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.
Citation: Martincová E, Voleman L, Najdrová V, De Napoli M, Eshar S, Gualdron M, et al. (2012) Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology. PLoS ONE 7(4): e36314. https://doi.org/10.1371/journal.pone.0036314
Editor: Ross Frederick Waller, University of Melbourne, Australia
Received: January 21, 2012; Accepted: April 2, 2012; Published: April 27, 2012
Copyright: © 2012 Martincová et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by grants from the Czech Science Agency P305/10/0651 and LC07032 and from the Ministry of Education of the Czech Republic MSM 0021620858 and from the funders of the Biology of Parasitism course in Woods Hole (Burroughs Wellcome Fund, Howard Hughes Medical Institute and Wellcome Trust). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In recent years studies of anaerobic protists such as Giardia intestinalis and Trichomonas vaginalis have revealed a number of exciting aspects of their cell biology, including cytoskeleton structures, vesicular transport and organelle biogenesis –. Besides unique cellular structures –, many of the common eukaryotic processes have been stripped to their essentials in these protists e.g. , . The combination of their parasitic lifestyle, anaerobic metabolism and their evolutionary position  makes them attractive objects to study.
One of the features typical to anaerobic protists is the absence of ‘classical’ mitochondria, herein represented by organelles called mitosomes in G. intestinalis and hydrogenosomes in T. vaginalis . Mitosomes, the simplest mitochondria-related compartments, seem to have lost all but the single pathway of iron-sulfur cluster assembly . Compared to mitosomes, hydrogenosomes are more elaborate organelles, possessing substrate level ATP synthesis as well as iron and amino acid metabolism , . Moreover, recent proteomic studies of hydrogenosomes suggest that many more pathways are yet to be described , .
Characterization of cellular organelles and their dynamics strongly relies on the concerted action of reverse genetics and live cell imaging. While particular advancements have been achieved in the former (e.g. gene silencing and protein overexpression) –, only limited technical innovations have been introduced into the latter , .
GFP and its derivatives are the first choice of reporters for live imaging in aerobic eukaryotes. They offer great protein stability as well as a broad range of spectral variants that enable multichannel studies. However the major drawback for their widespread use in anaerobic protists is the formation of the GFP fluorophore , : upon translation and protein folding the fluorophore is formed from the tripeptide Ser 65-Tyr 66-Gly 67 by an intramolecular cyclization, which requires the presence of molecular oxygen , . This reaction does not require additional proteins and occurs spontaneously in all eukaryotic compartments, except within anaerobic cells, which employ oxygen scavenging pathways in order to limit its toxic effects , . Cells can be temporarily oxygenated and observed under the microscope ,  . While this approach has proven to be efficient for large cellular structures such as the cytoskeleton , , the organelles like mitosomes and hydrogenosomes exhibit only very weak labeling. Additionally, the double membrane surrounding the organelles may have limited capacity to import GFP.
Alternative approaches for live cell imaging exploit the use of chemical fluorescent tags, which form covalent or noncovalent bonds with the reporter protein or peptide . Of these, SNAP and CLIP tags are commonly used for both extra- and intracellular labeling , . The SNAP tag was successfully used to track the distribution of G. intestinalis RabA homologue in the live parasite . However, the use of the tag has been limited to this single study so far.
In this work, we decided to test a newly developed tag termed HaloTag, which utilizes a mutant form of haloalkane dehalogenase as a reporter protein. While the original enzyme hydrolyzes alkylhalides into a free halide and a primary alcohol, the H289Q mutant form of the protein (HaloTag) leaves free halide but remains covalently bound to the alkyl chain . Thus, when a ligand with the alkylhalide chain is exposed to the native HaloTag, it is specifically bound by a covalent bond. The lack of dehalogenase activity among eukaryotes guarantees very low unspecific background labeling.
Here, we report the successful introduction of the HaloTag into vectors for stable expression in G. intestinalis and T. vaginalis. Moreover, using a TMR-halo ligand we were able to show live images of mitochondria-related compartments in these two anaerobic protists for the first time.
