The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

Journal of Structural Biology 154 (2006) 89–99

www.elsevier.com/locate/yjsbi

The Xagellar attachment zone of Trypanosoma cruzi epimastigote forms

Gustavo Miranda Rocha a, Bruno Alves Brandão a, Renato Arruda Mortara b, Márcia Attias a, Wanderley de Souza a, Tecia M.U. Carvalho a,¤

a Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, llha do Fundão, Rio de Janeiro, RJ 21949-900, Brazil

b Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP—Escola Paulista de Medicina, Rua Botucatu, 862 6° andar, São Paulo, SP 04023-062, Brazil

Received 26 August 2005; received in revised form 16 November 2005; accepted 22 November 2005Available online 22 December 2005

Abstract

The Xagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the Xagellum emergesfrom the Xagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear seriesof apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groupsappear to be important in the FAZ region: a membrane glycoprotein of 72 kDa and several high molecular weight proteins. To gain abetter understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDasurface glycoprotein (Gp72), involved in cell body-Xagellum adhesion, had been deleted by target gene replacement. Using immunoXuo-rescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence ofGp72, other proteins involved in the formation of FAZ remain concentrated in the Xagellar pocket region. The analysis of a 3-D recon-struction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association withFAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.© 2005 Elsevier Inc. All rights reserved.

Keywords: Trypanosoma cruzi; Flagellum; Flagellum attachment zone; Gp72

1. Introduction

The Xagellum is a specialized structure involved in cellmotility as well as other biological processes such as cellrecognition and attachment. It is responsible for establish-ing the contact of Xagellated parasites with potential hostcells. One characteristic feature of the trypanosomatidsXagella is that a signiWcant segment of its length is stronglyattached to the cell body and this region is hence designatedXagellar attachment zone (FAZ)1. Transmission electronmicroscopy of thin sections and freeze-fracture replicas

* Corresponding author. Fax: +55 21 2260 2364.E-mail address: [email protected] (T.M.U. Carvalho).

1 Abbreviations used: FAZ, Xagellar attachment zone; ER, endoplasmicreticulum; FP, Xagellar pocket; FESEM, Weld emission scanning electronmicroscopy.

1047-8477/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2005.11.008

have shown that this portion is a specialized domain of thecell surface (De Souza, 1995, 2002). It is more evident inepimastigote and trypomastigote developmental stageswhere after the emergence from the Xagellar pocket theXagellum remains attached along the cell body for most ofits length, except for the distal tip.

The FAZ region of trypomastigote form of Trypano-soma brucei has been well characterized and available datashow that it is a complex system of membrane connections,Wlaments, and specialized microtubules formed by tightcontact and Xanked by structures originated from both theXagellum and the cell body membranes (Bastin et al., 2000).FAZ also links the axoneme to the paraXagellar rod, andboth to the junction of the Xagellum and plasma membrane(Sherwin and Gull, 1989). A specialized protoWlament occu-pies a speciWc position in the subpellicular array of microtu-bules and is associated with a quartet of microtubules that

mailto: [email protected]

mailto: [email protected]

90 G.M. Rocha et al. / Journal of Structural Biology 154 (2006) 89–99

are very stable and seem to be anchored close to the base ofthe Xagellum, at the expected position of the Xagellarpocket (Sherwin and Gull, 1989). At the side of the Xagel-lum, the FAZ displays Wlamentous elements connecting theparaXagellar rod proximal domain to its associated mem-brane (Sherwin and Gull, 1989).

Transmission electron microscopy of T. cruzi epimasti-gote FAZ showed a junctional complex formed by a linearseries of apposed macular structures of 25 nm in diameter,separated by amorphous material periodically spaced at90 nm intervals (Martinez-Palomo et al., 1976). Freeze-frac-ture studies in diVerent trypanosomes revealed a specializa-tion of both Xagellar and cell body membranes at the FAZregion with clusters of intramembranous particles (DeSouza et al., 1978; Gull, 1999; Martinez-Palomo et al., 1976;Vickerman, 1969).

Before cytokinesis, FAZ is duplicated and provides abase between the single basal body, the kinetoplast, theXagellar pocket, and the initial cleavage sites at the anteriorregion of the parasite (reviewed in Gull, 1999; Khol andBastin, 2005). These observations suggest that the FAZregion communicates with a directional cellular templatefor cytokinesis (Gull, 1999; Khol et al., 2003; Robinsonet al., 1995; reviewed in Khol and Bastin, 2005). Otherreports show that the absence of a single protein in thisregion may lead to impaired motility of the parasite, and itwas shown to be lethal for T. brucei (Cooper et al., 1993;Nozaki et al., 1996).

The nature of the proteins involved in the FAZ regionhas not been completely described yet, but two groups ofproteins appear to be important: an immunodominantmembrane glycoprotein of 72 kDa (Gp72) and severalhigh molecular weight proteins. Although concentrated inthe FAZ region, Gp72 is also distributed over the surfaceof the cell body and the Xagellar pocket membrane. Usingtarget gene replacement, Cooper et al. (1993) have devel-oped a T. cruzi gp72 epimastigote null mutant that pre-sents an unexpected morphology characterized by thedetachment of the Xagellum from the cell body, leading tomajor alterations in the overall shape of the parasite(Cooper et al., 1993; De Jesus et al., 1993). Furthermore,the mutants are less infective to both Triatoma infestansvectors (De Jesus et al., 1993) and mice (Basombrio et al.,2002). A Gp72 homologue was identiWed in T. brucei(Nozaki et al., 1996) but its function could not be deter-mined as null mutants were not viable. In promastigotesof Leishmania mexicana, the inhibition of Xagellumformation by a knock-out of some components of theXagellum assembly machinery has no blockage eVect inthe cytokinesis process (Adhiambo et al., 2005). Weiseet al. (2000) using also L. mexicana showed the presenceof a extremely short FAZ region in promastigote form. Ina recent review, Khol and Bastin (2005) pointed out thata homologue of gp72 also exists in Leishmania. Therefore,T. cruzi epimastigote null mutant represents an excellentmodel to further the studies of the FAZ region of thisparasite.

