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cytoplasmic DNA-containing vesicles. Using TEM and. DNA-specific stains, we found DNA-bearing vesicles organized in doughnut-like bodies in the nuclei, and.

Mol Genet Genomics (2002) 267: 622–628 DOI 10.1007/s00438-002-0693-2


F. Solı´ s Æ E. Orozco Æ L. Co´rdova Æ B. Rivera J.P. Luna-Arias Æ E. Go´mez-Conde Æ M.A. Rodrı´ guez

Entamoeba histolytica: DNA carrier vesicles in nuclei and kinetoplast-like organelles (EhkOs) Received: 21 December 2001 / Accepted: 8 May 2002 / Published online: 25 June 2002  Springer-Verlag 2002

Abstract Entamoeba histolytica, the protozoan responsible for human amoebiasis, has a complex genome, whose linear chromosomes and DNA circles have so far eluded detailed analysis. We report the detection by transmission electron microscopy of nuclear vesicles (0.05–0.3 lm in diameter) carrying DNA in E. histolytica trophozoites. In late anaphase many of these nuclear vesicles were found to be organized in structures of 2.5·1 lm, in association with chromosomes and microtubules. In glutaraldehyde-fixed and detergenttreated trophozoites, nuclear vesicles displayed a nonmembranous envelope. Binding of phosphotungstate stain and recognition by serum from patients with systemic lupus erythematosus indicated that these vesicles contain DNA. Similar DNA carrier vesicles were found in the cytoplasm and in the E. histolytica kinetoplast-like Communicated by W. Goebel F. Solı´ s Æ L. Co´rdova Faculty of Medicine, Autonomous University of Chihuahua, A.P. 1090, Chihuahua, Chih. 31000, Mexico E. Orozco (&) Æ M.A. Rodrı´ guez Department of Experimental Pathology, CINVESTAV-IPN., A.P. 14-740, Mexico, D.F. 07300, Mexico E-mail: [email protected] Tel.: +52-55-7473800 ext. 5642 Fax: +52-55-7477108 B. Rivera Faculty of Chemistry, Autonomous University of Chihuahua, Ciudad Universitaria, Chihuahua, Chih. 31000, Mexico J.P. Luna-Arias Department of Molecular Biomedicine, CINVESTAV-IPN., A.P. 14-740, Mexico, D.F. 07300, Mexico E. Go´mez-Conde Mexican Institute of Social Security, CIBIOR, Laboratory of Comparative Pathology, Puebla 7200, Mexico

organelle (EhkO). By Feulgen staining, we detected DNA carrier vesicles entering or leaving the nuclei, suggesting a structural relationship between the nuclear vesicles and the vesicles present in the EhkOs. Keywords Entamoeba histolytica Æ DNA-carrying vesicles Æ Nuclear division Æ Cytoplasmic DNA Æ Kinetoplast-like organelles

Introduction Entamoeba histolytica, the protozoan responsible for human amoebiasis, has a worldwide distribution. It infects fifty million people and causes 100,000 deaths anually (Walsh 1986). Our knowledge of the organization of its DNA is incomplete. It has linear and circular DNA molecules in the nuclei (Zurita et al. 1991; Dhar et al. 1995), but DNA circles have also been detected in the E. histolytica kinetoplast-like organelle (EhkO) (Baez-Camargo et al. 1996a, 1997). In EhkOs, the DNA seems to be organized in networks similar to those described in the kinetoplasts of Trypanosomatidae. EhkOs also have DNA-containing vesicles (Baez-Camargo et al. 1996a). EhkOs are mitochondrion-like organelles found in the cytoplasm of the trophozoites (Baez-Camargo et al. 1996a). They contain the enzyme pyruvate ferredoxin oxidoredutase (PFO; Rodrı´ guez et al. 1998) and may correspond to the mitosome (Tovar et al. 1999) and crypton (Mai at al. 1999) structures described recently. Interestingly, earlier reports had suggested the presence of DNA-containing vesicles in the nucleus (MarquezMonter et al. 1990; Solis and Barrios 1991; Solis and Adams 1997). The mode of organization of the nuclear and EhkO DNA has not been clarified, and their functional and structural relationships remain unknown. The chromosomes of E. histolytica show only weak condensation during mitosis. Studies using transmission electron microscopy (TEM) have revealed 12–16 chromatin bodies in dividing nuclei of colchicine-synchronized trophozoites (Orozco et al. 1988), but it could


