetika MeSH
- hydrolasy genetika MeSH
- ligandy MeSH
- mitochondrie metabolismus MeSH
- molekulární zobrazování metody MeSH
- organely metabolismus MeSH
- protozoální proteiny genetika MeSH
- rekombinantní fúzní proteiny genetika MeSH
- reportérové geny genetika MeSH
- Trichomonas vaginalis cytologie genetika MeSH
- viabilita buněk MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal. Copyright 2009 Elsevier Inc. All rights reserved.
- MeSH
- afidikolin farmakologie MeSH
- bromodeoxyuridin metabolismus MeSH
- buněčný cyklus účinky léků MeSH
- časové faktory MeSH
- cyklin B analýza MeSH
- DNA-dependentní DNA-polymerasy MeSH
- fluorescenční protilátková technika MeSH
- fosforylace účinky léků MeSH
- Giardia lamblia cytologie genetika účinky léků MeSH
- histony metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory syntézy nukleových kyselin MeSH
- mitotický index MeSH
- poškození DNA účinky léků MeSH
- protozoální DNA biosyntéza účinky léků MeSH
- průtoková cytometrie MeSH
- replikace DNA účinky léků MeSH
- trofozoiti cytologie účinky léků MeSH
- Publikační typ
- práce podpořená grantem MeSH
Trophozoites of Giardia are equipped with a special organelle of attachment, essential for parasite survival and pathogenicity, the ventral disc. Although its basic structure is well established, its reorganization and assembly during cell replication is poorly understood. We addressed some of these problems with aid of conventional, confocal and electron microscopy. We found that dividing Giardia alternates attached and free swimming phases in accordance with functional competence of the parent or newly assembled discs. The division started in attached cells by detachment of the disc microtubules from basal bodies. Shortening and eventual loss of the giardin microribbons, and unfolding of the microtubular layer resulting in collapse of the disc chamber and parasite detachment underlined gradual disassembly of the parent disc skeleton. Two daughter discs assembled on the dorsal side of the attached cell, with their ventral sides exposed on the parent cell surface and their microtubular skeletons growing in counter-clockwise direction. A depression between the assembling discs marked the cleavage plane. The splitting continued during the free-swimming phase with ventral-ventral axial symmetry in a plane of the daughter discs. Finally, the daughter cells with fully developed discs but still connected tail to tail by a cytoplasmic bridge, attached to a substrate and terminated the division by a process resembling adhesion-dependent cytokinesis. The mode of assembly of the daughter discs and plane of the division is compatible with maintenance of the left-right asymmetry of the Giardia cytoskeleton in progeny, which cannot be satisfactorily explained by alternative models proposed so far. Copyright (c) 2007 Wiley-Liss, Inc.
- MeSH
- buněčné dělení fyziologie genetika MeSH
- cytokineze fyziologie MeSH
- dělení bunečného jádra fyziologie genetika MeSH
- financování organizované MeSH
- flagella fyziologie parazitologie ultrastruktura MeSH
- fluorescenční mikroskopie metody MeSH
- Giardia lamblia cytologie fyziologie genetika MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mikrotubuly fyziologie genetika ultrastruktura MeSH
- transmisní elektronová mikroskopie metody MeSH
- trofozoiti parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.
