Trophozoites of Giardia lamblia may have a Golgi-like structure

A. Lanfredi-Rangel a;b, W.M. Kattenbach c, J.A. Diniz Jr. b;d, W. de Souza a;b;*a Laboratorio de Biologia Celular e Tecidual, Centro de Biocieªncias e Biotecnologia, Universidade Estadual do Norte Fluminense,

Av. Alberto Lamego, 2000, 28015-620, Campos dos Goytacazes, Rio de Janeiro, Brazilb Laboratorio de Ultraestrutura Celular Hertha Meyer, Programa de Biologia Celular e Parasitologia, Instituto de Biof|sica Carlos Chagas

Filho, Universidade Federal do Rio de Janeiro, Av. Brig. Trompowski s/n., bloco G, Cidade Universitaria, CEP 21949-900 Rio de Janeiro, Brazilc Departamento de Histologia e Embriologia, Universidade Estadual do Rio de Janeiro, Av. Prof. Manoel de Abreu 48,

20550-170 Rio de Janeiro, Brazild Laboratorio de Microscopia Eletroªnica, Instituto Evandro Chagas, Avenida Almirante Barroso 492, 66090-000 Belem, Brazil

Received 20 July 1999; received in revised form 8 September 1999; accepted 16 October 1999

Abstract

Trophozoites of the primitive protozoan Giardia lamblia have been considered as cells which do not present the Golgicomplex. Using C6-NBD ceramide, which has been shown to label the Golgi complex of mammalian cells, labelling of theperinuclear region of G. lamblia was observed by confocal laser scanning microscopy. Transmission electron microscopy of thinsections and of replicas of freeze-fractured cells revealed the presence of concentric perinuclear membranes resembling theGolgi complex. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rightsreserved.

Keywords: Golgi structure; Endoplasmic reticulum; Electron microscopy; Cytochemistry; Giardia lamblia

1. Introduction

Eukaryotic cells are usually highly compartmental-ized, containing the nucleus and several organellessuch as mitochondria, peroxisomes, lysosomes, etc.In addition, an elaborate endomembrane systemformed by the endoplasmic reticulum (ER) and theGolgi cisternae and vesicles give to the cell cytoplasma certain complexity in its structural organization.The analysis of the ¢ne structure of several anaerobicprotozoa has shown that some of them lack typical

organelles such as peroxisomes and mitochondria, asoccurs with Entamoeba, Trichomonas and Giardia [1^3]. In addition, Entamoeba and Giardia lack a typicalGolgi complex with organized and parallel cisternae,although a membrane system resembling this organ-elle was described in Entamoeba [13]. Members ofthis group of organisms have been considered to beliving relics of the earliest phase of eukaryotic evo-lution and it was postulated that they have divergedfrom a common eukaryotic ancestor before the sym-biotic origination of mitochondria or other energy-converting organelles [4]. Other specialized structuressuch as the hydrogenosome in Trichomonas [5^7] andthe peripheral vacuolar system in Giardia [8] arealso of interest from an evolutionary point of view.

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 4 6 - 7

* Corresponding author. Tel. : +55 (21) 2602364;Fax: +55 (21) 2602364; E-mail: wsouza@domain.com.br

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Giardia and Trichomonas have been considered asexcellent models for evolutionary studies becauseTrichomonas may have a modi¢ed mitochondrion,the hydrogenosome, and Giardia may have gainedand lost a mitochondrion structure [9,10]. Even theendomembrane system formed by the ER and theGolgi varies signi¢cantly among these cells. For in-stance, a highly developed Golgi is seen in Tricho-monas [11,12] while a typical Golgi is not observed inEntamoeba and Giardia trophozoites [13^15].

Previous morphological studies indicate that thetrophozoite form of the most primitive eukaryoteGiardia lamblia does not present a typical Golgi[16,17], although this organelle can be visualized dur-ing the process of transformation of trophozoitesinto cystic forms [15,18]. In this study, we studiedsome aspects of endomembrane structures of an im-portant intestinal parasite of humans, G. lamblia.Confocal laser scanning microscopy of cells labelledwith C6-NBD ceramide, a probe widely used to iden-tify the Golgi complex of mammalian cells [19], con-¢rmed the presence of a Golgi-like structure introphozoites. Transmission electron microscopy ofthin sections and replicas of quick frozen, freeze-fractured and deep-etched cells revealed the endo-membrane system of trophozoites of G. lamblia.The results obtained are described in this paper.