not be determined whether these bodies corresponded to distinct molecules. Gomez-Conde et al. (1998), using fluorescence microscopy, unequivocally demonstrated the presence of six chromosomes in dividing trophozoites. Thus, six is the minimal number of bona fide chromosomes in E. histolytica. However, the possibility that other unidentified chromosomes exist can not be discounted. The molecular karyotypes of E. histolytica obtained by pulsed-field gel electrophoresis (PFGE) exhibited 8–16 bands (Valdes et al. 1990; Orozco et al. 1993; Petter et al. 1993; Willhoeft and Tannich 1999). Six bands between 170–1,600 kb hybridized with a Tetrahymena sp. telomeric probe (Baez-Camargo et al. 1996b). However, no telomeric sequences have been found in E. histolytica yet, and two or more chromosomes could be present in the broad PFGE bands. Using three distinct PFGE methodologies we have identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb, whose reorientation times and migration velocities under various experimental conditions, using Saccharomyces cerevisiae chromosomes as a control, confirmed their linearity and approximate molecular sizes (Riveron et al. 2000). Their exact lengths will be accurately known after sequencing of the E. histolytica genome [now in progress at The Institute for Genomic Research, Rockville, Md. (http://www.tigr.org)] has been completed. PFGE and hybridization with several gene probes has suggested the presence of 14 chromosomes in E. histolytica trophozoites (Willhoeft and Tannich 1999). However, this number may not be precise, because of the aberrant migration of many DNA molecules with different topology in PFGE assays. E. histolytica contains DNA circles between of 4 and 60 kb in length (Dhar et al. 1995; Lioutas et al. 1995), and complex DNA structures formed by concatenated circles (Orozco et al. 1997). There are about 200 copies per cell of the 24.5-kb ribosomal episome (rDNA) located in the so-called peripheral chromatin (Bhattacharya et al. 1989; Huber et al. 1989; Zurita et al. 1991), but rDNA sequences also exist in EhkOs (Orozco et al. 1997). Many rDNA concatamers are resolved by PFGE using pulse times longer than 120 s (Baez-Camargo et al. 1996b), and overlap with other DNA molecules, giving the broad bands obtained in PFGE assays. In the work reported here, we have studied the nuclear DNA-bearing vesicles in E. histolytica and analyzed the structural relationship between nuclear and cytoplasmic DNA-containing vesicles. Using TEM and DNA-specific stains, we found DNA-bearing vesicles organized in doughnut-like bodies in the nuclei, and forming the EhkOs in the cytoplasm.

Materials and methods E. histolytica cultures Trophozoites of clone A strain HM1:IMSS (Orozco et al. 1983) were axenically cultured in TYI-S-33 medium and harvested during the logarithmic growth phase (Diamond et al. 1978).

Transmission electron microscopy Trophozoites were fixed for 1 h at 37C with 2.5% glutaraldehyde and post-fixed with 1% osmium tetroxide (both prepared in 0.1 M cacodylate buffer). Cells were dehydrated using a graded series ethanol followed by propylene oxide, and embedded in Spurr’s resin. For phosphotungstic acid (PTA) staining (Esquivel et al. 1987), the trophozoites were rinsed three times, after glutaraldehyde fixation, in 0.2 N HCl (for 15 min each), and stained with 3% PTA in 0.2 N HCl (pH 2.3) for 1 h at room temperature (RT). Trophozoites were also simultaneously treated with glutaraldehyde and 0.1% Triton X-100. Thin sections were stained with aqueous uranyl acetate and lead citrate. Samples were processed and observed with a JEOL 1010 TEM. Immunolocation of DNA in trophozoites Trophozoites were fixed with 4% paraformaldehyde for 15 min at 37C and incubated for 20 min in 0.1% Triton X-100 in phosphatebuffered saline (PBS, pH 6.8). Then the cells were incubated with systemic lupus erythematosus (SLE)-positive serum, diluted 1:20 in PBS, for 1 h at RT. The trophozoites were then washed with PBS and incubated with Protein A coupled to horseradish peroxidase (Sigma) for 1 h at RT and then with 3,3¢-diaminobenzidine (Sigma) to visualize binding. Cells were observed with a light microscope. Negative controls were carried out using DNase-treated (40 U/ml, 37C, 1 h) cells, or by incubating the cells with normal human serum instead of the SLE serum, or omitting the SLE serum. Feulgen staining of trophozoites Trophozoites were fixed with 4% paraformaldehyde for 3 h at 37C, washed in water for 5 min and incubated in 0.5 N HCl for 1 h. Then the cells were stained with Feulgen stain (Cell Analysis Systems) for 1 h at RT, dehydrated in absolute ethanol, cleared in xylene, mounted with histological mounting media and observed through a phase-contrast microscope (Argu¨ello et al. 1992). The sizes of stained structures were measured from photographic prints.