Materials and Methods
The G. intestinalis strain WB (ATCC 30957) was grown in TYI-S-33 medium supplemented with 10% heat-inactivated bovine serum, 0.1% bovine bile, and antibiotics. The T. vaginalis strain T1 was grown in TYM pH 6,2 medium supplemented with 10% heat inactivated horse serum. Both organisms were cultured at 37°C.
Preparation of cell fractions
G. intestinalis trophozoites were harvested in ice-cold PBS, washed once in ST buffer (250 mM sucrose, 0.5 mM KCl, 10 mM Tris [pH 7.2]) and suspended in ST buffer with protease inhibitors 50 µg/ml N-a-tosyl-L-lysine chloromethyl ketone and 10 µg/ml of leupeptin. Cells were lysed on ice using sonication, during which the cell integrity was checked under the microscope. The lysate was centrifuged twice at 2450× g for 10 minutes to remove unbroken cells, nuclei and residual cytoskeleton. Supernatant was transferred to a new tube and the centrifugation step repeated twice. The resulting supernatant was spun down at 180 000× g for 30 minutes. Final supernatant and pellet contained the cytosolic and high-speed pellet fraction, respectively.
T. vaginalis cells were harvested, washed once in ST buffer and suspended in ST buffer containing protease inhibitors (see above). Cells were sonicated on ice and the lysate was twice centrifuged at 2450× g (see above). Supernatant was spun down at 180 000× g for 30 minutes. The final supernatant corresponded to the cytosolic fraction. The pellet was resuspended in 1 ml of ST buffer, transferred to a new microcentrifuge tube and spun down at 30 000× g for 10 minutes. The Resulting pellet contained a white layer of lysosomes resting on top of a brown pellet of hydrogenosomes. Lysosomes were carefully removed using a pipette and this step was repeated once more. The final pellet corresponded to the hydrogenosomal fraction.
Cloning and stable cell transformation
G. intestinalis .
First, pTG vector (gift from Francis D. Gillin, ) was modified to contain NdeI PstI sites. The polylinker containing EcoRV, NdeI, XhoI, PstI, NsiI, MluI and ApaI sites was introduced into the vector using 5′-CATGGATATCCATATGCTCGAGCTGCAGATGCATACGCGTATGGTGAGCAAGGGCGAGGAG-3′ and 5′-GATCGGGCCCTCACTTGTACAGCTCGTCCAT-3′ primers. The PCR product was digested by EcoRV and ApaI and ligated into EcoRV/ApaI linearized pTG vector. The 300 bp of 5′UTR of G. intestinalis ornithine carbamoyl transferase (OCT) DNA sequence was amplified using 5′-CATGGATATCGAATTCGATGCTTCG-3′ and 5′-CATGCATATGTTTAATTTTCAGCCTCTACTG-3′ primers, digested by EcoRV and NdeI primers and ligated into modified pTG vector. The HaloTag DNA sequence was amplified from pHT2 vector (Promega) using 5′-ATGCTGCAGATGGGATCCGAAATCGGTACA-3′and 5′-CATGGGGCCCTTAGCCGGCCAGCCCGGGGAG-3′ oligonucleotides. The resulting PCR product was digested by PstI and ApaI and ligated into modified pTG vector. G. intestinalis IscU was amplified from genomic DNA using 5′-CTAGCATATGATGACTTCTGATGCCGCAGAT-3′ and 5′-GACTATGCATAGAAGACTTTGATACCTGTAT-3′ oligonucleotides. The product was digested by NdeI and NsiI and ligated into modified pTG vector containing HaloTag coding sequence.
T. vaginalis .
For expression in T. vaginalis, the HaloTag DNA sequence was amplified from pHT2 vector using 5′-CATGAGATCTATGGGATCCGAAATCGGTACA-3′ and 5′-GCTACTCGAGTTAAGCGTAATCTGGAACATCGTATGGGTAGCCGGCCAGCCCGGGGAGCCA-3′. The C-terminal hemagglutinin (HA)-tag was introduced into the construct as a part of the reverse primer. The PCR product was digested by BglII and XhoI and ligated into BamHI/XhoI linearized TagVag2 vector containing a gene encoding hydrogenosomal frataxin. Both organism were electroporated using modified protocols published in , . Briefly, three hundred micro liters of T. vaginalis and G. intestinalis at approximate concentration 2,5×108 cells/ml and 3,3×108 cells/ml, respectively, were electroporated with 50 ug of the plasmid using a Biorad Gene Pulser under the time constant protocol (Tc = 175 ms, U = 350 V). Transfectants were maintained under pressure of selective antibiotics (57 ug/ml of puromycin for G. intestinalis and 200 ug/ml for T. vaginalis).
