Experimental Parasitology 119 (2008) 411–417

Contents lists available at ScienceDirect

Experimental Parasitology

journal homepage: www.elsevier .com/ locate/yexpr

Acanthamoeba castellanii: Identification and distribution of actin cytoskeleton

Arturo González-Robles a,*, Guadalupe Castañón a, Verónica Ivonne Hernández-Ramírez a,Lizbeth Salazar-Villatoro a, Mónica González-Lázaro b, Maritza Omaña-Molina c, Patricia Talamás-Rohana a,Adolfo Martínez-Palomo a

a Department of Experimental Pathology, Center for Research and Advanced Studies, Mexico City, Mexicob Department of Cell Biology, Center for Research and Advanced Studies, Mexico City, Mexicoc Faculty of Superior Studies, UNAM, Iztacala, Tlalnepantla State of Mexico, Mexico

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 October 2007Received in revised form 28 February 2008Accepted 7 April 2008Available online 12 April 2008

Index Descriptors and Abbreviations:Acanthamoeba castellaniiFree-living amoebaeCytoskeletonActinCryo-electronmicroscopy

0014-4894/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.exppara.2008.04.004

* Corresponding author. Address: CINVESTAV-IPNNacional 2508, San Pedro Zacatenco, 07360 Mexico5061 3396.

E-mail address: goroa@cinvestav.mx (A. González-

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmi-croscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks ofactin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endo-cytic structures. In addition, the comparision between total actin content in whole extracts among differ-ent amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa forNaegleria fowleri and Entamoeba histolytica.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Acanthamoebae are ubiquitous free-living amoebae. Acantha-moeba castellanii is recognized as the etiological agent of chronicgranulomatous amebic encephalitis, a fatal disease of the centralnervous system (Martínez, 1980) and amoebic keratitis, a progres-sive and painful sight-threatening infection of the eyes (Butleret al. 2005; Illingworth and Cook, 1998; Niederkorn et al., 1992).Cytoskeleton in eukaryotic cells is formed by actin containing fila-ments, microtubules formed by tubulin, and intermediate fila-ments constituted by diverse proteins. Actin filaments andmicrotubules form the basis of almost all major cell movements.

Particularly, it is accepted that actin cytoskeleton plays a struc-tural and dynamic role in diverse functions of the cell such as adhe-sion (Gumbiner, 1996), motility (Bretscher, 1991; Condeelis, 1993;Kabsch and Vandekerckhove, 1992; Mitchison, 1995), and phago-cytosis (Swanson and Baer, 1995) and other processes. The actin-rich cytoskeleton of the trophozoites allows rapid morphologicalchanges in response to signals from outside stimuli. During loco-motion, coordinated actin assembly occurs near the plasma mem-brane creating lamellipodia and filopodia; these structures, alongwith the movements of the amoebae play an important function

ll rights reserved.

, Av. Instituto Politécnico, D.F., Mexico. Fax: +52 55

Robles).

in the first steps of the cytopathic effect. Here, by means of lightand electron microscopy, we describe the localization and distribu-tion of actin in A. castellanii trophozoites, and compare the actincontent in total extracts from different amoebae.

2. Materials and methods

2.1. Cells

2.1.1. AmoebaeAcanthamoeba castellanii trophozoites, were originally isolated

from a contact lens associated with a human case of keratitis(Association to prevent blindness in Mexico, Luis Sánchez BulnesHospital, Mexico City). The sample was grown and maintained inaxenic culture in 2% Bacto Casitone (Pancreatic digest of casein,Becton–Dickinson, Sparks, MD) supplemented with 10% fetal bo-vine serum (Gibco, Grand Islands, NY). Cultures were incubatedat 26 �C in borosilicate tubes (Pyrex, Mexico).

