The median body of Giardia lamblia: an ultrastructural study

The median body of Giardia lamblia: an ultrastructural study

Bruno Piva, Marlene Benchimol *

Universidade Santa Úrsula, Laboratório de Ultraestrutura Celular, Rio de Janeiro, Brazil

Received 1 March 2004; accepted 28 May 2004

Available online 05 October 2004

Abstract

Giardia lamblia is an intestinal parasite of several mammals. The most striking feature of Giardia is the presence of a complex and uniquecytoskeleton, and among its components the median body (MB) is the least defined microtubular structure. In the present study, we used atechnique that allowed the removal of the plasma membrane and observation of cytoskeletal structures by both routine scanning electronmicroscopy (SEM) and field emission high resolution SEM. This technique permitted new observations such as details and insights of themedian bodies, not previously described or controversial in the literature. Light microscopy after Panotic staining, immunofluorescencemicroscopy using several antibodies, and thin sections were also used to better characterized the Giardia MB. The new observationsconcerning the median bodies were : (1) they are not one or two structures, but varied in number, shape and position ; (2) they were found inmitotic and interphasic trophozoites, in disagreement with previous works ; (3) they were present in about 80 % of the cells, and not in 50 %of the cells, as previously described ; (4) they could be connected either to the plasma membrane, to the adhesive disc, and caudal flagella, andthus they are not completely free in the cells, as published before ; (5) they can protrude the cell surface ; (6) their microtubules react withseveral anti-tubulin and -beta giardin antibodies. These observations add new data on the scarce literature and to this largely understudied cellstructure.© 2004 Elsevier SAS. All rights reserved.

Keywords: Giardia lamblia; Median body; Scanning electron microscopy; Field emission scanning electron microscopy; Cytoskeleton

1. Introduction

The diplomonad Giardia lamblia is a parasitic protozoancell that infects thousands of people all over the world,causing a disease known as giardiasis. The trophozoite formof this protist lacks organelles found in higher eukaryotes,such as mitochondria and peroxisomes (Gillin et al., 1996).Even structures such as the Golgi complex are absent (orcontroversial) in trophozoites (Lanfredi-Rangel et al., 1999;Lujan et al., 1995; Marti and Hehl, 2003; Reiner et al., 1990).The Giardia cell possesses cytoskeletal structures composedof microtubules (Brugerolle, 1991; Kulda and Nohýnková,1995). In the interphase, these include the basal bodies andaxonemes of the eight flagella, microtubules accompanyingthe caudal axonemes—the funis—made up of sheets of mi-

crotubules following the axonemes of the caudal flagella,(Erlandsen and Feely, 1984; Kulda and Nohýnková, 1995),the median body (MB), formed by an irregular set of micro-tubules, and the ventral adhesive disc built on a helicoidallyturned layer of parallel microtubules. Although most of theultrastructural studies have focused on the cytoskeleton andadhesive disc of Giardia, up to now, sparse data are availablein the literature on the MB structure and behavior. Its func-tion is also unknown, although some workers have proposedit as a store of prepolymerized tubulin for the disc assembly(Feely et al., 1990) or a novel microtubule-organizing center(MTOC) in Giardia (Meng et al., 1996). The MB appears inall species and the exact shape and position vary among thespecies and can be used as a taxonomic tool, since it differsslightly in position and shape in each of the three Giardiaspecies as defined by Filice (1952). The MB has been de-scribed in G. lamblia as one or two roughly aligned fasciclesof microtubules situated transversely to the axonemes (Fil-ice, 1952; Kulda and Nohýnková, 1995).

Previous studies reported that the is about 2 µm in diam-eter, has a crescent shape when observed in whole cells and is

* Corresponding author. Universidade Santa Úrsula, Laboratório deUltraestrutura, Rua Jornalista Orlando Dantas, 59, Botafogo, Rio deJaneiro,RJ CEP 222-31-010, Brazil. Tél./Fax : +55 21 553 1615.

E-mail address: [email protected] (M. Benchimol).

Biology of the Cell 96 (2004) 735–746

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generally ovoid when seen in sections (Friend, 1966). Thisauthor also stated that the MB does not have origin or inser-tion into any other structure, and that the MB microtubulesare randomly arranged.

In the present work, we show for the first time images ofthe MB by scanning electron microscopy (SEM) and fieldemission scanning electron microscopy (FESEM) addingnew data on the knowledge of this cytoskeletal structure.

2. Results

2.1. Light microscopy

Giardia stained with Panotic kit and analyzed by brightfield light microscopy exhibited intense staining of the nu-clei, flagella, and MB (Fig. 1). This procedure allowed thestudy of MB number, size, and shape. This stain kit is consti-tuted by two stains and one fixative. One of the stains hasaffinity for the nuclei, which stains in deep blue, and the otherstain has affinity for the cytosol, which stains pink. Oneproblem concerning the use of this kit is that some cells arenot well stained, and thus some information is eventuallylost. The MB was situated perpendicular to Giardia centralaxis, and sometimes could present a small deflection(Fig. 1b–f). Cells that apparently did not have an, in factdisplayed either a small MB (Fig. 1a) or it was superimposedto the caudal axonemes, and thus difficult to be recognized.The MB presented a variable position (Fig. 1) and thickness(Fig. 2). When in dorsal views, MB bundles could be seenrunning between the caudal axonemes and the right nucleus(Fig. 1b).