Halo-labeling and immunofluorescence microscopy
Cell were incubated for 30 minutes in regular growth media supplemented with HaloTag TMR Ligand (1: 500 dilution) at 37°C. After the incubation the cells were pelleted at 1500× g and washed twice with fresh media. Cells were then incubated for 60 minutes at 37°C, pelleted and resuspended in fresh media or PBS. For immunofluorescence, the cells were incubated on slides for 15 minutes, fixed in −20°C methanol for 5 minutes and transferred to −20°C acetone for 5 minutes. Blocking and immunolabeling was performed in 0,25% Gelatin, 0,25% BSA, 0,05%.
Tween20 in PBS 7,4 using specific rabbit polyclonal antibodies raised against T. vaginalis malic enzyme and G.intestinalis Tom40 . Primary antibodies were decorated by Alexa Fluor 488 anti-rabbit antibody. Slides were mounted in hard set Vectashield containing DAPI. For live cell imaging, labeled G. intestinalis cells were allowed to attach to the surface of 96 well optical bottom plates and imaged directly. Labeled T. vaginalis cells were mounted in low temperature-melting 2% agarose dissolved in PBS and analyzed by microscopy. Cells were observed using an OLYMPUS Cell-R, IX81 microscope system and images processed by Fiji (http://fiji.sc/wiki/index.php/Fiji). During all steps cells were protected from light.
Results and Discussion
Mitosomes and hydrogenosomes can be found in anaerobic protists from different eukaryotic lineages. Recent phylogenetic and functional data have shown that these double membrane bound organelles represent long evolved mitochondrial forms adjusted to anaerobic environments . While devoid of many typical mitochondrial functions, they contain unique metabolic adaptations as well as simplified versions of intricate molecular processes occurring in mitochondria –. To date only limited information is available on their biogenesis, inheritance and related membrane dynamics . In order to follow these processes in living cells we have introduced HaloTag technology into both G. intestinalis and T. vaginalis.
The coding sequence of HaloTag was introduced into G. intestinalis and T. vaginalis episomal vectors pTG and TagVag2, respectively ,  . Transcription from these vectors is driven by promotor regions in 5′ UTRs of highly expressed ornithine carbamoyl transferase and succinyl-CoA thiokinase , , respectively, which ensure strong constitutive protein expression in both organisms. For specific labeling of mitochondria-related organelles in these anaerobic protists, the HaloTag was inserted as a C-terminal fusion to the mitosomal and hydrogenosomal marker proteins GiIscU and TvFtx, respectively , .
Expression of proteins fused to the HaloTag was determined on western blots of cellular fractions (Figure 1). G. intestinalis IscU-HaloTag fusion was detected by specific polyclonal antibody raised against mitosomal IscU. Two dominant protein bands of approximately 15 kDa and 50 kDa were detected, which is consistent with the expected molecular weights (the size of HaloTag is 33 kDa) (Figure 1A). While the lower band corresponded to the mature form of nuclear encoded IscU, the upper band represented IscU-HaloTag fusion. The specific signal was present in the lysate and high speed pellet fraction, which is in addition to other vesicular structures enriched for mitosomes. Additional weak protein bands were detected, which likely corresponded to partially proteolytically degraded protein forms.
Western blot analyses of cellular fractions of G. intestinalis and T. vaginalis transformants expressing GiIscU-Halo and TvFtx-Halo fusions, respectively. A) GiIscU-Halo was detected by specific anti-IscU polyclonal antibodies in cell lysate and high-speed pellet (HSP). Two bands in these fractions represent the nuclear encoded (GiIscU) and episomally encoded HaloTag fusion (GiIscU-Halo). B) TvFtx-Halo product was detected by anti-HA monoclonal antibodies in T. vaginalis cellular fractions. The fusion protein was found exclusively in cell lysate and in hydrogenosomes. The upper panels demonstrate the protein profile on the coomassie stained SDS-PAGE gel. Lys-lysate, Cyt-cytosol, HSP-high-speed pellet, Hyd-hydrogenosomes.