Results Detection of nuclear vesicles in interphase and dividing trophozoites In the course of our previous studies on nuclear division in E. histolytica, we observed the presence of nuclear vesicles in trophozoites (Solis and Barrios 1991; Solis and Adams 1997), as previously described by others (Rosenbaum and Wittner 1970; Chevez et al. 1972; Feria-Velasco and Trevino 1972; Marquez-Monter et al. 1990). To study these vesicles we analyzed 150 thin sections of nuclei in interphase and 150 in mitosis. Thin sections of trophozoites stained with lead citrate and uranyl acetate revealed few nuclear vesicles, ranging from 0.05 to 0.3 lm in diameter, in interphase nuclei; these were located close to the peripheral material, previously described as consisting of the nucleolus and chromatin (Albach and Booden 1978) (Fig. 1a, arrowhead). Nuclei in prophase, distinguished by the clustering of the chromosomes in the center (Solis and Adams 1997), also showed few nuclear vesicles (Fig. 1b, arrowhead). Interestingly, in anaphase trophozoites, identified by the presence of elongated nuclei (Solis and Adams 1997), the number of nuclear vesicles was


Fig. 2a, b. Nuclear vesicles close to the mitotic spindle and to chromosomes. Dividing trophozoites were processed by TEM, and nuclear vesicles were found close to the microtubules of the mitotic spindle (a, arrow) and chromosomes (b, arrowheads). The arrow in b shows a vesicle bridging the gap between two chromosomes; the asterisk marks a nuclear vesicle that is made up of smaller vesicles

Fig. 1a–d. Nuclear vesicles in interphase and dividing trophozoites. The nuclei of interphase and dividing trophozoites were analyzed by TEM. a Interphase. b Prophase (the arrows indicate the chromosomes. c Anaphase. The arrowheads in panels a–c indicate nuclear vesicles. Panel d shows a higher magnification of the nut-shaped structure (boxed) in c. The arrows indicate nuclear vesicles

increased considerably and they appeared to be associated with one of the nuclear poles, forming structures of about 2.5·1 lm (Fig. 1c, arrowhead, d). In dividing nuclei, the nuclear vesicles were located close to the microtubules of the mitotic spindle (Fig. 2a, arrow) and proximate to condensed chromatin bodies, previously described as chromosomes (Orozco et al. 1988; Solis and Adams 1997) (Figs. 2b and 3a, arrowheads). In some cases, smaller vesicles formed bridges between nuclear vesicles of two chromatin bodies (Fig. 2b, arrow). Other nuclear vesicles appeared to be formed from smaller ones (Fig. 2b, asterisk), suggesting a vesicle fusion process. Ultrastructural characterization of the nuclear vesicles In general, the nuclear vesicles found in mitotic nuclei ranged between 0.05 to 0.3 lm in diameter, but smaller

Fig. 3a–d. Ultrastructure of nuclear vesicles. a Part of an anaphase nucleus with nuclear vesicles (arrow); the arrowhead indicates a chromosome. b Higher magnification of the boxed area in a. c Nuclear vesicles stained with PTA. d Nuclear vesicles in detergenttreated trophozoites: The arrowheads indicate electrodense bodies, the arrow points to the peripheral envelope, and the asterisks indicate the diffuse material inside the nuclear vesicles

and larger vesicles were also detected. Electrodense material was associated with the inner surface of the envelope of nuclear vesicles and was found embedded in more diffuse material within them (Fig. 3a–c). The periphery of the nuclear vesicles differed from the nuclear and plasma membranes. To investigate the nature of the envelope of nuclear vesicles, trophozoites were treated with glutaraldehyde and detergent to fix the cells and extract membranous material at the same time, avoiding damage to other structures. Using this technique, the cell membranes were dissolved, but the morphology of the remaining structures was altered minimally or not at all. In these assays, the nuclear vesicles appeared oval or