2.2. Median body size

In order to estimate the MB size, we analyzed 250 cellsstained by Panotic kit and made measurements using photo-graphic material or computer acquired images. The MB size

Fig. 1. Gallery of G. lamblia images after Panotic staining observed in bright field light microscopy. The diversity of MB size, shape and locations can be noted(arrows). (a) shows a small MB whereas (d,e) display MB protruding at the plasma membrane. (1b) displays a MB with nucleus proximity. (1c) shows a cellpresenting three median bodies. Bars = 5 µm.

Fig. 2. Morphometry of median bodies performed in 250 cells stained byPanotic kit. The measurements were made using photographic material orcomputer acquired images, in order to compare the cells’ and median bodieswidth and length, respectively. The MB size varied between 0.2 µm and1.8 µm (average of 0.84 µm) in width and 0.8 µm to 8.0 µm (average of3.34 µm) in length. About 60 % of the cell width is occupied by the MB,whereas only 8.4 % of cell length is occupied by it.

736 B. Piva, M. Benchimol / Biology of the Cell 96 (2004) 735–746

was extremely variable among the cells (Fig. 1). Some ofthem displayed a large size, sometimes reaching the plasmamembrane boundaries (Fig. 1d,e). It could form a protuber-ance in the cells’periphery (Fig. 1e), and in this case, the cellswere generally oversized. On the other hand, other cells haveso small median bodies that at first glance this structure

seemed absent (Fig. 1a). We observed that small medianbodies were generally located superimposed to the caudalaxonemes. The MB size varied between 0.2 µm and 1.8 µm(average of 0.84 µm) in thickness and 0.8 µm to 8.0 µm(average of 3.34 µm) in length (Fig. 2). About 60 % of the cellwidth is occupied by the MB, whereas only 8.4 % of celllength is occupied by it (Fig. 2).

The percentage of organisms in mid-logarithmic growthphase with visible median bodies was determined. Mor-phometry performed in Panotic stained cells showed that in1820 cells, 1448 exhibited the median bodies, which repre-sented almost 80 % of the cells’ population (Table 1). Divid-ing cells also displayed median bodies (Fig. 3). In 1470 mi-totic cells, 1257 presented median bodies (Table 1). They

Table 1Morphometry performed in Panotic stained cells showing that in 1820 cells,1448 exhibited the median bodies, which represented almost 80 % of theinterphasic cells’ population. In 1470 mitotic cells, 1257 presented medianbodies. It corresponds to 85.5 % of MB presence in dividing cells

Cell number Cells presenting MB %Interphasic cells 1820 1448 79.5 %Dividing cells 1470 1257 85.5 %

Fig. 3. Localization of tubulin in Giardia by immunofluorescence microscopy after staining with monoclonal #357 antibody with the purpose to label the MB.Cells are seen from interphase (row A), to different progressive phases of mitosis (B–D) in immunofluorescence and DIC labeling was observed in the medianbodies (arrowheads), axonemes, flagella and disc, in all phases of the cell division. Note the mirror-symmetry of the median bodies in the late mitosis phase(Fig. 3 heures–k). Bar = 5 µm.

737B. Piva, M. Benchimol / Biology of the Cell 96 (2004) 735–746

could be observed in both daughter cells (Fig. 3), or in onlyone. It corresponds to 85.5 % of MB presence in dividingcells (Table 1). When both daughter cells exhibited medianbodies, they were in a mirror-symmetry (Fig. 3).

2.3. Immunofluorescence microscopy

Monoclonal anti-tubulin and -giardin antibodies wereused (Table 2). The monoclonal antibody TAT–1 which rec-ognizes a-tubulin allowed an intense labeling in medianbodies and a less intense staining in the axonemes, flagella,and adhesive disc (not shown). The #357 monoclonal anti-tubulin antibody was used with the purpose to label the MB,as previously demonstrated (Campanati et al., 2003). Label-ing was observed in the median bodies, axonemes, flagellaand disc, in all phases of the cell division (Fig. 3). The#GT335 antibody which recognizes glutamylated tubulinwas also used, and although labeling in the axonemes wasobserved, it did not decorate either the disc or the medianbodies (not shown). The median bodies were also negativefor c-tubulin, whereas two stained dots observed in the kine-tosomal complex were seen in non dividing cells (notshown). When either 7G9 or 2G3 anti-b-giardin monoclonalantibodies were used, some cells displayed positive labelingin the MB, whereas all cells exhibited labeling of the adhe-sive disc (Fig. 4).

2.4. The median body by routine SEM and TEM

The morphology of Giardia when viewed by routine SEMis like a pear half (Fig. 5a). The ventral disc is seen in the

anterior ventral half of the cell and is surrounded laterally andanteriorly by the marginal groove and the ventro-lateralflange. It also presents four pairs of flagella, namely theanterior, posterior-lateral, caudal, and ventral (Fig. 5a).

When the MB was analyzed by transmission electronmicroscopy, it was seen as a microtubular structure where themicrotubules run perpendicular to the main cell axis(Fig. 5b–g). Several microtubules were also positioned andtilted to the caudal axonemes (Fig. 5b,c). Some cells exhib-ited microtubules in apparent interaction with the plasmamembrane or at least in close proximity with it (Fig. 5e,f).Microribbons were occasionally observed among the MBmicrotubules (Fig. 5e). In some cells, which the disc disas-sembly was seen, continuity between the disc microtubulesand MB microtubules was observed (Fig. 5e).