To obtain further information on the FAZ region ofT. cruzi epimastigotes a comparative analysis was per-formed using antibodies that recognize diVerent proteins onboth the Xagellum (anti-FRA) and cell body (anti-4D9)sides. For this, we used a wild-type and a gp72 null mutantof T. cruzi that presented a detached Xagellum from the cellbody. We decided to analyze whether this altered pheno-type, with no expression of gp72, also showed an alterationon the distribution of some proteins present in this region.Additional information on the structural organization ofthe FAZ region was obtained using electron microscopytechniques: freeze-fracture, cytochemistry, and three-dimensional reconstruction of serial ultra-thin sections.

2. Materials and methods

2.1. Parasites

Epimastigotes forms of T. cruzi Y strain (wild-type) anda gp72 null mutant of T. cruzi (gp72-null, kindly suppliedby Dr. George Cross, Rockfeller University, USA) wereused throughout this study. Wild-type and gp72-nullepimastigotes were cultivated as previously described (Coo-per et al., 1993) in liver-infusion tryptose (LIT) mediumsupplemented with 10% fetal bovine serum and 1% heminfor 3–6 days at 28 °C. G418 (400 �g/mL) was added to gp72-null epimastigote cultures. T. brucei (472 strain) procyclicforms, used as a positive control in the immunoXuorescenceassays, were grown in SDM 79 medium supplemented with10% fetal bovine serum for 2 days at 27 °C.

2.2. Antibodies

The following antibodies were used: DOT1 and L3B2,monoclonal antibodies reactive to T. brucei FAZ (kindlysupplied by Dr. Keith Gull, University of Oxford, UK);4D9 mAb, reactive towards a high molecular weight com-ponent localized on the cell body side of the Xagellar-cellbody attachment zone (Cotrim et al., 1995; Mortara et al.,2001); FRA polyclonal antibody raised in rabbit (a giftfrom Dr. Marcos Krieger, TECPAR, IBMBP, FIOCRUZ,Curitiba, Paraná, Brazil) against T. cruzi Xagellar protein(Lafaille et al., 1989).

2.3. ImmunoXuorescence confocal microscopy

Wild-type and gp72 null mutant epimastigote 4 days oldcultures were washed twice with PBS pH 7.2, and Wxed with2% paraformaldehyde in phosphate buVer for 20 min.Samples were then washed twice in PBS and settled ontopoly-L-lysine-coated coverslips. Adhered cells were madepermeable either with acetone at ¡20 °C for 10 min (for fur-ther incubation with 4D9 and FRA antibodies) or a mix-ture of 1:4 acetone/methanol at ¡20 °C for 10 min (forDOT1 and L3B2 antibodies). After the permeable cellswere rehydrated in PBS and non-speciWc antigen-bindingsites were blocked with 2% BSA in PBS, pH 8.0, for 1 h at

G.M. Rocha et al. / Journal of Structural Biology 154 (2006) 89–99 91

room temperature. Samples were incubated for 60 min atroom temperature with 4D9 and FRA antibodies (asciticXuid or sera diluted 1:300) or DOT1 and L3B2 (undiluted).After washing, bound immunoglobulins were detected byincubation with secondary antibody conjugated with Alexa488 or 543 (Molecular Probes, Eugene, OR, USA, diluted1:400). For control the same procedure was followed,except that the primary antibody was omitted. Slides weremounted in N-propylgalate (Sigma P130) to reduce photo-bleaching and observed on a 310 Zeiss Confocal LaserScanning Microscope, using Argon and He/Ne Lasers andLP510 and LP590 Wlters.

2.4. Field-emission scanning electron microscopy

Cells were Wxed in 2.5% glutaraldehyde in 0.1 M phos-phate buVer, pH 7.2, for 60 min, washed in the same buVer,and settled onto poly-L-lysine-coated coverslips. Cells werethen post-Wxed in 1% OsO4, 0.8% potassium ferrocyanide in0.1 M sodium cacodylate buVer at room temperature for30 min, washed in 0.1 M phosphate buVer, dehydrated in anethanol series, and critical-point dried with CO2. Sampleswere ion-sputtered with 2–3 nm carbon layer using a Gatanmodel 681 to avoid charge eVect. Secondary electronimages were obtained in a JEOL JSM 6340 F FESEM at a5.0 kV accelerating voltage.

2.5. Transmission electron microscopy

Cells were harvested by centrifugation and washed inPBS. They were then Wxed in 2.5% glutaraldehyde in 0.1Mphosphate buVer, pH 7.2, for 60 min, washed in the samebuVer, post-Wxed in 1% OsO4, 0.8% potassium ferrocyanidein 0.1 M sodium cacodylate buVer at room temperature for40 min, washed in 0.1 M phosphate buVer, dehydrated in ace-tone, and embedded in Polybed resin. Ultrathin sections werestained with uranyl acetate and lead citrate and observed in aZEISS EM900 transmission electron microscope.