In order to detect HaloTagged hydrogenosomal frataxin in T. vaginalis, an additional single hemagglutinin (HA)-tag was introduced to the C-terminus of the HaloTag sequence. Using anti-HA antibodies the protein band of about 47 kDa, corresponding to the expected protein fusion size, was detected in the cell lysate and hydrogenosomal fractions (Figure 1B).
In both organisms, the HaloTag fusion proteins were expressed at a moderate level with no growth defect obvious in daily culturing, indicating that the tag does not interfere with the cellular metabolism of the anaerobic eukaryotes, similar to what has been shown in mammalian cells .
In order to confirm that the fusion protein is targeted to mitochondria-related compartments of G. intestinalis and T. vaginalis, cells were labeled with HaloTag TMR ligand, fixed and immunolabeled with specific antibodies raised against organellar marker proteins. In G. intestinalis, mitosomes were labeled by Tom40-specific antibody . Tom40 is a conserved protein of the outer mitochondrial/mitosomal membrane and its detection revealed typical mitosomal distribution within G. intestinalis cells: the central array of mitosomes between the two nuclei as well as the peripheral ones scattered throughout the cytoplasm. The HaloTag signal from GiIscU was found to be in perfect agreement with Tom40, revealing highly specific mitosomal compartment labeling in G. intestinalis (Figure 2A).
Immunofluorescence analyses of G. intestinalis and T. vaginalis transformants expressing GiIscU-Halo and TvFtx-Halo fusion, respectively. Cells were incubated with TMR-Halo ligand (red), washed and fixed for immunofluorescence analysis. A) TMR-Halo labeled G. intestinalis cells were fixed and labeled by anti-Tom40 specific polyclonal antibodies (green). B) TMR-Halo labeled T. vaginalis cells were fixed and decorated by anti-malic enzyme specific polyclonal antibodies (green). Nuclei were stained with DAPI (blue).
In contrast to mitosomes, which are scarce, T. vaginalis hydrogenosomes are abundant organelles distributed along the major cytoskeletal structures such as the costa and axostyle. Malic enzyme is the most dominant hydrogenosomal protein  and its detection in fixed TMR-Halo ligand-labeled cells revealed typical hydrogenosomal distribution. The same pattern was obtained with TMR labeled HaloTag, as indicated in the merged image (Figure 2B).
These experiments showed that the HaloTag TMR ligand is a membrane-permeable ligand in both organisms, capable of diffusing across the cell membrane as well as the two membranes surrounding the mitosomes and hydrogenosomes. Although some background labeling was detected using HaloTag in mammalian cells , no such signal was found in two anaerobic organisms used in this study.
Following the co-localization experiments, labeled cells were observed live for various time periods (Figure 3). While attached G. intestinalis trophozoites could be observed directly in optical bottom plates filled with medium (Figure 3A, Supplementary Movie S1 and S2), T. vaginalis were mounted in 2% low melting agarose in order to slow down the rapidly moving cells (Figure 3B, Supplementary Movie S3 and S4). In both parasites, the specific fluorescence signal could be followed visually for more than 60 minutes. Notably, for prolonged cell observation an anaerobic chamber would be necessary.
Halo-TMR labeled organelles were followed in living cells. A) Labeled G. intestinalis cells were allowed to attach to the bottom of the well and directly observed while B) the labeled T. vaginalis cells were mounted in 2% agarose and then submitted to microscopy. Five different snapshots in time are shown. The original movies are part of the supplementary data.
In summary, these experiments demonstrate the applicability of HaloTag in labeling the mitochondria-related organelles of G. intestinalis and T. vaginalis. These tiny double membrane bound organelles have been some of the most challenging cellular structures for live imaging in anaerobic eukaryotes, and to our knowledge this work is the first report of its kind.