2.5. The median body by SEM and FESEM after detergentextraction

Here, we have used a procedure to examine by SEM thecytoskeletal structures underlying the plasma membrane af-ter membrane extraction by detergents (Fig. 6). Ultrastruc-tural examination of G. lamblia by conventional SEM or/andby FESEM allowed us a new visualization of the all micro-tubular cytoskeleton, the MB included (Figs. 6 and 7). Withthis procedure, new data were added to the MB structure, andas it is situated dorsally to the caudal axonemes, it was betterobserved by SEM when the cells were adhered by the ventralsurface (Fig. 6).

One interesting observation was that when the plasmamembrane was completely removed the median bodies werenot visualized. Only when plasma membrane remnants wereleft, the MB appeared. It let us think in a linkage between theMB and the plasma membrane. Thus, different times anddetergent concentrations were tried until the adequate MBobservation was achieved. We concluded that for a goodobservation of the MB by SEM the cell must be in a dorsalview (disc down) and still with plasma membrane remnants(Fig. 6). In addition, if the cells were spin down after deter-gent treatment, the median bodies were never observed. Theywere completely removed during the centrifugation proce-

Table 2Description of the Antibodies used in G. lamblia

Antibody Specificity Source Monoclonal/polyclonal

Immunogen

#357 b-Tubulin Amershan/England Monoclonal Chicken embryo brain tubulinKMX b-Tubulin Provided by Dr. K. Gull Monoclonal P. polycephalum amoebal tubulin

(Woods et al., 1989)TAT-1 a-Tubulin Provided by Dr. K. Gull Monoclonal Cytoskeleton extraction of T. brucei

(Woods et al., 1989)#GT335 Glutamylated tubulin terminal

C epitopeProvided by Dr. Wolff Monoclonal Brain mouse tubulin(Wolf et al., 1992)

T 6793 (clone no. 6-11B-1) Acetylated tubulin Sigma/St. Louis, États-Unis Monoclonal Sea urchin sperm axonemes7G9 and 2G3 Anti-giardin Provided by Dr. S. Svard Monoclonal Giardia

(Palm et al., unpublished)

Fig. 4. Immunofluorescence microscopy localization of b-giardin in Giar-dia, using the monoclonal antibody 7G9. The disc presents positive labelingas well the MB (arrowheads). a, DIC visualization ; b, immunofluorescence ;c, overlay. Bar = 5 µm.


dure. Thus, the better results were obtained when living cellswere first adhered to coated coverslips, detergent treated, andthen fixed.

The FESEM revealed that the MB was constituted byseveral small fascicles formed by microtubules forminglarger bundles (Fig. 7). The bundle number was variable aswell the microtubules number found in each fascicle. Thisalso varied in different organisms. Each fascicle was formed

by parallel microtubules with different lengths, but the dif-ferent fascicles were not parallel (Fig. 7a,b). Some fasciclesappeared to be tightly connected, leading a misunderstandingby light microscopy of the presence of only one or twomedian bodies (Fig. 6e,f). In addition, all bundles could alsobe observed together, seeming to be formed by a singlethicker bundle when visualized by light microscopy(Fig. 6e).

Fig. 5. Routine preparation for SEM (5a) and transmission electron microscopy (b–g) of G. lamblia trophozoites showing the anterior (A), posterior-lateral (P),ventral (V) and caudal (C) flagella. Note that in this routine SEM preparation no internal structures are observed. Giardia in longitudinal section seen by TEM(5b) shows the nuclei (N), MB and peripheral vesicles (V). (5c–g) displays different aspects and localization of the MBs. It can protrude at the plasma membrane(5e,f). In Fig. 5d, the MB is situated close to the disc, and continuity between MB and disc microtubules is observed (arrowhead in inset).Bars = 1 µm.


Some MB microtubules have their ends towards to thecitosol (Fig. 6c), whereas others were directed towards thecells’ anterior region (Fig. 7c). Connections with the ventral

disc (Fig. 7a–c) and funis microtubules were also observed(Fig. 7a–d). A higher concentration of microtubules fascicleswas observed in the caudal axonemes region.

Fig. 6. The SEM of several Giardia in dorsal views after detergent treatment. The plasma membrane was partially removed allowing the observation of themedian bodies (arrows). In this gallery of images is possible to note the different sizes, disposition, and a variable number of fascicles of the median bodies,which are not possible to observe in routine preparations as in Fig. 5a. The MB (arrows) can be seen protruding on the plasma membrane (6a), or in closeproximity with one of the nuclei (6d). Also, two or more fascicles are clearly seen in all figures. Note in Fig. 6e that the MB occupies the whole cell width. N,nucleus. Bars = 1 µm.


3. Discussion

The MB of Giardia is perhaps the least defined microtu-bular structure, and its function is largely unknown. It hasbeen used as criterion of speciation (Filice, 1952), and it wassuggested that its main function would be as a store ofprepolymerized tubulin thus providing templates for forma-tion of the disc fibers required for fast assembly of daughteradhesive discs during cytokinesis (Kulda and Nohýnková,

1995). However, this was only a speculation, since no datawere presented for this hypothesis.