Detection of basic proteins was carried out as describedelsewhere (Gordon and Bensch, 1968). BrieXy, cells wereWxed in 2.5% glutaraldehyde in 0.1 M phosphate buVer, pH7.2, for 60 min, dehydrated in ethanol, and incubated for120 min at room temperature in 2% phosphotungstic acidin 100% absolute ethanol. Samples were then washed inethanol, embedded in Polybed resin, and ultrathin sectionswere observed unstained in a ZEISS EM900 transmissionelectron microscopy.

2.6. Freeze-fracture replicas

Cells were Wxed in 2.5% glutaraldehyde in 0.1 M phos-phate buVer, pH 7.2, for 60 min, washed in phosphatebuVer, and then placed in increasing glycerol concentra-tions. Samples were left in 15% glycerol overnight, har-vested by centrifugation, and resuspended in 30% glycerolin PBS. After 3 h, cells were placed on Balzers support golddisks, rapidly frozen in Freon 22 for 15 s and transferred to

liquid nitrogen. Fracture was carried out at ¡115 °C in aBalzers apparatus. Specimens were shadowed with plati-num at a Wxed angle of 45° and with carbon at 90°. Replicasobtained were cleaned in a 5% hypochloride solution,washed in distilled water, mounted on 300 mesh nickelgrids and observed in a ZEISS EM900 transmissionelectron microscope.

2.7. Three-dimensional reconstruction

Epoxy-embedded serial sections of wild-type and gp72null mutant epimastigote forms of T. cruzi Y strain wereused to carry out three-dimensional reconstructions of theXagellar-cell body attachment zone. Samples were trimmedand sectioned as described elsewhere (Fahrenbach, 1984;Attias et al., 1996). Serial micrographs were printed at aWnal magniWcation 200,000£. Cells, Xagellar membrane,axoneme, endoplasmic reticulum, and mitochondrion wereoutlined on each micrograph with distinct colors, and eachplane was separately traced using a graphic tablet (Numon-ics 2205). A 3-D reconstruction and morphometry programwas used (Young et al., 1987). This program provides theperimeter and area of the digitized proWles, and these datawere used for calculations of relative volumes of cell struc-tures. The resulting data Wles consisted of contour outlinesrepresenting cross-sections of the objects of interest withinthe volume. The Wles were transferred to a Silicon Graphicsworkstation, and surfaces between planes were generatedusing the software package SYNU (synthetic universe)(Hessler et al., 1992). In the Silicon Graphics workstation,images rotating at 6° steps were rendered and all imageswere recorded in order to obtain an animation of the recon-struction.

3. Results

3.1. ImmunoXuorescence microscopy

Several monoclonal antibodies that recognize the Xagel-lum-cell body attachment region of trypanosomatids wereused. In T. brucei trypomastigotes, monoclonal antibodiesL3B2 and DOT1 showed a continuous immunoXuorescencestaining at the FAZ region (data not shown). The length ofthe labeled region in this parasite, with L3B2 was about17.5�m. L3B2 antibody showed an intense labeling patternin the initial portion of the Xagellar-cell body adhesion zoneof wild-type T. cruzi epimastigotes (Figs. 1A and A�). Thelabeled region reached a length of 11�m, and was thicker atits most basal portion. A short labeled region, of about 2�m,was observed in gp72 null mutants but was restricted to theregion of emergence of the Xagellum from the Xagellarpocket (Figs. 1B and B�). DOT1 antibody intensely labeledthe T. brucei FAZ region and so it was used as positive con-trol, but did not recognize either the wild-type form nor themutant T. cruzi epimastigote forms (data not shown).

We also tested antibodies that have been shown to rec-ognize high molecular weight proteins located on the cell


body (Mab 4D9, Cotrim et al., 1995; Mortara et al., 2001)or Xagellum (polyclonal FRA, Lafaille et al., 1989) side ofthe FAZ region. Both antibodies labeled the FAZ regionof wild-type epimastigotes (Figs. 1C and C� for 4D9 andFigs. 1E and E� for FRA) but labeling with 4D9 antibodywas more intense in the region where wild-type epimasti-gotes emerged from the Xagellar pocket (Figs. 1C and C�).This was similar to the pattern obtained with L3B2. Ingp72 null mutants, 4D9 reactive components wererestricted to the point of emergence of the Xagellum fromthe Xagellar pocket (Figs. 1D and D�). Dividing gp72 nullmutant cells with two Xagella showed a double labelingnear the Xagellar pocket opening (Fig. 1D�). FRA stainingof wild-type cells was restricted to the Xagellar-cell bodyattachment zone but in gp72 null mutants the labeling wasobserved along the detached Xagellum and in the cyto-plasm (Figs. 1F and F�).

3.2. Field emission scanning electron microscopy

The Xagellum of wild-type Trypanosoma cruzi epimasti-gote forms emerges from the Xagellar pocket (FP) andremains tightly attached to the cell body along its length,which is about 11.5�m, except for its distal tip (Fig. 2A).Projections of the Xagellar membrane could be seen par-

tially covering the cell body in Fig. 2A and in high magniW-cation in Fig. 2B. The cytostome, which appears as asurface opening localized close to the FP, was visualized inabout 9% of the examined epimastigotes (not shown). TheXagellum of gp72 null mutant was detached from the cellbody (Fig. 2C). The FP displayed a large loose cup likeopening (Figs. 2C and D). We did not observe the Xagellarmembrane projection over the cell body as seen in wild-typeparasites. The cytostome was observed in about 20% of thenull mutant cells examined (Figs. 2E and F).