2.1.2. MDCK cellsMonolayers of epithelial cells of the established MDCK line of

canine kidney origin (Madin Darby Canine Kidney) were grownon 25 cm2 cell culture flasks (Corning Incorporated, NY) in Dul-becco’s modified Eagle’s medium (Microlab, Mexico). They weresupplemented with 10% fetal bovine serum (Gibco, Grand Islands,NY) and antibiotics in a 5% CO2 atmosphere at 37 �C.

412 A. González-Robles et al. / Experimental Parasitology 119 (2008) 411–417

Confluent monolayers were incubated in the same conditions ina mixture of Bactocasitone and Dulbecco’s modified Eagle’s med-ium in equal proportions and A. castellanii trophozoites were addedin a 1:2 target cell: amoeba ratio.

In a parallel assay, freshly trypsinized MDCK cells were incu-bated for 1–2 h with the amoebae in a 1:1 ratio.

2.2. Electron microscopy

Fast freeze-fixation followed by freeze substitution. Axenicallycultured amoebae in logarithmic phase of growth were pelletedand placed into the hole of a 7 mm diameter anti-adhesive plasticring, positioned on a foam rubber support and rapidly frozen on acopper mirror pre-cooled at liquid nitrogen temperature (�197 �C)using a Reichert KF-80 unit. Freeze substitution was achieved witha Reichert CS Auto System in acetone containing 4% osmiumtetroxide for 48 h at �80 �C. Afterwards, samples were slowlybrought to room temperature at a rate of 4 �C/h, transferred toabsolute ethanol, infiltrated and embedded in LR white resins. Thinsections were stained with uranyl acetate and lead citrate.

2.2.1. Cryo-fixationTrophozoites were fixed with a mixture of 4% paraformalde-

hyde/0.5% glutaraldehyde for 1 h at room temperature, embeddedin 2.3 M sucrose in 2.5% polyvinylpyrrolidone and frozen in liquidnitrogen.

2.2.2. Immunogold labelingCryo-fixed and freeze-substituted thin sections were placed on

formvar-coated nickel grids and incubated for 1 h in 10% fetal bo-vine serum and 0.12%. glycine. Then, sections were washed 5�with Dulbecco’s Phosphate Buffered Saline (DPBS) and 0.12%, gly-cine and incubated with anti-actin antibody (Amersham Biosci-ences, USA) diluted 1:100 in 5% bovine serum albumin for 2 h.Afterwards, sections were washed 5�with DPBS and 0.12% glycine,and incubated with goat-anti mouse IgG antibody (1:40) conju-gated with 15 nm colloidal gold particles (Ted Pella Redding, CAUSA) for 1 h. Finally, sections were washed 5� with DPBS and0.12% glycine, deeply rinsed in distilled water and stained withuranyl acetate and lead citrate.

Observations were performed with a Morgagni 268 D transmis-sion electron microscope, FEI Company, Eindhoven, TheNetherlands.

2.2.3. Scanning electron microscopySamples were fixed with 2.5% glutaraldehyde in 0.1 M sodium

cacodylate buffer pH 7.2, dehydrated with increasing concentra-tions of ethanol and critical point dried (31 �C and 1100 psi) usinga Samdri apparatus (Tousimis), and coated with gold particles in anion sputtering device (JEOL JFC-1100). They were then examinedwith a Philips XL-30 ESEM scanning electron microscope, FEI Com-pany, Eindhoven, The Netherlands.

2.3. Fluorescence microscopy

Amoebae cultured for 48 h were chilled in an ice-water mixturefor 5 min, pelleted by centrifugation and fixed with 4% paraformal-dehyde for 1 h, then permeabilized 15 min with 0.2% Triton X-100,washed with DPBS and blocked 30 min with fetal bovine serum di-luted in DPBS. Afterwards, the cells were washed again and treatedwith 1:40 phalloidin-tagged rhodamine complex (MolecularProbes, Eugene, OR, USA) for 20 min washed exhaustively withDPBS, and mounted on glass slides with Vectashield resin (VectorLaboratories Inc., Burlingame, CA, USA). To observe trophozoitesattached to the substratum, some cells were placed on square glasscover slips and after 1 h samples were treated as mentioned above.