3.1. Median body size, number, and shape

In the literature, the MB has been described as one or twocytoskeletal structure (Filice, 1952) present only in ca. 50 %of the cells (Bertram et al., 1984). In addition, these authorsalso found isolates in which the MB was absent. In the

Fig. 7. The FESEM of Giardia after detergent extraction. The plasma membrane was partially removed, allowing observation of the cytoskeleton, the MBincluded. The ventral disc (D), the two nuclei (N), MB, anterior (A), caudal (C) and posterior-lateral flagella (P) are seen. In Fig. 7(a,b), every fascicle thatconstitutes the MB is observed. In Fig. 7(c,d), the median bodies are seen curved, and towards the cells’ anterior region. The fascicles number is variable as wellas their disposition and location. Posterior-lateral axoneme, P ; caudal axoneme, C. Bars = 1 µm.


present study, we took advantage of the Panotic staining andshowed that at least 80 % of the cells displayed medianbodies. Our results are contradictory with the literature andthe possible cause may be the methodology used. In fact,some cells presented median bodies difficult to visualize,since (1) they can be in a vertical position and could behidden by the axonemes of the caudal flagella, when ob-served by light microscopy, or (2) they are so small that werenot easily seen. The absence of MB in some cells led to theassumption that these structures are not permanent but rathertemporary storage of tubulin. It is possible that Giardia hasdifferent MB sub-populations, or the size of these structuresdid not allow its visualization by routine methods. On theother hand, the absence of the MB in 20 % of the cellsobserved in the present work could be due by problems in thePanotic staining. It is possible that this proportion could behigher if a better staining method is found. Another possibil-ity is that the lack of MB could be related with genetic errorsin some cells or the existence of a sub-population, whichdoes not present a typical MB.

Using high resolution SEM in detergent treated cells, theMB was clearly visible. The MB was seen as a structureformed by a variable number of fascicles, which were formedby different microtubules number. The fascicles were gath-ered and formed a tight bundle. Our observations by SEMand FESEM demonstrated that there is not one or two medianbodies as published in the literature, but several fascicles, anda variable number between one and six fascicles were foundin the present study. It is important to point out that themedian bodies were seen by the first time using SEM in thepresent paper. The SEM herein used can achieve a resolutionof 3.5 nm at 30 kV, whereas the FESEM shows a resolution of1.2 nm at 15 kV.

Several previous reports have used whole mounts of cy-toskeletons for high resolution electron microscopy (Bell,1995; Bell et al., 1988; Braet et al., 1996; Chen et al., 1995;Lindroth et al., 1988; Ris, 1985). There are several problemsassociated with the preparation of whole mounts, in whichdetergent extraction has been made, and the researcher mustbe aware for effects of critical point drying, and type offixatives used on the morphology of Triton-resistant cytosk-eletons when observed by SEM and TEM (Bell et al., 1988;Lindroth et al., 1988). In the last years, a number of studiesusing these techniques called the attention to the possibledistortions that can arise during CPD procedure and how toavoid them (Chen et al., 1995; Ris, 1985). In order to increaseSEM signal yield, enhance contrast and reduce chargingproblems, coating of the specimens with a metal layer isnecessary (Pawley, 1990). Other important factors in highresolution SEM are contamination, beam damage, and me-chanical stability (Chen et al., 1995). The effects of CPD andfreeze-drying on the morphology of microtubules by scan-ning and transmission electron microscopy were presentedby some authors (Lindroth et al., 1988), showing that cytosk-eletons attached to Formvar films suffer less structural dam-age than cells attached to glass, because the Formvar film

absorbs some of the stress associated with shrinkage duringdrying. In addition, if an effort is made to exclude water andethanol from CO2, microtubules can be well preserved (Ris,1985). We have tried to overcome these problems, and we areaware of the possible artefacts inherent to the method hereinused. Thus, we present our results of imaging the structure ofthe cytoskeletal elements such as the MB of Giardia, show-ing views not presented before, and adding new informationto this structure.

Campanati et al. (2003) noticed a heterogeneous labelingof MB when antibodies anti-tubulin were used. They demon-strated using fluorescence microscopy that the central regiondisplayed less (or absence) of reactivity than the rest of thestructure. In our opinion, this observation was due to a spaceamong the fascicles, which of course did not react withantibodies. The advantage of using high resolution SEM wasto discern each fascicle, which has not been done until thepresent study, and show that the MB number is variable.

Friend (1966) published that the MB measured 2 µmdiameter, and Cheissin (1964) that it was 5 µm long. Ourmeasurements showed that it varied between 0.2 µm and1.8 µm (average of 0.84 µm) in thickness and 0.8 µm to8.0 µm (average of 3.34 µm) in length. There is a large MBsize variation that could be explained by different degrees ofpolymerization of microtubules. Also, the strain used in thepresent study (WB) could be different from those used be-fore.

Campanati et al. (2003) using antibodies anti-tubulin withdifferent specificities suggested that a mixed population ofmicrotubules, both stable and unstable, could form the me-dian bodies.

3.2. The median body in mitotic cells

The MB was visible by us throughout cell cycle phases. Itwas located dorsally in the cytoplasm behind the disc. Solov-iev (1963) reported the absence of the median bodies in thelate phases of division and in young daughter individuals,whereas Filice (1952) claimed for the absence of the MB infreshly excysted trophozoites. Cerva and Nohýnková (1992)reported that the median bodies disappear in a early stage ofGiardia mitosis, before the karyokinesis. In the presentstudy, we found trophozoites with MB in all phases of the cellcycle, in early and late phases of the mitosis, or during thecytokinesis. These data are contradictory with those of theliterature (Cerva and Nohýnková, 1992; Soloviev, 1963). Inrelation to Filices’ observations, we have studied only tro-phozoites and studies are in course in order to determine ifexcysting cells do not present median bodies, as suggested bythis author. Marshall and Holberton (1993) described a newcoiled coil protein as a specific marker for the MB. Thisstructure was described as a three times larger than a giardinsubunit, and these authors have suggested a possible role inimmobilizing the microtubules between cell divisions.