3.3. Transmission electron microscopy

Analysis of ultrathin sections of T. cruzi epimastigotesconWrmed a close adhesion of the Xagellum to the cell bodymembrane (Fig. 3). A 9 (nine) nm space separated theXagellar and cell body membranes. On the cytoplasmic sideof sections cut near the Xagellar pocket, three microtubulesappeared close to the FAZ region and an electron densemass was observed between the axoneme and the Xagellarmembrane (Fig. 3B). ProWles of the ER were seen in closeassociation with subpellicular microtubules and mitochon-drion (Figs. 3A–D). Finger-like projections of the Xagellarmembrane were seen in contact with the cell body (Fig. 3E).Transversal sections of the wild-type epimastigotes at the

Fig. 1. ImmunoXuorescence microscopy of epimastigote forms of wild-type (A, C, and E) and gp72 null mutant (B, D, and F) cells of T. cruzi labeled withL3B2 (A� and B�), 4D9 (C and D) and FRA (E and F) antibodies that recognize FAZ region proteins. In wild-type, the staining with L3B2 (A and A�) and4D9 (C and C�) are continuous along the FAZ region and concentrate near the emergence of the Xagellum (arrows). But with null mutants cells (B and B�

for L3B2 and D and D� for 4D9) the labeling is restricted to the area near the emergence of the Xagellum from the Xagellar pocket. We observed dividingnull mutant cells with a double labeling near the Xagellar pocket opening using 4D9 antibody (D�, arrow). FRA labeling of wild-type cells (E and E�)shows a continuous staining along the FAZ region (arrow) but in gp72 null mutant (F and F�) labeling was observed all along the whole cell body andXagellum. A–F, phase contrast; A�–F�, Xuorescence microscopy. Bars: 10 �m.


FAZ region revealed that two of the subpellicularmicrotubules were replaced by Wlamentous material (Figs.3E and F).

None of the structures forming the FAZ region wereobserved in gp72 null mutant parasites that also displayedopened Xagellar pockets (Fig. 4).

Incubation of cells with ethanolic phosphotungstic acid(E-PTA) that reacts with basic proteins, revealed the pres-ence of an electron dense reaction at the cell body-Xagellumattachment region of wild-type epimastigotes (Figs. 5A–D).In favorable sections, punctation and regularly spaceddense labeling was evident, especially underneath the cellbody membrane (Figs. 5C and D). Neither the cell body northe Xagellum of gp72 null mutant epimastigotes werelabeled under these conditions (Figs. 5E and F). In bothwild-type and gp72 null epimastigotes, E-PTA labeling ofthe nuclear chromatin, the kinetoplast periphery, the glyco-somes and the acidocalcisomes was observed.

3.4. Freeze-fracture studies

Analysis of wild-type T. cruzi epimastigote replicasrevealed that most Xagellar membranes present few intra-membranous particles (IMPs). However, the FAZ region ofwild-type epimastigotes showed a row of IMPs clusters(Figs. 6A and B) and a linear array of IMPs on both PFand EF faces (Figs. 6C and D), that could be involved withthe FAZ region. The IMPs were longitudinally oriented inrelation to the main axis of the Xagellum (Figs. 6A–D). Thecytostome was observed as a region with no IMPs delim-ited by a linear array of IMPs (Fig. 6C).

Freeze-fracture replicas of gp72 null mutants showedonly a few randomly distributed IMPs at the initial portionof the Xagellum (Figs. 6E and F). The Xagellar necklace,which is formed by an aggregation of IMPs at the base ofthe Xagellum, was present in gp72 null mutant (arrows inFigs. 6E and F).

Fig. 2. Field emission scanning electron microscopy of T. cruzi wild-type and gp72 null mutant cells. In wild-type cells, the Xagellum is tightly attached tothe cell body along the FAZ region (A) and a projection of the Xagellar membrane partially covers the cell body membrane (B, arrow). In gp72 null mutantepimastigotes, the Xagellum appears detached from the cell body (C, arrow). A deformation of the Xagellar pocket opening was observed (C and D, ¤). Thecytostome is more visible in the null mutant cells (arrows in E and F). Bars: A–E D 1 �m; FD 0.1 �m.


makes contact with the cell body surface, could be clearly

3.5. Three-dimensional reconstruction

In wild-type T. cruzi epimastigotes, the mitochondrionand ER appear in close association to the FAZ region, asevidenced by the three-dimensional reconstruction of theserial sections. Mitochondria were always localized lateralto the Xagellum and ER segments were closely associated tothe FAZ region (Figs. 7A–D). In gp72 null mutant, mito-chondrion but not endoplasmic reticulum was observed inthe same region as described for wild-type (Figs. 7E and F).

A Xagellar membrane projected over the parasite cellbody, as seen by FESEM, was also evident (Figs. 7A, C,and D). This projection, as well as the region where it

Fig. 4. Transmission electron micrograph of T. cruzi gp72 null mutantshowing the Xagellum detached from the cell body (arrow). No otherultrastructural alterations were detected in these cells. (f) Flagellum, (k)kinetoplast, (N) nucleus. Bars: 1 �m.

seen in a sequence of diVerent angles of the reconstructedarea in an animated video, showing the mitochondrion andER associated to the FAZ region and membrane projection(Video 1).

4. Discussion

The site of attachment of the Xagellum to the cell bodyin trypanosomes comprises a specialized region that isknown as the Xagellum attachment zone (FAZ). Thisregion has been studied in detail in T. brucei (Bastin et al.,2000). We took advantage of the fact that the target genedeletion of a 72 kDa glycoprotein impairs attachment ofthe Xagellum to the cell body of T. cruzi (Cooper et al.,1993). This suggests that this protein is an essential compo-nent of the FAZ region, and made a comparative study ofthe wild-type and the mutant.