Observations were done in a fluorescence equipped Axiophotphoto-microscope (Carl Zeiss, Germany). Photographs were ob-tained with a Zeiss AxioCam MRc digital camera (Carl Zeiss VisionGmbH, Germany).

2.4. Preparation of cell lysates, electrophoresis and Western blot

Total extracts prepared from an equivalent number of cellswere run in 10% SDS–PAGE electrophoresis gel (Bio-Rad, Hercules,CA) under reducing conditions. For lysates preparation, trophozo-ites (1 � 106) were washed with phosphate-buffered saline, andlysed with 1 ml of lysis buffer (10 mM Tris–HCl, pH 7.4,150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 3 mM NEM, IA, andTLCK and 10 lM phalloidin). Proteins were then transferred ontonitrocellulose membranes (Bio-Rad, Hercules, CA), blocked with5% non-fat dry milk in TBS-T for 1 h at room temperature, thenwashed and incubated 2 h at room temperature with an anti-ac-tin antibody (Lessard, 1988) (1:5000, clone C4, Chemicon). Mem-branes were washed with TBS-T, and then incubated with goat-anti mouse IgG (H + L) conjugated to horseradish peroxidase(1:1000) for 1 h at room temperature. After washing with TBS-T, antibody-reactive actin was detected by chemiluminescenceusing the substrate Super Signal (Pierce, Rockford, IL) accordingto the manufacture’s instructions. Different concentrations (2, 4,6 and 10 lg) of rabbit muscle actin (Worthington, Lakewood,NJ) were analyzed by SDS–PAGE 10% and Western blot. The stan-dard curve was incubated with monoclonal antibody C4 (1/5000)under the same conditions described above. The complex anti-body–antigen was developed using chemiluminescence. The den-sitometric analysis of rabbit muscle actin was used to calculatethe total actin in the whole extracts of amoebae.

2.5. Cytoskeleton preparation

The amoebae were disrupted with 40 mM Tris–HCl pH 6.8 con-taining 3 mM NEM, IA, and TLCK and 1 mM PMSF, 2.5% Triton X-100 and 10 lm phalloidin. The cell suspension was centrifuged at10,000g for 10 min at 4 �C. Soluble fraction was separated fromthe pellet (cytoskeleton); the pellet was washed three times with500 ll of washing buffer (same as above except Triton X-100 andphalloidin) and was resuspended in 50 ll of the same buffer. Alic-uots were separated and used to determine protein concentrationby the DC method (Bio-Rad).

3. Results

3.1. Transmission electron microscopy

Fast-freeze fixation followed by cryo-substitution, an im-proved preservation of the whole cell, made it possible to estab-lish the ultrastructural characterization of the cytoplasm ofaxenically cultured trophozoites of A. castellanii. The presenceof a densely packed fibrogranular matrix, evenly distributed allover the cytoplasm, and the appearance of a finely granular con-tent in many vesicles and vacuoles, most of which showed asharply circular outline, was observed. In many ectoplasmicareas corresponding to lamellae, a fine mesh of fibrogranularmaterial formed by bundles and networks of microfilamentsapproximately 8 nm in diameter was found (Fig. 1A). Samples,either cryo-substituted or cryo-sectioned and treated with theanti-actin monoclonal antibody, were positive mainly in theectoplasmic areas near the plasma membrane and in the acan-thopodia where actin was present (Fig. 1B and D). No labelwas found when samples were incubated only with the second-ary antibody.