3.3. The median body and its association with other cellstructures

We observed that the MB displayed different positions :they can be seen transversally located, as a comma, or invariable degrees of tilting, even in a vertical position, super-imposed to the axonemes of the caudal flagella. In someoccasions, one of its extremities is seen up-righted towardsthe disc and could even contact the microtubules of theadhesive disc. These observations are new and were madepossible due to the new technique herein used.

A previous report discussed the MB attachment (Crossleyet al., 1986). It was published that the MB is loosely attachedto the funis and caudal axonemes, transversely oriented inone or more compact bundles. In the present study, weverified that the MB is not only linked to the structuresreported by these authors, but also to the plasma membraneand also occasionally to the disc. The role for the associationof the MB to these structures is not known, but we speculatethat the microtubules that constitute the MB may have itsorigin from the funis or disc microtubules. They could beoriginated from the microtubules of the caudal flagella,which fan out towards the posterior-lateral flagella, as dem-onstrated recently by us (Benchimol et al., 2004). We pro-pose that some microtubules in their way towards theposterior-lateral flagella do not anchor properly to the denserods of the axonemes of the posterior-lateral flagella and thuskeep growing during polymerization phase. Some of thesemicrotubules reach the plasma membrane and are anchoredto it by a proteinaceous underlying material as previouslydemonstrated (Benchimol et al., 2004). Thus, it would ex-plain why the MB disappears when the plasma membrane iscompletely extracted by detergent treatment. We observedthat when cytoskeletons are isolated by suspending cells inTriton without first attaching them to a surface such as acoverslip, the median bodies are always removed and nor-mally left into the supernatant. Similar observation has beenmade by Crossley et al. (1986). This fact corroborate with ourhypothesis that the MB is associated with the plasma mem-brane, and/or is connected to other cells’ structures by fragilelinks that are disrupted during detergent treatment.

Previous studies related the MB with the disc progenesisbecause both structures were labeled with antibodies tob-giardin (Crossley et al., 1986). Giardin is a predominantprotein found in the microribbons present in the dorsal faceof the adhesive disc. In addition, Brugerolle (1975) demon-strated the presence of small appendages on the MB micro-tubules similar to the dorsal ribbons of the disc, and disc.(Holberton et al. 1981) observed in tubulin nucleation experi-ments an extensive microtubule growth from the MB. Wehave also observed positive reaction in some median bodieswhen two anti-b-giardin monoclonal antibodies were used,confirming the possible presence of microribbons among themicrotubules of the MB. However, the MB labeling was notfound in all cells, whereas the disc was always stained.Cross-sections of the MB exhibited associated structures

similar to disc microribbons, in agreement with (Brugerolle1975) observations. We speculate that during the formationof new disc in the course of cell division, the bridges thatmaintain the disc microtubules linked are disrupted and somemicrotubules are directed towards the cytosol. These micro-tubules could originate the MB. This hypothesis would ex-plain the location of b-giardin on MB observed by us in thepresent paper, and others (Crossley et al., 1986) and alsowould explain why several median bodies present links withthe adhesive disc.

In some TEM images, we observed an apparent discdisassembly was observed, with disc fragments in continuitywith microtubules of the median bodies, suggesting thatthese structures may be related. In addition, we have recentlydemonstrated the presence of a new set of vesicles in closeproximity with the MB (Benchimol, 2002). These associa-tions suggest that a functional relationship may exist betweencontractile proteins and the MB.

Other components have been described in the medianbodies besides microtubules. Feely et al. (1982) has reportedby immunofluorescence microscopy the presence of actinand a-actinin in the MB of trophozoites, suggesting a func-tional relationship between these proteins. The immunocy-tochemical localization of centrin was also reported in theMB (Belhadri, 1995; Corrêa et al., 2004; Meng et al., 1996).Meng et al. (1996) proposed that since the anti-centrin anti-bodies reacted weakly with mature adhesive disc, centrincould be incorporated into the disc via the MB. In addition,the finding of centrin staining in the MB led to a suggestionof a novel MTOC in Giardia (Elmendorf et al., 2003). How-ever, c-tubulin, a protein found in MTOCs, was not found inthe median bodies by us (present work) and others (Nohýnk-ová et al., 2000).

3.4. Immunofluorescence microscopy

Previous studies showed the diversity of tubulin in G. lam-blia by immunofluorescence microscopy (Campanati et al.,1999, 2003; Soltys and Gupta, 1994). Interestingly, the me-dian bodies were labeled by several different anti-tubulinantibodies, except by using the GT335 antibody to detectglutamylated tubulin (present work, Boggild et al., 2002),and c-tubulin (Nohýnková et al., 2000). The MB presentsacetylated tubulin (Campanati et al., 2003; Soltys and Gupta,1994), mono and polyglycylated tubulin (Campanati et al.,1999), and tyrosinated tubulin (Campanati et al., 2003). Ourresults are in agreement with these authors. In addition, wealso found labeling using anti-b-giardin. Giardins are a groupof 29–38 kDa proteins present in the microribbons, which arepart of the ventral disc (Crossley and Holberton, 1985).