In contrast to previous reports in which no overall mor-phological alterations were observed in mutant trypano-somes (Bastin et al., 1999), we observed that the FP openingwas altered in T. cruzi gp72 null mutants. The FP is locatedat the anterior end of the cell, encircling the base of theXagellum. Its opening is restricted by desmosome-likestructures (reviewed in De Souza, 1995). In Leishmaniapromastigotes, Xagellar beating aVects pocket opening

Fig. 3. Transmission electron micrographs of ultrathin sections of T. cruzi wild-type epimastigote forms of T. cruzi. (A) The Xagellum is tightly attached tothe cell body and the endoplasmic reticulum is closely together to the FAZ region (white arrows). (B) Electron dense structures within the Xagellum arelocated between the axoneme and the Xagellar membrane (¤) and also noticeable are three microtubules associated to the FAZ region (black arrow).Transversal ultrathin sections of wild-type cells show the localization of the endoplasmic reticulum (arrows in C and D) and mitochondria (m) in the vicin-ity of FAZ region. A projection of the Xagellar membrane over the cell body membrane (black arrow in E) and electron dense Wlamentous structuresextending along the cell body and Xagellar membranes (arrowheads in E and F) are visible. (f) Flagellum, (k) kinetoplast. Bars: 1 �m.


(Webster and Russell, 1993). The gp72 null mutants display despite the short length of the FAZ region of promastigote
a free Xagellum emerging from a loose FP opening, whichresults in altered motility and gp72 depleted parasites arenot able to swim and tend to sink towards the bottom ofthe culture Xask (not shown). It was shown that RNAisilencing of FLA1 (a gp72 homologue in T. brucei) and ofcomponents of the Xagellum assembly machinery in procy-clic trypomastigote of T. brucei blocks cytokinesis (LaCount et al., 2002; Khol et al., 2003; reviewed in Khol andBastin, 2005). The epimastigote gp72 null mutant used inour experiments showed a growth inhibition of around 50%and no multinucleated cells (data not shown). Also,Adhiambo et al. (2005) showed no cytokinesis blockage inL. mexicana promastigote after inhibition using a knockout model of components of Xagellum assembly machinery.Concerning this, Khol and Bastin (2005) described the pres-ence of a gp72/FLA1 homologue in Leishmania database
forms. These results described in Leishmania is in agree-ment with ours results in epimastigote of T. cruzi showing acomplete process of cytokinesis and the presence of viableparasites. Khol and Bastin (2005) described that division“proceeds in an helical manner toward the posterior end,following the path of the Xagellum.” The motility of thegp72 null mutant cell is completely altered (data notshown), and we could speculate that this altered motilitymight delay cytokinesis process leading to a growth inhibi-tion. De Jesus et al. (1993) also showed that this gp72 nullmutant cells of T. cruzi is poorly competent to producetrypomastigote forms. The enlargement of the FP openingmay also aVect the parasite by means of unrestricted accessof molecules to the FP milieu. Acquisition of nutrients inthese cells is accomplished by the FP and the cytostome(De Souza, 2002). Okuda et al. (1999) showed that the

Fig. 5. Detection of basic proteins using E-PTA cytochemistry. Wild-type T. cruzi epimastigotes presented intense labeling at the Xagellum-cell body adhe-sion area (small arrows in A–C). Punctation and regularly spaced labeling is observed in some cells (arrowheads in C and D). In gp72 null mutants, thelabel is absent in the region of the adhesion (small arrows in E and F). Both cells were also labeled in the nucleus (N), kinetoplast (k) periphery andorganelles resembling glycosomes and acidocalcisomes (large arrows in A, E, and F). Bars: 1 �m.


T. cruzi epimastigote cytostome is also associated with theXagellar complex. In the T. cruzi gp72 null mutant, the cyto-stome was observed in about 20% of the cells examined byhigh resolution scanning microscopy while in control cellsthis structure was visible in only 9% of the epimastigotes(data not shown), thus indicating possible changes in thespeciWc organization of the anterior region of the proto-zoan.

ImmunoXuorescence images in T. cruzi showed thatlabeling with monoclonal antibodies DOT1 and L3B2 pre-sented the same punctuated pattern previously describedfor T. brucei trypomastigotes (Kohl et al., 1999; Woodset al., 1989). Interestingly, L3B2 antibody also labeled T.cruzi epimastigotes, conWrming the idea that this region isconserved among these species. Antibodies 4D9 and FRA

revealed a linear labeling at the FAZ region of T. cruziepimastigotes, distinct from the L3B2 staining pattern.Monoclonal antibody 4D9 was described to recognize ahigh molecular weight component localized in the cell bodyof the FAZ region (Cotrim et al., 1995; Mortara et al., 2001)while FRA, employed in immunodiagnosis assays, labeledthe Xagellum near the FAZ region (Krieger et al., 1992;Lafaille et al., 1989). Labeling in gp72 null mutants wasobserved only in the region near the FP. Interestingly, FRAantigen appeared as a more intense labeling in these cells.Ours results suggest that the lack of Gp72 may somehowhave altered the distribution and assembly of the proteincomplex in the FAZ region. This hypothesis could explainthe apparent concentration of some components close tothe site of emergence of the Xagellum in the null mutants.