Fig. 1. Transmission electron microscopy of A. castellanii trophozoites treated with different cryo-techniques. (A–C) Cryo-substitution. (D–E) Cryo-sections. (A) The cytoplasmof the amoeba was observed with an excellent preservation, where small bundles of actin filaments were present at the periphery of the cell (arrowheads). Bar = 0.5 lm. (B,D)Actin labeled with monoclonal anti-actin antibody followed by secondary antibody conjugated to gold particles. (B) Bar = 0.1 lm. (D) Bar = 0.5 lm. (C,E) Control cryo-sectionsthat were incubated only with the secondary antibody. (C) Bar = 0.1 lm. (E) Bar = 0.5 lm.

A. González-Robles et al. / Experimental Parasitology 119 (2008) 411–417 413

3.2. Immunofluorescence microscopy

Trophozoites attached to the substratum treated with phalloi-din–rhodamine complex showed a strong reaction to this markerin wide cytoplasmic projections such as lamellae and acanthopodiawhich form during cell motion (Fig. 2A). Meanwhile, in roundedamoebae the reaction to the specific probe was observed as irreg-ular ‘‘patches” uniformly distributed all over the cell surface(Fig. 2C).

3.3. Interaction with MDCK cells

When trophozoites were co-incubated for 1 h with freshly tryp-sinized epithelial MDCK cells or monolayers grown to confluence,trophozoites interacted with them in different ways. A characteris-tic feature of the interaction with individualized cells was the for-mation of slender phagocytic cylindrical structures in which actinwas present (Fig. 3A), that formed few seconds after the interaction

between the amoeba and the target cell. Frequently, trophozoitesadhered, to the cell pulled out and ingested small portions of thetarget cell (Fig. 3B, E and F).

In contrast, when amoebae were co-incubated with MDCK epi-thelial cell monolayers, trophozoites made intimate contact withthe surface of target cells, sometimes detaching and damagingthem, and by means of food-cups, ingested injured cells. In addi-tion, actin was detected by immunofluorescence in the areas thatwere in close contact with the target cells (Fig. 3C). Based on trans-mission electron microscopy observations in longitudinal sectionsof phagocytic channels, actin became visible as a smooth compo-nent of fibrogranular material (Fig. 3F).

3.4. Electrophoresis and immunoblotting

Fig. 4 shows the profile of proteins from total extracts of the dif-ferent amoebae analyzed in this study; the pattern of each oneshowed significant differences. E. histolytica presented a marked

Fig. 2. (A,C) Fluorescence microscopy of A. castellanii trophozoites after co-incubation with anti-actin antibody conjugated to phalloidin/rhodamine complex. (A) Amoebaeattached to the substratum presented a strong positive reaction in the lamellae and acanthopodia (arrow). (C) Rounded trophozoite showed the reaction all over the cellsurface. (B,D) Phase contrast images. Bar = 10 lm.

414 A. González-Robles et al. / Experimental Parasitology 119 (2008) 411–417

complexity in the concentration and molecular weight of the pro-teins observed by Coomassie stain. In free living amoebae, severalhigh molecular weight proteins were present; nevertheless a44 kDa protein predominated in A. castellanii. Similar results werefound for N. fowleri, however the molecular weight of the predom-inant protein was 45 kDa. The analysis of the replica of this mate-rial by Western blot using the C4 anti-actin monoclonal antibody isshown in Fig. 4B. Proteins with molecular weights from 44 to45 kDa were recognized with the C4 antibody. Surprisingly, Naegle-ria lovaniensis was not positive to the C4 antibody.

The total concentration of actin present in each one of theorganisms studied was quantified by comparison of densitometricanalysis of a Western blot of commercial actin as well as the Wes-tern blot of the different species of amoebae. The concentration ofactin present in E. histolytica was 10 lg/15 � 103 cells, 9.2 lg/10 � 103 cells in N. fowleri and 8.8 lg/15 � 103 cells in A. castellanii.