3.5. Possible roles for the median bodies

No specific function has been ascribed to the MB, al-though it has been speculated to serve : (1) as microtubulereserve of the cell ; (2) disc progenesis ; (3) immobilization


of microtubules between cell divisions ; (4) microtubulenucleation site (Holberton et al., 1981). Here, we add apossible new role : participation in vertical tail flexion. Webased this proposal in the following aspects : (a) presence inthe MB of contractile proteins and calcium-binding proteins,such as actin, a-actinin (Feely et al., 1982) and centrin(Belhadri, 1995; Corrêa et al., 2004; Meng et al., 1996) ; (b)interactions with other microtubular structures, such as disc,axonemes of the caudal flagella, and funis ; (c) plasmamembrane connections ; (d) location on the middle-posteriorregion of the cell body, where the Giardia tail flexion occurs.Observation of the MB in living cells during tail movementwould allow to confirm or discard this hypothesis. However,the other hypothesis as a microtubule reserve for the cell isalso possible and still needs future studies.

4. Materials and methods

4.1. Organisms and culture

G. lamblia strain WB (American Type Culture Collection,N0. 30957) was cultivated in TYI-S-33 medium enrichedwith 10 % heat-inactivated fetal bovine serum (Diamond etal., 1978) at pH 7.05, without added vitamins, iron, or anti-biotics (Gillin et al., 1989), but supplemented with 0.1 %bovine bile (Keister, 1983) for 48–72 heures, at 37 °C.

4.2. Panotic staining

Living cells were allowed to adhere to poly-L-lisinecoated coverslips. Cell fixation and staining were performedusing the Panotic staining kit (Laborclin, PR, Brazil), follow-ing manufacturer’s instructions. The images obtained wereprocessed in the Adobe PhotoShop (États-Unis).

4.3. Immunofluorescence

Cells were allowed to adhere to coverslips previouslycoated with poly-L-lysine. They were fixed at room tempera-ture in 4 % paraformaldehyde (v/v) in 0.1 M cacodylatebuffer (pH 7.2) or PHEM buffer (50 mM MgCl2, 70 mMKCl, 10 mM EGTA, 20 mM Hepes, 60 mM Pipes, pH 6.8) orMTSB (microtubule stabilizer buffer, 4 M glycerol, 100 mMPipes, pH 6.8, 1 mM EGTA, 5 mM MgCl2) (Gordon et al.,2001). Thereafter, the cells were permeabilized with 2 %Nonidet (NP-40) (Sigma), for 40 minutes Alternatively, thecells were fixed and permeabilized with methanol/acetone at–20 °C for 10 minutes The cells were quenched using 50 mMammonium chloride solution and 3 % (w/v) bovine serumalbumin (BSA) in PBS for the next 30 minutes Severalmonoclonal antibodies were used (see Table 1). The cover-slips were covered with each antibody and incubated in ahumidified chamber for 1–3 heures at room temperature.After thorough washing in PBS, the samples were incubatedfor 1 heure in the dark, with TRITC or Alexa-conjugated

anti-mouse antibody (Sigma or Molecular Probes, respec-tively, États-Unis) or Alexa anti-IgM antibody (MolecularProbes, États-Unis) diluted 1:100 in 1 % BSA in PBS. Insome experiments, the nuclei were stained with 10 µg/mlDAPI (4’-6-diamidino-2-2-phenylindole, MolecularProbes). Cells were examined with ana Zeiss Axiophot 2 mi-croscope (Zeiss). Images were acquired and processed usinga chilled C5985-10 CCD camera (Hamamatsu, Japan).

4.4. Primary antibodies and conjugates

Several anti-tubulin and anti-giardin antibodies were used(see Table 2).

4.5. Transmission electron microscopy

Cells were fixed overnight at room temperature in 2.5 %(v/v) glutaraldehyde (Sigma, États-Unis) in 0,1 M cacodylate(Sigma, États-Unis) buffer (pH 7.2). After that, the cells werewashed twice in phosphate buffer saline (PBS). Post-fixationwas performed in 1 % OsO4 (Sigma) in cacodylate buffercontaining 5 mM CaCl2 and 0.8 % potassium ferricyanide inthe dark, for 30 minutes Cells were washed in PBS, dehy-drated in acetone and embedded in Epon. Ultrathin sectionswere stained with uranyl acetate for 20 minutes and leadcitrate for 5 minutes and observed in a JEOL 1210 electronmicroscope operating at 80 kV.

4.6. Conventional scanning electron microscopy

The cells were adhered on coverslips, fixed in 2.5 %glutaraldehyde in cacodylate buffer, post-fixed for 5 minutesin 1 % OsO4, dehydrated in ethanol, critical point dried withCO2, sputter-coated with gold-palladium and examined in aJeol 5800X scanning electron microscope operating at 12 kV.

4.7. Field emission high resolution scanning electronmicroscopy

Cells were adhered to poly-L-lysine-coated glass cover-slips and then treated with permeabilization buffer (0.5 %Nonidet-40 P-40, 0.1 M Pipes, 1 mM MgSO4, 2 mM glyc-erol, 2 mM EGTA, 1 mM PMSF (phenylmethylsulfonylfluoride), 0.5 % Triton X-100) for different times (10 min-utes–2 h). As Giardia plasma membrane was very resistant toextraction, the best results were obtained when two deter-gents were simultaneously used. After several trials, the bestresults were achieved with the above protocol. Although bothdetergents are interchangeable, only when they were used,the plasma membrane was completely removed. The cellswere washed in PBS and then fixed in 2.5 % glutaraldehydein phosphate buffer, post-fixed for 5 minutes in 1 % OsO4,dehydrated in ethanol, critical point dried with CO2, andsputter-coated only with carbon. The samples were examinedin a Jeol JSM-6340F FESEM operating at an acceleratingvoltage of 5 kV, using the standard SEI and BEI detectors.