HaloTag technology is relatively new to the cell biology. It exhibits excellent specificity and fast chemistry but as true for other large protein tags such as the fluorescent proteins or SNAP-tag, its major drawback is the size, which may interfere with the function of the carrier protein . When possible the imaging studies rely on GFP and other recently characterized fluorescent proteins e.g. , . In these cases, the chemical tags such as HaloTag, SNAP-tag or tetracystein helix motif  offer additional customizable labeling, especially suitable for pulse-chase  or FRET experiments. In the anaerobic unicellular organisms or the anaerobic tissues of some invertebrates the GFP maturation requires an extra oxygenation step, which may perturb narrow physiological conditions. In these cases, the chemical tags may be the first choice protein-labeling approach. Moreover, the speed and the specificity of the formation of the covalent bond between the HaloTag and the ligand provides new means of protein purification from not easily tractable organisms .
Mitochondria are known to be very dynamic organelles undergoing constant antagonist fusion and fission reactions . Several GTPases drive these opposing reactions in a highly regulated manner and the defects in the fusion or fission result in disintegration or collapse of the organelles, respectively. So far no information has been obtained on the machinery controlling the dynamics of mitosomes and hydrogenosomes. Given that neither the components of the mitochondrial division cycle nor the homologues of bacterial division proteins were found in the genomes of mitosome- and hydrogenosome-bearing eukaryotes, the HaloTag has the potential to be a means of identifying the different components driving these processes in these protists.
This opens up more fundamental questions regarding the evolution of the mitochondrial division apparatus, the transition from a FtsZ- to a dynamin-based system as well as the origin of mitochondrial fusion. We believe that the introduction of HaloTag technology to the cell biology of anaerobic protists will be of assistance in the process of answering these questions.
Giardia intestinalis expressing mitosomal IscU-HaloTag fusion was labeled with TMR-Halo ligand. Images were taken every at 10 second intervals, movie is displayed at 2 frames per second.
Nomarski differential contrast of the same visual field as in Movie S1.
Trichomonas vaginalis expressing hydrogenosomal frataxin-HaloTag fusion was labeled with TMR-Halo ligand. Images were taken every at 10 second intervals, movie is displayed at 2 frames per second.
We would like to thank the organizers (Boris Striepen and Dan Godlberg) of the Biology of Parasitism course in Woods Hole.
Conceived and designed the experiments: JT PD. Performed the experiments: EM LM VN MN SE MG CH DS DT DT DW MM. Analyzed the data: EM LV. Wrote the paper: EM LV PD.
- 1. Hehl AB, Marti M (2004) Secretory protein trafficking in Giardia intestinalis. Mol Microbiol 53: 19–28.AB HehlM. Marti2004Secretory protein trafficking in Giardia intestinalis.Mol Microbiol531928
- 2. Paredez AR, Assaf ZJ, Sept D, Timofejeva L, Dawson SC, et al. (2011) An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins. Proc Natl Acad Sci U S A 108: 6151–6156.AR ParedezZJ AssafD. SeptL. TimofejevaSC Dawson2011An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.Proc Natl Acad Sci U S A10861516156
- 3. Poxleitner MK, Carpenter ML, Mancuso JJ, Wang C-JR, Dawson SC, et al. (2008) Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 319: 1530–1533.MK PoxleitnerML CarpenterJJ MancusoC-JR WangSC Dawson2008Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis.Science31915301533
- 4. Tovar J, León-Avila G, Sánchez LB, Sutak R, Tachezy J, et al. (2003) Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature 426: 172–176.J. TovarG. León-AvilaLB SánchezR. SutakJ. Tachezy2003Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation.Nature426172176