Fig. 6. Freeze-fracture micrographs of T. cruzi wild-type (A–D) and gp72 null mutant (E–F) epimastigotes. An IMP linear clustering was observed in theXagellum (arrows in A and B) on both PF and EF faces (arrowheads in C and D). The cytostome was clearly visible (C). No specializations were observedin T. cruzi gp72 null mutants (E and F); the Xagellar necklace was not altered (arrow in E and F). Xagellum (f); cell body (c); cytostome (cy). Bars: A–C andE and F, 1 �m; D, 0.1 �m.


It is important to point out that detection of basic pro- dense region that may correspond to arrays of intraXagellar
teins revealed a punctate labeling encircling the FAZ regionthat may correspond to the punctuate electron-denseregion described by Bastin et al. (2000). In gp72 nullmutants, we were not able to observe E-PTA reaction forbasic proteins in the FAZ region, reinforcing the idea thatthese proteins are important for the normal function of thisregion. At present the nature of the basic proteins involvedin Xagellum-cell body attachment is not known.
The microtubule quartet observed in the FAZ region ofT. brucei trypomastigotes (Gull, 1999) could not bedetected in our studies. We only observed a microtubuletriplet. Okuda et al. (1999) observed three to four microtu-bules related to the T. cruzi FAZ region, in agreement withthe present observations. We also observed an electron-

transport particles (IFT) (Nogales, 2000; reviewed inRosenbaum and Witman, 2002).

Freeze-fracture replicas of wild-type epimastigotes con-Wrmed previous studies (De Souza, 1995; Martinez-Palomoet al., 1976) in which a specialization of typical IMPs wereobserved on both P and E faces of the Xagellar membrane(Figs. 6A–D). The IMPs, presumed to be integral mem-brane proteins (De Souza et al., 1978), appeared as a lineararray of particles longitudinally oriented in relation to themain axis of the Xagellum. However, they were signiWcantlyscarce in the gp72 null mutants corroborating the immuno-Xuorescence and cytochemistry assays, carried in oursexperiments, in which Gp72 and basic proteins are absentfrom the FAZ. Adhesion of the Xagellum to the cell body

Fig. 7. Three-dimensional reconstruction of the FAZ region of T. cruzi wild-type (A–D) and gp72 null mutant (E and F) epimastigotes. In wild-type cells,proWles of the endoplasmic reticulum (green) and mitochondrion (yellow) show a distinct association to the FAZ region, and a projection of the Xagellarmembrane (blue) over the cell body (white or opaque) is also noticeable. In gp72 null mutant epimastigote forms, mitochondrion proWles (yellow) showeda similar association as in the wild-type cells, but neither association of the ER to FAZ region nor the projection membrane were visualized. Axonemaland subpellicular microtubules (red); kinetoplast (pink).


was observed only in wild-type parasites (Cooper et al.,1993).

The detailed analysis of the organelles in the FAZ regionby transmission electron microscopy techniques (ultrathinsection and three-dimensional reconstruction) clearly dem-onstrated a speciWc distribution of the single mitochon-drion and smooth ER near the FAZ region. This is similarto the intimate relation of ER to the FAZ region asdescribed in T. brucei (Bastin et al., 2000). In gp72 nullmutant, the mitochondrion was evident, in contrast to theabsence of ER, near the FAZ region. The present observa-tions lead us to propose that the presence of ER proWlesand mitochondria in the vicinity of the FAZ region mayfacilitate protein traYc and energy exchange. In regards tothis, mitochondria also concentrate in the Xagellar regionof spermatozoan cells, providing energy intermediates forXagellar beating (Cardullo and Baltz, 1991). Interestingly,ER loops were described between the regularly spaced sub-pellicular microtubules of Leishmania parasites (Pimentaand De Souza, 1985). These proWles appear to interact withthe plasma membrane but no function has as yet beenascribed for this association. Association of the ER withthe specialized microtubules adjacent to the FAZ Wlamentsmay also play a role in Ca2+ availability to the Xagellum.Calcium-binding proteins (Engman et al., 1989) and Ca2+-dependent adenylate cyclase activity (Paindavoine et al.,1992) have been described in trypanosome Xagellar mem-brane but the downstream signaling components of theassociated pathway have not been identiWed. Their localiza-tion to the Xagellar membrane may however, suggest a rolein parasite motility, as observed in Crithidia oncopelti(Holwill and McGregor, 1976).

ProWles of the Xagellar membrane observed in ultrathinsections of some wild-type parasites were shown to bemembrane projections in the three-dimensional reconstruc-tions from serial thin sections and by Weld emission scan-ning electron microscopy. This Xagellar membraneprojections appear in around 13% of wild-type cells (datanot shown). These structures were Wrst reported byCarvalho and de Souza (1987) that observed many Wlopo-dium-like projections from the Xagellar membrane inT. cruzi epimastigotes and speculated that they could rein-force the Xagellum adhesion to the cell body. Supportingthis hypothesis, no membrane projection was observed ingp72 null mutants.

Studies of the FAZ region in trypanosomes may pro-vide not only important information on basic cell biologyand motility of these parasites but may also lead to theidentiWcation of components that may be important can-didates for immunodiagnosis and immunoprotection. Inregards to this, several Xagellum-restricted antigens con-ferring variable degrees of protection were described intrypanosomes (Olenick et al., 1988; Pereira et al., 2003a,b;Souto-Padron et al., 1989). Also, the altered motilityobserved in FAZ-defected cells restricts infection. Alto-gether these results outline the importance of the FAZregion of trypanosomes.