4.8. Conventional scanning electron microscopy

The cells were adhered, fixed in 2.5 % glutaraldehyde incacodylate buffer, post-fixed for 5 minutes in 1 % OsO4,dehydrated in ethanol, critical point dried with CO2, sputter-coated with gold-palladium and examined in a Jeol 5800Xscanning electron microscope operating at 12 kV.

Acknowledgements

This work was supported by the Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq),Fundação Carlos Chagas Filho de Amparo à Pesquisa doEstado do Rio de Janeiro (FAPERJ), Programa de Núcleos deExcelência (PRONEX), Coordenação de Aperfeiçoamentode Pessoal de Ensino Superior (CAPES) and AssociaçãoUniversitária Santa Úrsula (AUSU). The authors thank thetechnical support of William Christian Molêdo Lopes, theLaboratório de Ultraestrutura Celular Hertha Meyer (UFRJ)for the use of the FESEM, and Dr. Staffan Svard by kindlyprovided the anti-giardin antibodies.

References

Belhadri, A., 1995. Presence of centrin in the human parasite Giardia: afurther indication of its ubiquity in eukaryotes. Biochem. Biophys. Res.Commun. 214, 597–601.

Bell, P.B., 1995. The application of scanning electron microscopy to thestudy of the cytoskeleton of cells in culture. J. Microsc. 179, 67–76.

Bell, P.B., Lindroth, M., Fredrilsson, B.A., 1988. Preparation of cytoskel-etons of cells in culture for high resolution scanning and scanningtransmission electron microscopy. Scanning Microsc. 2, 1647–1661.

Benchimol, M., 2002. A new set of vesicles in Giardia lamblia. Exp.Parasitol. 102, 30–37.

Benchimol, M., Piva, B., Campanati, L., DeSouza, W., 2004. Visualizationof the funis of Giardia lamblia by high-resolution field emission scan-ning electron microscopy—new insights. J. Struct. Biol. 147, 102–115.

Bertram, M.A., Meyer, E.A., Anderson, D.L., Jones, C.T., 1984. A morpho-metric comparison of five axenic Giardia isolates. J. Parasitol. 70,530–535.

Boggild, A.K., Sundermann, C.A., Estridge, B.H., 2002. Post-translationalglutamylation and tyrosination in tubulin of tritrichomonads and thediplomonad Giardia intestinalis. Parasitol. Res. 88, 58–62.

Braet, F., De Zanger, R., Kalle, W., Raap, A., Tanke, H., Wisse, E., 1996.Comparative scanning, transmission, and atomic force microscopy of themicrotubular cytoskeleton in fenestrated liver endothelial cells. Scan-ning Microsc. Suppl. 10, 225–236.

Brugerolle, G., 1975. Contribution a l’étude cytologique et phylétique desdiplozoaires (Zoomastigophorea, Diplozoa Dangeard 1910). V. nouvelleinterprétation de l´organisation cellulaire de Giardia. Protistologica 11,99–109.

Brugerolle, G., 1991. Flagellar and cytoskeleton systems in amitochondrialflagellates: Archamoeba, Metamonada and Parabasala. Protoplasma164, 70–90.

Campanati, L., Bre, M.H., Levilliers, N., DeSouza, W., 1999. Expression oftubulin polyglycylation in Giardia lamblia. Biol. Cell. 91, 499–506.

Campanati, L., Troester, H., Monteiro-Leal, L.H., Spring, H., Trendelen-burg, M.F., DeSouza, W., 2003. Tubulin diversity in trophozoites ofGiardia lamblia. Histochem. Cell Biol. 119, 323–331.

Chen, Y., Centonze, V.E., Verkhovsky, A., Borisy, G.G., 1995. Imaging ofcytoskeletal elements by low-temperature high-resolution scanning elec-tron microscopy. J. Microsc. 179, 67–76.

Cerva, L., Nohýnková, E., 1992. A light microscopic study of the course ofcellular division of Giardia intestinalis trophozoites grown in vitro.Folia Parasitol. (Prague) 39, 97–104.

Cheissin, E.M., 1964. Ultrastructure of Lamblia duodenalis. I. Body sur-face, sucking disc and median bodies. J. Protozool. 11, 91–98.

Corrêa, G., Diaz-Morgado, J., Benchimol, M., 2004. Centrin in Giardialamblia—ultrastructural localization. FEMs Microbiol. Lett. 233,91–96.

Crossley, R., Holberton, D.V., 1985. Assembly of 2.5 nm filaments fromgiardin, a protein associated with cytoskeletal microtubules in Giardia. J.Cell Sci. 76, 205–231.

Crossley, R., Marshall, J., Clark, J.T., Holberton, D.V., 1986. Immunocy-tochemical differentiation of microtubules in the cytoskeleton of Giardialamblia using monoclonal antibodies to a-tubulin and polyclonal anti-bodies to associated low molecular weight proteins. J. Cell Sci. 80,233–252.

Diamond, L.S., Harlow, D., Cunnick, C.C., 1978. A new medium for theaxenic cultivation of Entamoeba histolytica and other Entamoeba.Trans. R. Soc. Trop. Med. Hyg. 72, 431–432.