- 5. Ankarklev J, Jerlström-Hultqvist J, Ringqvist E, Troell K, Svärd SG (2010) Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 8: 413–422.J. AnkarklevJ. Jerlström-HultqvistE. RingqvistK. TroellSG Svärd2010Behind the smile: cell biology and disease mechanisms of Giardia species.Nat Rev Microbiol8413422
- 6. Elmendorf HG, Dawson SC, McCaffery JM (2003) The cytoskeleton of Giardia lamblia. Int J Parasitol 33: 3–28.HG ElmendorfSC DawsonJM McCaffery2003The cytoskeleton of Giardia lamblia.Int J Parasitol33328
- 7. Konrad C, Spycher C, Hehl AB (2010) Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia. PLoS Pathog 6: e1000835.C. KonradC. SpycherAB Hehl2010Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia.PLoS Pathog6e1000835
- 8. Prucca CG, Slavin I, Quiroga R, Elías EV, Rivero FD, et al. (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754.CG PruccaI. SlavinR. QuirogaEV ElíasFD Rivero2008Antigenic variation in Giardia lamblia is regulated by RNA interference.Nature456750754
- 9. Jedelský PL, Doležal P, Rada P, Pyrih J, Smíd O, et al. (2011) The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis. PLoS One 6: e17285.PL JedelskýP. DoležalP. RadaJ. PyrihO. Smíd2011The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis.PLoS One6e17285
- 10. Rada P, Doležal P, Jedelský PL, Bursac D, Perry AJ, et al. (2011) The Core Components of Organelle Biogenesis and Membrane Transport in the Hydrogenosomes of Trichomonas vaginalis. PLoS ONE 6: e24428.P. RadaP. DoležalPL JedelskýD. BursacAJ Perry2011The Core Components of Organelle Biogenesis and Membrane Transport in the Hydrogenosomes of Trichomonas vaginalis.PLoS ONE6e24428
- 11. Simpson AGB (2003) Cytoskeletal organization, phylogenetic affinities and systematics in the contentious taxon Excavata (Eukaryota). Int J Syst Evol Microbiol 53: 1759–1777.AGB Simpson2003Cytoskeletal organization, phylogenetic affinities and systematics in the contentious taxon Excavata (Eukaryota).Int J Syst Evol Microbiol5317591777
- 12. Embley TM, Martin W (2006) Eukaryotic evolution, changes and challenges. Nature 440: 623–630.TM EmbleyW. Martin2006Eukaryotic evolution, changes and challenges.Nature440623630
- 13. Dolezal P, Dancis A, Lesuisse E, Sutak R, Hrdý I, et al. (2007) Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis. Eukaryot Cell 6: 1431–1438.P. DolezalA. DancisE. LesuisseR. SutakI. Hrdý2007Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis.Eukaryot Cell614311438
- 14. Mukherjee M, Brown MT, McArthur AG, Johnson PJ (2006) Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis. Eukaryot Cell 5: 2062–2071.M. MukherjeeMT BrownAG McArthurPJ Johnson2006Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis.Eukaryot Cell520622071
- 15. Schneider RE, Brown MT, Shiflett AM, Dyall SD, Hayes RD, et al. (2011) The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes. Int J Parasitol 41: 1421–1434.RE SchneiderMT BrownAM ShiflettSD DyallRD Hayes2011The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes.Int J Parasitol4114211434
- 16. Carpenter ML, Cande WZ (2009) Using morpholinos for gene knockdown in Giardia intestinalis. Eukaryot Cell 8: 916–919.ML CarpenterWZ Cande2009Using morpholinos for gene knockdown in Giardia intestinalis.Eukaryot Cell8916919
- 17. Davis-Hayman SR, Nash TE (2002) Genetic manipulation of Giardia lamblia. Mol Biochem Parasitol 122: 1–7.SR Davis-HaymanTE Nash2002Genetic manipulation of Giardia lamblia.Mol Biochem Parasitol12217
- 18. Gourguechon S, Cande WZ (2011) Rapid tagging and integration of genes in Giardia intestinalis. Eukaryot Cell 10: 142–145.S. GourguechonWZ Cande2011Rapid tagging and integration of genes in Giardia intestinalis.Eukaryot Cell10142145
- 19. Saraiya AA, Wang CC (2008) snoRNA, a novel precursor of microRNA in Giardia lamblia. PLoS Pathog 4: e1000224.AA SaraiyaCC Wang2008snoRNA, a novel precursor of microRNA in Giardia lamblia.PLoS Pathog4e1000224
- 20. Yu DC, Wang AL, Wu CH, Wang CC (1995) Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia. Mol Cell Biol 15: 4867–4872.DC YuAL WangCH WuCC Wang1995Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.Mol Cell Biol1548674872