2. Materials and methods

2.1. Parasites

Trophozoites of the Portland-1 strain of G. lamblia[20] were cultivated in TYI-S-33 medium supple-mented with 10% heat-inactivated bovine serumand 0.1% bovine bile at 37³C [21]. Subcultureswere made twice a week and vials containing cellsthat had grown for 72 h (late exponential phase)were incubated on ice at 4³C for 15 min. The freeparasites were harvested by centrifugation at 500Ugfor 7 min and washed three times in 0.1 M phos-phate bu¡er (pH 7.2) plus 0.85% NaCl (PBS).

2.2. Fluorescence microscopy

Trophozoites were collected by centrifugation,washed in 199 medium without bovine serum as de-

scribed above and allowed to adhere to coverslipspreviously coated with 0.1% poly-L-lysine in PBS.Then, the living cells were incubated in a solutioncontaining 0.9 Wl ml31 C6-NBD ceramide (MolecularProbes), 0.68 Wg ml31 bovine albumin (Sigma Chem-ical) in 199 medium, pH 7.2, for 10 min at 37³C inthe dark. Subsequently, the cells were washed severaltimes in 199 medium and incubated again for 20 minas described above. Then, the coverslips containingthe cells were mounted and observed in a ZeissConfocal Laser Scanning Microscope (Laser 488/LP515).

2.3. Transmission electron microscopy

The cells were collected by centrifugation at500Ug for 7 min, washed three times in PBS (pH7.2) and ¢xed in a solution containing 2.5% glutar-aldehyde in 0.1 M cacodylate bu¡er, pH 7.2. There-after, the cells were washed as described above andpost-¢xed for 60 min at 4³C in a solution containing1% osmium tetroxide, 0.8% potassium ferrocyanidein 0.1 M cacodylate bu¡er. Subsequently, the cellswere washed three times in bu¡er, dehydrated inacetone and embedded in Epon. Thin sections werestained with uranyl acetate and lead citrate and ob-served in a Zeiss 900 transmission electron micro-scope.

2.4. Localization of glucose-6-phosphatase

The cells were collected by centrifugation, washedand then ¢xed for 30 min at 4³C in a solution con-taining 1% glutaraldehyde and 1% formaldehyde in0.1 M cacodylate bu¡er, pH 7.2. After that, the cellswere softly scraped o¡ and incubated for 30 min at37³C in medium designed for localization of glucose-6-phosphatase activity, which consisted of 5 mMMgCl2, 4 mM CeCl2, 5 mM glucose-6-phosphatasein a 0.06-M Tris-maleate bu¡er, pH 6.5 [22]. Afterincubation, the cells were washed with cacodylatebu¡er, post-¢xed with osmium tetroxide and proc-essed for transmission electron microscopy as de-scribed above.

2.5. Quick-freezing, freeze-fracture and deep-etching

The cells were collected by centrifugation at

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1500Ug for 10 min and ¢xed in 2.5% glutaraldehydein 0.1 M cacodylate bu¡er (pH 7.2) for 2 h at roomtemperature. After that, the cells were rinsed sixtimes in distilled water followed by centrifugationand small drops containing the cells were placed ona specimen support disk that was designed for theBalzers freeze-etch apparatus. The specimen diskswere a¤xed to the plunger of a Med Vac rapid freez-ing device assembled for `slam' freezing against acopper block which was cooled by liquid nitrogen.Subsequently, the specimen disk was rapidly trans-ferred to the Balzers apparatus operating at a vac-uum of 2U1036 Torr. Only one pass with the micro-tome knife was made so that the fracture plane wasabout 10 Wm from the surface of the impact to thecopper block. After fracturing at 3115³C, the tem-perature was raised to 3100³C for 5^10 min foretching. Platinum was evaporated onto the specimenat an angle of 15³ when the specimen was rotated.Carbon was evaporated at an angle of 90³. Replicaswere cleaned in a series of bleach and distilled water,mounted on 200-mesh grids and observed in a Zeiss900 electron microscope. Figures illustrating thistechnique were printed so that regions containingevaporated platinum were light and regions lackingmetal were dark [23].

3. Results and discussion

We used the £uorescent dye C6-NBD ceramide tolabel the trophozoites of G. lamblia. It has beenshown that it is internalized by the cells and cleavedand the £uorescent derivative ceramide accumulatesin the Golgi in higher eukaryotes, demonstrating evi-dence that sphingomyelin and glycosphingolipid syn-thesis take place in the Golgi [19,24]. In most of thecells examined up to now, C6-NBD ceramide stainedthe perinuclear region, where the Golgi complex isusually located. In the case of trophozoites ofG. lamblia, we observed a heavy staining in the re-gion around the two nuclei (Fig. 1). In some cells, acrescent staining pattern, resembling that reportedfor other cells which present a typical Golgi complex,was observed [25,26]. Lujan et al. [15], working with¢xed trophozoites, did not observe signi¢cant stain-ing of G. lamblia trophozoites incubated in the pres-ence of NBD ceramide. However, staining was ob-served in ¢xed encysting trophozoites. At present, wedo not have a clear explanation for the fact that weobserved staining of typical trophozoite forms. Weworked with trophozoites at the exponential phase ofgrowth. For this reason, we consider that most of thecells at this phase would not be in the encysting