As the total extract of N. lovaniensis was negative with the C4anti-actin antibody, we decided to further explore the reasonwhy the antibody does not recognize any protein in this extract.In order to determine whether the absence of signal was proteinconcentration dependent, and to enrich actin content, actin cyto-skeleton fractions were prepared from increasing number of cells(104,105 and 106). As shown in Fig. 4C, actin was present in cyto-skeleton fractions of N. lovaniensis, whereas actin was absent fromsoluble fractions (data not shown). Densitometric analysis con-firmed the protein concentration dependence for actin detectionwith the C4 anti-actin antibody.

4. Discussion

The pathogenesis of Acanthamoeba infections and the biochem-ical determinants responsible for the virulence of this pathogen re-main poorly understood. Adhesion of Acanthamoeba trophozoitesto host cells is thought to be a primary step mediated by a man-nose-binding protein (MBP) expressed on the surface of the cell(Garate et al., 2004). However, to date there are no available re-ports regarding the role of the actin cytoskeleton in the adhesionprocess.

Actin is described as one of the most abundant and highly con-served proteins in eukaryotic cells. This protein is involved in manyvital cellular functions such as cell motility (Pollard and Borisy,2003), organelle transport (Snider et al., 2004), endocytosis (Baderet al., 2004) and exocytosis (Yarar et al., 2005). In eukaryotic cellsthe cytoskeleton is a dynamic group of proteinaceous structuresthat continuously change as cells move and divide. During cellmovement, prearranged actin assembled close to the plasma mem-brane as bundles and networks providing a framework that allowsrapid changes in morphology which produce cell protrusions suchas lamellipodia and filopodia. Early work on the characterization ofcytoplasmic actin isolated from A. castellanii reported that 20% ofthis protein was recovered from cells extracts (Gordon et al., 1976).

A previous report on A. castellanii ameboid locomotion pointedout the participation of filopodia and acanthopodia in adhesionprocesses (Preston and King, 1984). However the contribution ofactin in these structures had not been proposed previously.

In this study, we have shown the presence and distribution ofthe actin cytoskeleton in A. castellanii by means of specific anti-ac-tin antibodies using fluorescence and transmission electronmicroscopy techniques. Actin fibers and networks were foundmainly in cytoplasmic locomotion structures as lamellipodia (flat-tened fan-shaped pseudopodia) and acanthopodia, (long slendercytoplasmic processes) and in diverse endocytic structures thatsometimes extend far away from the cell body.

Additionally, we detected actin in total extracts in A. castellaniiand in other amoebae species, as E. histolytica and N. fowleri.Although the apparent molecular weight of A. castellanii is slightlyhigher than the one of E. histolytica, these amoebae have similarconcentrations of actin, suggesting that this protein in A. castellaniicould play an important role in many cellular processes. The C4anti-actin antibody was able to recognize a protein with differentmolecular weight in the samples tested; in E. histolytica and N. fow-leri it recognized a 45 kDa molecule whereas in A. castellanii a44 kDa protein was observed. Surprisingly, the C4 anti-actin anti-body was not able to detect a protein in the total extract of N.lovaniensis; however when actin enriched fractions were analyzed,the antibody recognized a band of the expected molecular weight,

Fig. 3. (A,C) Fluorescence pictures of interaction between A. castellanii trophozoites and MDCK cells in culture. When fresh trypsinized MDCK cells (A) and confluent cellmonolayers (C) were co-incubated with amoebae, the reaction to actin was clearly detected in the contact areas and also in the endocytic structures (arrowheads). (B,D) Phasecontrast images. (A–D) Bar = 20 lm. (E) Scanning electron microscopy. Low magnification of the interaction between low platted MDCK target cells and trophozoites after 1 h.When slender phagocytic cylindrical structures of the amoeba touched the epithelial cell, it usually ingested a portion of the cell surface. Bar = 10 lm. (F) Similar image to (E)by transmission electron microscopy where the phagocytic channel of the trophozoite is clearly composed of a fine fibrogranular material. Bar = 0.5 lm. Ac, Acanthamoebacastellanii trophozoites; MDCK, target epithelial cells in culture.