- 21. Dawson SC, House SA (2010) Imaging and analysis of the microtubule cytoskeleton in giardia. Methods Cell Biol 97: 307–339.SC DawsonSA House2010Imaging and analysis of the microtubule cytoskeleton in giardia.Methods Cell Biol97307339
- 22. Regoes A, Hehl A (2005) SNAP-tag™ mediated live cell labeling as an alternative to GFP in anaerobic organisms. Biotechniques 39: 809–812.A. RegoesA. Hehl2005SNAP-tag™ mediated live cell labeling as an alternative to GFP in anaerobic organisms.Biotechniques39809812
- 23. Elias EV, Quiroga R, Gottig N, Nakanishi H, Nash TE, et al. (2008) Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia. J Biol Chem 283: 35996–36010.EV EliasR. QuirogaN. GottigH. NakanishiTE Nash2008Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia.J Biol Chem2833599636010
- 24. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90: 1103–1163.DM ChudakovMV MatzS. LukyanovKA Lukyanov2010Fluorescent proteins and their applications in imaging living cells and tissues.Physiol Rev9011031163
- 25. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67: 509–544.RY Tsien1998The green fluorescent protein.Annu Rev Biochem67509544
- 26. Lloyd D, Harris JC, Maroulis S, Biagini GA, Wadley RB, et al. (2000) The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity. Microbiology 146 Pt 12: 3109–3118.D. LloydJC HarrisS. MaroulisGA BiaginiRB Wadley2000The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity.Microbiology146 Pt 1231093118
- 27. Smutná T, Gonçalves VL, Saraiva LM, Tachezy J, Teixeira M, et al. (2009) Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase. Eukaryot Cell 8: 47–55.T. SmutnáVL GonçalvesLM SaraivaJ. TachezyM. Teixeira2009Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase.Eukaryot Cell84755
- 28. Stefanic S, Morf L, Kulangara C, Regös A, Sonda S, et al. (2009) Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote. J Cell Sci 122: 2846–2856.S. StefanicL. MorfC. KulangaraA. RegösS. Sonda2009Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote.J Cell Sci12228462856
- 29. House S a, Richter DJ, Pham JK, Dawson SC (2011) Giardia flagellar motility is not directly required to maintain attachment to surfaces. PLoS Pathog 7: e1002167.a. House SDJ RichterJK PhamSC Dawson2011Giardia flagellar motility is not directly required to maintain attachment to surfaces.PLoS Pathog7e1002167
- 30. Wombacher R, Cornish VW (2011) Chemical tags: Applications in live cell fluorescence imaging. J Biophotonics 4: 391–402.R. WombacherVW Cornish2011Chemical tags: Applications in live cell fluorescence imaging.J Biophotonics4391402
- 31. Gautier A, Juillerat A, Heinis C, Corrêa IR, Kindermann M, et al. (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15: 128–136.A. GautierA. JuilleratC. HeinisIR CorrêaM. Kindermann2008An engineered protein tag for multiprotein labeling in living cells.Chem Biol15128136
- 32. Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K (2004) Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101: 9955–9959.A. KepplerH. PickC. ArrivoliH. VogelK. Johnsson2004Labeling of fusion proteins with synthetic fluorophores in live cells.Proc Natl Acad Sci U S A10199559959
- 33. Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, et al. (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3: 373–382.GV LosLP EncellMG McDougallDD HartzellN. Karassina2008HaloTag: a novel protein labeling technology for cell imaging and protein analysis.ACS Chem Biol3373382
- 34. Lauwaet T, Davids BJ, Torres-Escobar A, Birkeland SR, Cipriano MJ, et al. (2007) Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol Biochem Parasitol 152: 80–89.T. LauwaetBJ DavidsA. Torres-EscobarSR BirkelandMJ Cipriano2007Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation.Mol Biochem Parasitol1528089
- 35. Yu DC, Wang AL, Wang CC (1996) Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia. Mol Biochem Parasitol 83: 81–91.DC YuAL WangCC Wang1996Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia.Mol Biochem Parasitol838191