rounded, exhibiting a detergent-resistant envelope (Fig. 3d, arrow) with diffuse material inside (Fig. 3d, asterisks) and many smaller vesicles around the internal electrodense bodies (Fig. 3d, arrowheads). Not all nuclear vesicles displayed all the characteristics described here, suggesting that individual vesicles could be in different stages of formation, or that there is more than one type of vesicle in the nucleus. The nuclear vesicles contain DNA To investigate the contents of the nuclear vesicles, the trophozoites were treated with PTA, a DNA-specific stain (Esquivel et al. 1987), before they were processed for TEM. PTA stained both the nuclear chromatin (Fig. 4a, arrow) and the nuclear vesicles (Figs. 3c, 4a–c), showing that they contain DNA. PTA also stained doughnut-like bodies of 0.3–2 lm of diameter in many trophozoites. Vesicles could be distinguished within the doughnut-like bodies (Fig. 4a–c), the outer portions of which appeared compact, while the inner part appeared less dense (Fig. 4a–c). These vesicles have a similar morphology to those shown in Figs. 1d and 4c (arrows), suggesting that the doughnut-like bodies could represent transverse sections of these structures. DNA-containing vesicles are present in nuclei and in the cytoplasm Vesicles of similar morphology were detected both in the nucleus (Fig. 5a, arrows) and in the cytoplasm (Fig. 5b, arrows, inset) using PTA, demonstrating the presence of DNA in both. We also used antibodies against DNA, obtained from a patient with systemic lupus erythematosus (SLE), which showed a positive reaction with Fig. 4a–c. Nuclear vesicles forming doughnut-like bodies. Trophozoites were stained with PTA and processed by TEM. a Nucleus stained with PTA. The arrow indicates chromatin and the arrowhead a doughnut-like body. b Magnification of the doughnut-like body in a. The arrow shows a vesicle with a similar morphology to those shown in Fig. 1d. c Doughnut-like body stained by PTA. The arrows point to vesicles close to the doughnutlike body

nuclear chromatin (Fig. 5c, arrowhead) and with EhkOs composed of antibody-positive units (Fig. 5c, arrows), which might correspond to the vesicles carrying DNA. These results suggested that the DNA-bearing vesicles found in the cytoplasm might form part of EhkOs. We also found thin sections displaying vesicles with DNA in the nucleus, EhkOs and cytoplasm of the same trophozoite (Fig. 5d). These vesicles were all similar in morphology, and resembled the vesicles that form the structures shown in Figs. 1c and d and 4a–c. Differences between the EhkO envelope and the nuclear and cellular membranes can be observed in the trophozoite shown in Fig. 5d–g. Electrodense material, at least part of which may correspond to DNA, was transversally disposed in the EhkO (1.9 lm in diameter), whereas in the nucleus (5 lm in diameter) it appeared at the periphery or was condensed in the center (Fig. 5d, also see Fig. 1a and b). Whole trophozoites stained with the Feulgen stain, which is highly specific for DNA (Argu¨ello et al. 1992), revealed nuclei in which up to 20 pink, rounded structures between 0.2 and 2 lm in diameter could be observed (Fig. 6a–g), which were also present in the cytoplasm, forming the EhkOs (Fig. 6e–g). On the basis of their size, morphology and DNA content, these structures might correspond to the vesicles and doughnut-like bodies observed by TEM in the nuclei. In 300 trophozoites analyzed in three different experiments, we found DNA outside of the nuclei – forming one or more EhkOs per trophozoite, or entering or leaving the nucleus (Fig. 6b and f, arrows) – in 70±10% of the cells. However, EhkOs and nuclei frequently appeared in different planes in the cell. Observation of live trophozoites stained with acridine orange (Gomez-Conde et al. 1998) showed DNA leaving the nucleus instead of entering it (data not shown). Negative controls performed with DNase-treated trophozites did not show such structures (Fig. 6h).