A. González-Robles et al. / Experimental Parasitology 119 (2008) 411–417 415

and which concentration was dependent on the number of cells.Therefore, the absence of recognition in the case of the total ex-tracts of N. lovaniensis was due to a comparatively, very lowamount of this protein.

In E. histolytica, structures formed by polymerized actin areabundant especially when trophozoites are cultured on fibronectinsubstrates (Talamás-Rohana and Ríos, 2000). In the presence of tar-get cells, a fast polymerization of amoeba actin initiates at the siteof the target contact (Bailey et al., 1985). In addition actin polymer-ization is essential for cytolytic and phagocytic activities of thisparasite (Bailey et al., 1985). On the contrary, Wetzel et al.(2003) reported that Toxoplasma gondii uses mostly monomeric ac-tin to entry host cells.

The functional diversification of actin structures may be ac-counted for the diversification of actin. The number of actin genesin a species can vary widely (Hightower and Meagher, 1986). In

E. histolytica a number of actin and actin related proteins (ARP)genes have been reported (Loftus et al., 2005). In A. castellanii agene that codifies for actin (actin gene I) which is expressedin vivo (Nellen and Gallwitz, 1982) has been isolated and com-pletely sequenced.

In summary, our observations demonstrate that the A. castellaniicytoskeleton is largely made of fibers and networks of actin andwere primarily found in cytoplasmic locomotion structures suchas lamellipodia and in diverse endocytic structures.

In addition, we identified actin in total extracts in A. castellaniitrophozoites and found that regardless the difference in size com-pared with E. histolytica, these amoebae have similar concentra-tions of this protein. It will be necessary to study the externalsignals that trigger the initial steps in the organization and regula-tion of the cytoskeleton elements by means of which trophozoitesacquire motility, attachment and entrance in host cells.

N. lovaniensis

E. histolytic

a

A. castella

nii

N. fowleri

A

45

94

66

31

14

kDa

0

2

4

6

8

10

12

E. histolytic

a

A. castella

nii

N. fowleri

N. lovaniensis

To

tal a

ctin

(μg

/15

X 1

03 ce

lls)

E. histolytic

a

A. castella

nii

N. fowler

i

N. lova

niensis

Total extractsB

C Cytoskeleton fractions

1 2 3

N. lovaniensis

To

tal a

ctin

(μg

)

0

2

4

6

1 2 3

CellNumber

(1) 104

(2) 105

(3) 106

Total extracts

Fig. 4. Identification and characterization of actin in A. castellanii and in other amoebae. (A) Coomassie blue staining of total extracts of different amoebae. Molecular weightsmarkers are shown on the left column. (B) Western blot analysis of total extracts of different amoebae with the C4 anti-actin antibody. Actin concentration on these sampleswas quantified using a standard curve of commercial actin by densitometric analysis. The graph represents the average of three different experiments. (C). Western blotanalysis of cytoskeleton fractions of N. lovaniensis with the C4 anti-actin antibody. Actin concentration was determined as described in (B).

416 A. González-Robles et al. / Experimental Parasitology 119 (2008) 411–417

References

Bader, M.F., Doussau, F., Chasserot-Golaz, S., Vitale, N., Gasman, S., 2004. Couplingactin and membrane dynamics during calcium regulated exocytosis: a role forRho and ARF GTPases. Biochimica et Biophysica Acta 1742, 37–49.

Bailey, G.B., Day, D.B., Gasque, J.W., 1985. Rapid polymerization of E. histolytica actininduced by interaction with target cells. Journal of Experimental Medicine 162,546–558.

Bretscher, A., 1991. Microfilaments structure and function in the corticalcytoskeleton. Annual Review of Cell Biology 7, 337–374.

Butler, T.K., Males, J.J., Robinson, L.P., Wechsler, A.W., Sutton, G.L., Cheng, J., Taylor,P., McClellan, K., 2005. Clinical and Experimental Ophthalmology 33, 41–46.