- 36. Delgadillo MG, Liston DR, Niazi K, Johnson PJ (1997) Transient and selectable transformation of the parasitic protist Trichomonas vaginalis. Proc Natl Acad Sci U S A 94: 4716–4720.MG DelgadilloDR ListonK. NiaziPJ Johnson1997Transient and selectable transformation of the parasitic protist Trichomonas vaginalis.Proc Natl Acad Sci U S A9447164720
- 37. Dagley MJ, Dolezal P, Likic VA, Smid O, Purcell AW, et al. (2009) The protein import channel in the outer mitosomal membrane of Giardia intestinalis. Mol Biol Evol 26: 1941–1947.MJ DagleyP. DolezalVA LikicO. SmidAW Purcell2009The protein import channel in the outer mitosomal membrane of Giardia intestinalis.Mol Biol Evol2619411947
- 38. Dolezal P, Dagley MJ, Kono M, Wolynec P, Likić VA, et al. (2010) The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica. PLoS Pathog 6: e1000812.P. DolezalMJ DagleyM. KonoP. WolynecVA Likić2010The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica.PLoS Pathog6e1000812
- 39. Hrdy I, Hirt RP, Dolezal P, Bardonová L, Foster PG, et al. (2004) Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I. Nature 432: 618–622.I. HrdyRP HirtP. DolezalL. BardonováPG Foster2004Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I.Nature432618622
- 40. Shiflett AM, Johnson PJ (2010) Mitochondrion-related organelles in eukaryotic protists. Annu Rev Microbiol 64: 409–429.AM ShiflettPJ Johnson2010Mitochondrion-related organelles in eukaryotic protists.Annu Rev Microbiol64409429
- 41. Smith A, Johnson P (n.d.) Gene expression in the unicellular eukaryote Trichomonas vaginalis. Res Microbiol 162: 646–654.A. SmithP. Johnsonn.d.Gene expression in the unicellular eukaryote Trichomonas vaginalis.Res Microbiol162646654
- 42. Dolezal P, Smíd O, Rada P, Zubácová Z, Bursać D, et al. (2005) Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proc Natl Acad Sci U S A 102: 10924–10929.P. DolezalO. SmídP. RadaZ. ZubácováD. Bursać2005Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting.Proc Natl Acad Sci U S A1021092410929
- 43. Drmota T, Proost P, Van Ranst M, Weyda F, Kulda J, et al. (1996) Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis: purification and characterization. Mol Biochem Parasitol 83: 221–234.T. DrmotaP. ProostM. Van RanstF. WeydaJ. Kulda1996Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis: purification and characterization.Mol Biochem Parasitol83221234
- 44. Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, Chepurnykh TV, et al. (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24: 461–465.NG GurskayaVV VerkhushaAS ShcheglovDB StaroverovTV Chepurnykh2006Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.Nat Biotechnol24461465
- 45. Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, et al. (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5: 545–551.NC ShanerMZ LinMR McKeownPA SteinbachKL Hazelwood2008Improving the photostability of bright monomeric orange and red fluorescent proteins.Nat Methods5545551
- 46. Griffin BA, Adams SR, Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281: 269–272.BA GriffinSR AdamsRY Tsien1998Specific covalent labeling of recombinant protein molecules inside live cells.Science281269272
- 47. Yamaguchi K, Inoue S, Ohara O, Nagase T (2009) Pulse-chase experiment for the analysis of protein stability in cultured mammalian cells by covalent fluorescent labeling of fusion proteins. Methods Mol Biol 577: 121–131.K. YamaguchiS. InoueO. OharaT. Nagase2009Pulse-chase experiment for the analysis of protein stability in cultured mammalian cells by covalent fluorescent labeling of fusion proteins.Methods Mol Biol577121131
- 48. Urh M, Hartzell D, Mendez J, Klaubert DH, Wood K (2008) Methods for detection of protein-protein and protein-DNA interactions using HaloTag. Methods Mol Biol 421: 191–209.M. UrhD. HartzellJ. MendezDH KlaubertK. Wood2008Methods for detection of protein-protein and protein-DNA interactions using HaloTag.Methods Mol Biol421191209
- 49. Westermann B (2010) Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 11: 872–884.B. Westermann2010Mitochondrial fusion and fission in cell life and death.Nat Rev Mol Cell Biol11872884