Discussion The presence of nuclear vesicles in E. histolytica was demonstrated in earlier studies (Rosenbaum and Wittner 1970; Chevez et al. 1972; Feria-Velasco and Trevino 1972; Marquez-Monter et al. 1990). In these

626 b

Fig. 5a–g. Detection of DNA-containing vesicles carrying in nuclei, EhkOs and cytoplasm. a, b TEM of trophozoites showing nuclear vesicles (arrows in a) and cytoplasmic vesicles (arrows in b). The inset in b shows a cytoplasmic vesicle at higher magnification. c Trophozoites stained with anti-DNA antibodies and observed by light microscopy. The arrowhead indicates nuclear chromatin and the arrows EhkOs. d TEM of a single trophozoite with vesicles carrying DNA in EhkO (E), nuclei (n) and cytoplasm. Panels e–g show higher magnifications of the boxed areas in d. The arrows indicate DNA-bearing vesicles. PM, plasma membrane

reports, the authors did not associate the nuclear vesicles with the movement of DNA to the cytoplasm. Here, we characterized the nuclear vesicles and found them to be compacted, forming doughnut-like bodies. Similar vesicles, but even more compacted, were detected in the

EhkOs. Further experiments, such as in situ hybridization of DNA, or gene detection by FISH will help to determine the relationship among nuclear, cytoplasmic and EhkO vesicles. The interactions between the nuclear vesicles and the chromosomes have not been studied in detail. However, the presence of a larger number of DNAcarrying vesicles in dividing nuclei, and their proximity to chromosomes and to the microtubules of the mitotic spindle as seen by TEM, support the idea that the vesicles indeed contain nuclear DNA, as confirmed by staining with DNA-specific reagents (Figs. 4 and 6). In addition, the DNA-bearing vesicles may play a role in distributing the DNA between the daughter cells. The polar distribution of the DNA carrier vesicles seen in late anaphase nuclei suggests that these vesicles may be non-uniformly distributed between the nuclei formed in telophase. This uneven distribution of DNA content between the daughter cells could explain, at least in part, the variability of E. histolytica strains. The polar distribution of the DNA carrier vesicles seen in the late anaphase nuclei might, alternatively be attributed to the planes of section observed. However, we never detected doughnut-like bodies at both extremes of the nuclei. Based on these results, we favor the hypothesis that the vesicles play a role in the distribution of nuclear DNA, although further studies will be necessary to understand fully this novel process of DNA organization in E. histolytica. The different sizes of the vesicles and the presence of fibrils between them (Fig. 3) suggested that they may be fusing with each other, probably to assemble or disassemble the doughnut-like bodies. Intriguingly, fusion between vesicles would be facilitated by the presence of a membranous envelope, but we only detected a nonmembranous detergent-resistant coat, which may make vesicle fusion more difficult. The vesicle envelope resembles a bacterial cell wall more than a cell membrane. However, this does not preclude the existence of a typical membrane, which could not detected in the TEM experiments performed here. The vesicle envelope could protect the DNA during its transport from the nucleus to its final location, preventing its degradation by nucleases. DNA carrier vesicles could correspond to the previously described filamentous (0.01lm) or polyhedral (0.07–0.085 lm) E. histolytica viruses (Diamond et al. 1972), but vesicles (0.05–0.3 lm) are rounded or oval with the DNA in the periphery, whereas all known


nut-shaped structures in transverse sections (Fig. 1c and d), and the EhkOs in the cytoplasm. Acknowledgements The authors are deeply grateful for the excellent technical assistance of Mr. Alfredo Padilla-Barberi during the photographic work. The authors also thank to the Unit of Electron Microscopy at CINVESTAV-IPN. This work was also supported by a Howard Hughes Medical Institute Grant awarded to Dr. E. Orozco, and by CONACyT (Mexico).


Fig. 6a–h. Feulgen staining of trophozoites. a–g Feulgen-stained trophozoites, observed with a phase-contrast microscope. The arrows indicate DNA leaving the nuclei. h Trophozoite treated with DNase before Feulgen staining. n, nucleus; E, EhkOs. The scale bar for all panels is shown in g

viruses have their nucleic acids in the center of the virus particle. The experiments shown here do not rule out the possibility that DNA carrier vesicles could be virus-like particles. However, the presence of hydrogenosome-like proteins in the EhkOs (Rodriguez et al. 1998) makes it unlikely that they are of viral origin. In conclusion, we present in this paper experimental evidence for the existence of similar nuclear and cytoplasmic vesicles carrying DNA and forming doughnut-like structures in the nucleus, which appear as

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