Elmendorf, G.H., Dawson, S.C., McCaffery, J.M., 2003. The cytoskeleton ofGiardia lamblia. Int. J. Parasitol. 33, 3–28.

Erlandsen, S.L., Feely, D.E., 1984. Trophozoite motility and the mechanismof attachment. In: Erlandsen, S.L., Meyer, E.A. (Eds.), Giardia andGiardiasis. Plenum, New York and London, pp. 33–63.

Feely, D.E., Schollmeyer, J.V., Erlandsen, S.L., 1982. Giardia spp.: distri-bution of contractile proteins in the attachment organelle. Exp. Parasitol.53, 145–154.

Feely, D.E., Erlandsen, S.L., Chase, D.G., Holberton, D.V., Erlandsen, S.L.,1990. The biology of Giardia. In: Meyer, E.A. (Ed.), Giardiasis.Elsevier, Amsterdam, pp. 11–49.

Filice, F.P., 1952. Studies on the cytology and life history of a Giardia fromthe laboratory rat. Univ. Calif. Public. Zool. 57, 53–146.

Friend, D.S., 1966. The fine structure of Giardia muris. J. Cell Biol. 29,317–332.

Gillin, F.D., Boucher, S.E., Rossi, S.S., Reiner, D.S., 1989. Giardia lamblia.the roles of bile, lactic acid and pH in completion of the life cycle in vitro.Exp. Parasitol. 69, 164–174.

Gillin, F.D., Reiner, D.S., McCaffery, J.M., 1996. Cell biology of theprimitive eukaryote Giardia lamblia. Annu. Rev. Microbiol. 50, 679–705.

Gordon, B.M., Howard, L., Compton, D.A., 2001. Chromosome movementin mitosis requires microtubule anchorage at spindle poles. J. Cell. Biol.152, 425–434.

Holberton, D.V., Crossley, R., Marshall, J., 1981. Morphogenesis of the disccytoskeleton in Giardia. Studies of mictotubule assembly in vitro. 6thInt. Cong. Protozool. Warsaw.

Keister, D.B., 1983. Axenic cultivation of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc. Trop. Med. Hyg. 77,487–488.

Kulda, J., Nohýnková, E., 1995. Giardia in humans and animals. In:Kreier, J.P. (Ed.), Parasitic Protozoa, second ed. vol. 10. Academic Press,San Diego, pp. 225–422.

Lanfredi-Rangel, A., Kattenbach, W.M., Diniz, J.A., DeSouza, W., 1999.Trophozoites of Giardia lamblia may have a Golgi-like structure. FEMSMicrobiol. Lett. 181, 245–251.

Lindroth, M., Bell, P.B., Fredrikson, B.A., 1988. Comparison of the effectsof critical point-drying and freeze-drying on cytoskeleton and microtu-bules. J. Microsc. 151, 103–114.

Lujan, H.D., Marotta, A., Mowatt, M.R., Sciaky, N., Lippincott-Schwartz, J., 1995. Developmental induction of Golgi structure andfunction in the primitive eukaryote Giardia lamblia. J. Biol. Chem. 270,4612–4618.

Marshall, J., Holberton, D.V., 1993. Sequence and structure of a new coiledcoil protein from a microtubule bundle in Giardia. J. Mol. Biol. 231,521–530.


Marti, M., Hehl, A.B., 2003. Encystation-specific vesicles in Giardia: aprimordial Golgi or just another secretory compartment? Trend Parasi-tol. 19, 440–446.

Meng, T.C., Aley, S.B., Svard, S.G., Smith, M.W., Huang, B., Kim, J., et al.,1996. Immunolocalization and sequence of caltractin/centrin from theearly branching eukaryote Giardia lamblia. Mol. Biochem. Parasitol.79, 103–108.

Nohýnková, E., Draber, P., Reischig, J., Kulda, J., 2000. Localisation ofgamma-tubulin in interphase and mitotic cells of a unicellular eukaryote,Giardia intestinalis. Eur. J. Cell Biol. 79, 438–445.

Pawley, J.B., 1990. Practical aspects of high-resolution LVSEM. Scanning12, 247–252.

Reiner, D.S., McCaffery, M., Gillin, F.D., 1990. Sorting of cyst wall proteinsto a regulated secretory pathway during differentiation of the primitiveeukaryote, Giardia lamblia. Eur. J. Cell Biol. 53, 142–153.

Ris, H., 1985. The cytoplasmic filament system in critical point-dried wholemounts and plastic-embedded sections. J. Cell Biol. 100, 1474–1487.

Soloviev, M.M., 1963. Studies on division of Lamblia duodenalis in cul-tures. Med. Parasitol. Parazit. Bolezni. 32, 96–101 (in Russian).

Soltys, B.J., Gupta, R.S., 1994. Immunoelectron microscopy of Giardialamblia cytoskeleton using antibody to acetylated a-tubulin. J. Eukaryot.Microbiol. 41, 626–632.

Wolf, A., de Nechaud, B., Chillet, D., Mazarguil, H., Desbruyeres, E.,Audebert, S., et al., 1992. Distribuition of glutamylated a and b-tubulinin mouse tissues using a specific monoclonal antibody, GT 335. Eur. J.Cell Biol. 59, 425–432.

Woods, A., Sherwin, T., Sasse, R., MacRae, T.H., Baines, A.J., Gull, K.,1989. Definition of individual components within the cytoskeleton ofTrypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci.93, 491–500.


The median body of Giardia lamblia: an ultrastructural study

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