Fig. 1. Trophozoites were incubated in a solution containing C6-NBD ceramide and observed in a confocal laser scanning microscope.Labelling of the perinuclear region is observed. a, phase contrast ; b, £uorescence microscopy. Bar, 7 Wm.

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form. It is important to point out that activity forUDP N-acetyl-D-galactosamine transferase could bedetected in trophozoites [26].

Transmission electron microscopy of thin sectionsrevealed the presence of a parallel array of 5^8 cis-ternae resembling a Golgi complex in the perinuclearregion of many cells (Fig. 2a). Occasionally, theyshowed a concentric organization, leaving a lessdense region where glycogen particles concentrate

(Fig. 2b). Perinuclear Golgi-like stacks were also re-ported by McCa¡ery and Gillin [18].

We also observed the presence of a signi¢cantnumber of pro¢les of the ER that was especiallyevident when the cells were incubated in a mediumdesigned for the detection of glucose-6-phosphatase,a classical enzyme marker of the ER [22] (Fig. 2c).The ER cisternae were seen in connection with themembrane of the nuclei and irradiating towards the

Fig. 2. Thin sections of conventionally processed (a,b) and of cells incubated in a cytochemical medium designed for localization of glu-cose-6-phosphatase. In conventional thin sections, it was possible to observe cisternae in a parallel array (a), some of which in a concen-tric array, surrounding a less dense region rich in glycogen particles. Typical pro¢les of the ER can be seen after labelling for glucose-6-phosphatase (small arrows in c), even in the nuclear membrane. In some cells, strongly labelled cisternae in a parallel array are evident(arrowhead in c and arrow in d). a: Bar, 550 nm. b: Bar, 150 nm. c: Bar, 600 nm. d: Bar, 350 nm.

CFig. 3. Trophozoites of G. lamblia submitted to the quick frozen, freeze-fractured, deep-etched and rotary-replicated. Concentric mem-branes located around the nucleus could be well visualized. N, nucleus. Bars, 100 nm.

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dorsal and ventral portion of the protozoan. Prox-imity of the cisternae with the adhesive disc wasevident (Fig. 2d). At some regions, parallel and or-ganized glucose-6-phosphatase positive cisternaewere observed (Fig. 2c,d). They showed a organiza-tion typical of the Golgi complex. Although this en-zyme has been considered as a typical marker of theER of mammalian cells [22], it was also observed inthe Golgi complex of Tritrichomonas foetus [27].

The presence of a parallel array of perinuclearmembranes resembling a Golgi complex could bebetter visualized in replicas of quick frozen, freeze-fractured, deep-etched and rotary-replicated cells(Fig. 3a,b). In favorable fracture planes, connectionsbetween the cisternae, resembling those reported inthe Golgi of T. foetus [11], were observed.

Taken together, the available data indicate thattrophozoites of G. lamblia possess membrane cister-nae, mainly localized at the periphery of the nucleithat corresponds to elements of the Golgi complex.These cisternae seem to be more stable during theencystation process. At present, it is not completelyclear what factors are involved in the assembly andmaintenance of an intact Golgi complex. Studies us-ing drugs such as brefeldin A have shown that it ispossible to disorganize the Golgi complex of mam-malian cells into vesicles which are distributedthroughout the cytoplasm [28]. Even when cells entermitosis, the Golgi complex disassembles [29].GTPases such as rab 1 seem to be involved in themaintenance of the Golgi complex [30]. It is possiblethat organisms such as Entamoeba and Giardia lackmolecules involved in the maintenance of a morestable association of membranes which make a typ-ical Golgi complex.

Acknowledgements

We are very grateful to Joa¬o Carlos de AquinoAlmeida for the assistance to use the confocal laserscanning microscope. This work has been supportedby Conselho Nacional de Desenvolvimento Cient|¢-co e Tecnologico (CNPq), Financiadora de Estudose Projetos (FINEP), Fundac°a¬o de Amparo a© Pesqui-sa do Estado do Rio de Janeiro (FAPERJ), Progra-ma de Nucleos de Exceleªncia (PRONEX) and Fun-dac°a¬o Estadual do Norte Fluminense (FENORTE).

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