Acknowledgments

We thank Dr. George Cross for kindly supplying theT. cruzi null mutants. We also thank Dr. Keith Gull, Dr.Marco Krieger and Solange da Silva (UNIFESP-EPM)for providing the antibodies used in the present studyand Sebastião da Cruz, in memmorian and AntonioBosco for technical assistance. This work has beensupported by the Programa de Núcleo de Excelência(PRONEX), Conselho Nacional de DesenvolvimentoCientíWco e Tecnológico (CNPq) and Fundação CarlosChagas Filho de Amparo à Pesquisa do Estado do Riode Janeiro (FAPERJ).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.jsb.2005.11.008.

References

Adhiambo, C., Forney, J.D., Asai, D.J., LeBowitz, J.H., 2005. The twocytoplasmic dynein-2 isoforms in Leishmania mexicana perform sepa-rate functions. Mol. Biochem. Parasitol. 143, 216–225.

Attias, M., Vommaro, R.C., De Souza, W., 1996. Computer aided three-dimensional reconstruction of the free-living protozoan Bodo sp.(Kinetoplastida: Bodonidae). Cell Struct. Funct. 21, 297–306.

Basombrio, M.A., Gomez, L., Padilla, A.M., Ciaccio, M., Nozaki, T.,Cross, G.A., 2002. Targeted deletion of the gp72 gene decreases theinfectivity of Trypanosoma cruzi for mice and insect vectors. J. Parasi-tol. 88, 489–493.

Bastin, P., Pullen, T.J., Moreira-Leite, F.F., Gull, K., 2000. Inside and out-side of the trypanosome Xagellum: a multifunctional organelle.Microbes Infect. 2, 1865–1874.

Bastin, P., Pullen, T.J., Sherwin, T., Gull, K., 1999. Protein transport andXagellum assembly dynamics revealed by analysis of the paralysed try-panosome mutant snl-1. J. Cell Sci. 112, 3769–3777.

Cardullo, R.A., Baltz, J.M., 1991. Metabolic regulation in mammaliansperm: mitochondrial volume determines sperm length and Xagellarbeat frequency. Cell Motil. Cytoskeleton 19, 180–188.

Carvalho, T.U., de Souza, W., 1987. EVect of phorbol-12-myristate-13-acetate (PMA) on the Wne structure of Trypanosoma cruzi and itsinteraction with activated and resident macrophages. Parasitol. Res.74, 11–17.

Cooper, R., de Jesus, A.R., Cross, G.A.M., 1993. Deletion of an immuno-dominant Trypanosoma cruzi surface glycoprotein disrupts Xagellum-cell adhesion. J. Cell Biol. 122, 149–156.

Cotrim, P.C., Paranhos-Baccala, G., Santos, M.R., Mortensen, C., Cano,M.I., Jolivet, M., Camargo, M.E., Mortara, R.A., Da Silveira, J.F., 1995.Organization and expression of the gene encoding an immunodomi-nant repetitive antigen associated to the cytoskeleton of Trypanosomacruzi. Mol. Biochem. Parasitol. 71, 89–98.

De Souza, W., Martinez-Palomo, A., Gonzalez-Robles, A., 1978. The cellsurface of Trypanosoma cruzi: cytochemistry and freeze-fracture. J. CellSci. 33, 285–299.

De Souza, W., 1995. Structural organization of the cell surface of patho-genic protozoa. Micron 26, 405–430.

De Souza, W., 2002. Basic cell biology of Trypanosoma cruzi. Curr. Pharm.Design 8, 269–285.

De Jesus, A.R., Cooper, R., Espinosa, M., Gomes, J.E.P.L., Garcia, E.S.,Paul, S., Cross, G.A.M., 1993. Gene deletion suggests a role for Trypan-osoma cruzi surface glycoprotein gp72 in the insect and mammalianstages of the life cycle. J. Cell Sci. 106, 1023–1033.

http://dx.doi.org/10.1016/j.jsb.2005.11.008




Engman, D.M., Krause, K.H., Blumin, J.H., Kim, K.S., KirchhoV, L.V.,Donelson, J.E., 1989. A novel Xagellar Ca2+-binding protein in trypan-osomes. J. Biol. Chem. 264, 18627–18631.

Fahrenbach, W.H., 1984. Continuous serial sectioning for electron micros-copy. J. Electron Microsc. Techn. 1, 387–393.

Gordon, M., Bensch, K.G., 1968. Cytochemical diVerentiation of theguinea pig sperm Xagellum with phosphotungstic acid. J. Ultrastruct.Res. 24, 33–50.

Gull, K., 1999. The cytoskeleton of trypanosomatid parasites. Annu. Rev.Microbiol. 53, 629–653.

Hessler, D., Young, S.J., Carragher, B.O., Martone, M.E., Lamont, S.,Whittaker, M., Milligan, R.A., Masliah, E., Hinshaw, J.E., Ellisman,M.H., 1992. Programs for visualization in three-dimensional micros-copy. Neuroimage 1, 55–67.

Holwill, M.E., McGregor, J.L., 1976. EVects of calcium on Xagellar move-ment in the trypanosome Crithidia oncopelti. J. Exp. Biol. 65, 229–242.

Kohl, L., Sherwin, T., Gull, K., 1999. Assembly of the paraXagellar rod andthe Xagellum attachment zone complex during the Trypanosoma bruceicell cycle. J. Eukaryot. Microbiol. 46, 105–109.

Khol, L., Robinson, D., Bastin, P., 2003. Novel roles for the Xagellum incell morphogenesis and cytokinesis of trypanosomes. EMBO J. 22,5336–5346.

Khol, L., Bastin, P., 2005. The Xagellum of trypanosomes. Int. Rev. Cytol.244, 227–285.

Krieger, M.A., Almeida, E., Oellemann, W., Lafaille, J.J., Pereira, J.B.,Krieger, H., Carvalho, M.R., Goldenberg, S., 1992. Use of recombinantantigens for the accurate immunodiagnosis of Chagas disease. Am. J.Trop. Med. Hyg. 46, 427–434.

Lafaille, J.J., Linss, J., Krieger, M.A., Souto-Padron, T., de Souza, W.,Goldenberg, S., 1989. Structure and expression of two Trypanosomacruzi genes encoding antigenic proteins bearing repetitive epitopes.Mol. Biochem. Parasitol. 35, 127–136.

Martinez-Palomo, A., De Souza, W., Gonzalez-Robles, A., 1976. Topo-graphical diVerences in the distribution of surface coat componentsand intramembrane particles. A cytochemical and freeze-fracture studyin culture forms of Trypanosoma cruzi. J. Cell Biol. 69, 507–513.

Mortara, R.A., Minelli, L.M., Vandekerckhove, F., Nussenzweig, V.,Ramalho-Pinto, F.J., 2001. Phosphatidylinositol-speciWc phospholi-pase C (PI-PLC) cleavage of GPI-anchored surface molecules of Try-panosoma cruzi triggers in vitro morphological reorganization oftrypomastigotes. J. Eukaryot. Microbiol. 48, 27–37.

Nogales, E., 2000. Structural insights into microtubule function. Annu.Rev. Biochem. 69, 277–302.

Nozaki, T., Haynes, P.A., Cross, G.A.M., 1996. Characterization of theTrypanosoma brucei homologue of a Trypanosoma cruzi Xagellum-adhesion glycoprotein. Mol. Biochem. Parasitol. 82, 245–255.

Okuda, K., Esteva, M., Segura, E.L., Bijovsy, A.T., 1999. The cytostome ofTrypanosoma cruzi epimastigotes is associated with the Xagellar com-plex. Exp. Parasitol. 92, 223–231.

Olenick, J.G., WolV, R., Nauman, R.K., McLaughlin, J., 1988. A Xagellarpocket membrane fraction from Trypanosoma brucei rhodesiense:immunogold localization and nonvariant immunoprotection. Infect.Immun. 56, 92–98.

Paindavoine, P., Rolin, S., Van Assel, S., Geuskens, M., Jauniaux, J.C.,Dinsart, C., Huet, G., Pays, E., 1992. A gene from the variant surfaceglycoprotein expression site encodes one of several transmembraneadenylate cyclases located on the Xagellum of Trypanosoma brucei.Mol. Cell. Biol. 12, 1218–1225.

Pereira, V.R., Lorena, V.M., Nakazawa, M., da Silva, A.P., Montarroyos,U., Correa-Oliveira, R., Gomes, Y.M., 2003a. Evaluation of theimmune response to CRA and FRA recombinant antigens of Trypano-soma cruzi in C57BL/6 mice. Rev. Soc. Bras. Med. Trop. 36, 435–440.

Pereira, V.R., Lorena, V.M., Vercosa, A.F., Silva, E.D., Ferreira, A.G.,Montarroyos, U.R., Silva, A.P., Gomes, Y.M., 2003b. Antibody isotyperesponses in Balb/c mice immunized with the cytoplasmic repetitiveantigen and Xagellar repetitive antigen of Trypanosoma cruzi. Mem.Inst. Oswaldo Cruz. 98, 823–825.

Pimenta, P.F., De Souza, W., 1985. Fine structure and cytochemistry of theendoplasmic reticulum and its association with the plasma membrane ofLeishmania mexicana amazonensis. J. Submicrosc. Cytol. 17, 413–419.

Robinson, D.R., Sherwin, T., Ploubidou, A., Byard, E.H., Gull, K., 1995.Microtubule polarity and dynamics in the control of organelle posi-tioning, segregation, and cytokinesis in the trypanosome cell cycle. J.Cell Biol. 128, 1163–1172.

Rosenbaum, J.L., Witman, G.B., 2002. IntraXagellar transport. Mol. Cell.Biol. 3, 813–825.

Sherwin, T., Gull, K., 1989. The cell division cycle of Trypanosoma bruceibrucei: timing of event markers and cytoskeletal modulations. Philos.Trans. R. Soc. Lond. B. Biol. Sci. 323, 573–588.

Souto-Padron, T., Reyes, M.B., Leguizamon, S., Campetella, O.E., Frasch,A.C., de Souza, W., 1989. Trypanosoma cruzi proteins which are anti-genic during human infections are located in deWned regions of the par-asite. Eur. J. Cell Biol. 50, 272–278.

Vickerman, K., 1969. On the surface coat and the Xagellar adhesion in try-panosomes. J. Cell Sci. 5, 163–193.

Webster, P., Russell, D.G., 1993. The Xagellar pocket of trypanosomatids.Parasitol. Today 9, 201–206.

Weise, F., Stierhof, Y.-D., Kuhn, C., Weise, M., Overath, P., 2000. Distribu-tion of GPI-anchored proteins in the protozoan parasite Leishmania,based on an improved ultrastructural description using high-pressurefrozen cells. J. Cell Sci. 113, 4587–4603.

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

Young, S.J., Royer, S.M., Groves, P.M., Kinnamon, J.C., 1987. Three-dimensional reconstruction from serial micrographs using an IBM PC.J. Electron Microsc. Tech. 6, 207–215.

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

Documents

Transcript of The flagellar attachment zone of Trypanosoma cruzi epimastigote forms