Condeelis, J., 1993. Life at the leading edge: the formation of cell protrusions.Annual Review of Cell Biology 9, 411–444.

Garate, G., Cao, Z., Bateman, E., Panjwani, N., 2004. Cloning and characterization of anovel manose-binding protein of Acanthamoeba. Journal of Biological Chemistry279, 29849–29856.

Gordon, D.J., Eisenberg, E., Korn, E.D., 1976. Characterization of cytoplasmic actinisolated from A. castellanii by a new method. Journal of Biological Chemistry251, 4778–4786.

Gumbiner, B.M., 1996. Cell adhesion: the molecular basis of tissue architecture andmorphogenesis. Cell 84, 345–367.

Hightower, R.C., Meagher, R.B., 1986. The molecular evolution of actin. Genetics114, 315–332.

Illingworth, C.D., Cook, S.D., 1998. Acanthamoeba keratitis. Survey of Ophthalmology42, 493–508.

Kabsch, W., Vandekerckhove, J., 1992. Structure and function of actin. AnnualReview of Biophysics and Biomolecular Structure 21, 49–76.

Lessard, J.L., 1988. Two monoclonal antibodies to actin: one muscle selective andone generally reactive. Cell Motility and the Cytoskeleton 10, 349–362.

Loftus, B., Anderson, L., Davis, R., Alsmark, U.C.M., Samuelson, J., Amedeo, P.,Roncaglia, P., Berriman, M., Hirt, R.P., Mann, B.J., Nozaki, T., Suh, B., Pop, M.,Duchene, M., Ackers, J., Tannich, E., Leippe, M., Hofer, M., Bruchhaus, I.,Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z.,Harris, B., Harris, D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares,R., Whitehead, S., Quall, A.M., Rabbinowitsch, E., Norbertczak, H., Price, C.,Wang, Z., Guillén, N., Gilchrist, C., Stroup, S.E., Bhattacharya, S., Lohia, A.,Foster, P.G., Sicheritz-Ponten, T., Weber, C., Singh, U., Mukherjee, C., El-Sayed, N.M., Petri Jr., W.A., Clark, C.G., Embley, T.M., Barrel, B., Fraser, C.M.,Hall, N., 2005. The genome of the protist parasite Entamoeba histolytica.Nature 433, 865–868.

Martínez, A.J., 1980. Is Acanthamoeba encephalitis an opportunistic infection?Neurology 30, 567–574.

Mitchison, T.J., 1995. Evolution of a dynamic cytoskeleton. PhilosophicalTransactions of the Royal Society of London B 349, 299–304.

Niederkorn, J.Y., Ubelaker, J.E., McCulley, J.P., Stewart, G.L., Meyer, D.R., Mellon,J.A., Silvany, R.E., He, Y., Pidherney, M., Martin, J.H., Alizadeh, H., 1992.Susceptibility of corneas from various animal species to in vitro binding andinvasion by A. castellanii. Investigative Ophthalmology & Visual Science 33,104–112.

Nellen, W., Gallwitz, D., 1982. Actin genes and actin messenger RNA in A. castellanii.Nucleotide sequence of the split actin gene. Journal of Molecular Biology 159,1–18.

Preston, T.M., King, C.A., 1984. Amoeboid locomotion of Acanthamoeba castellaniiwith special reference to cell-substratum interactions. Journal of GeneralMicrobiology 130, 2317–2323.

Pollard, T.D., Borisy, G.G., 2003. Cellular motility driven by assembly anddisassembly of actin filaments. Cell 112, 453–465.

Talamás-Rohana, P., Ríos, A., 2000. Actin stress fibers in Entamoeba histolyticainduced by fibronectin. Archives of Medical Research 31, S131–S133.

A. González-Robles et al. / Experimental Parasitology 119 (2008) 411–417 417

Snider, J., Lin, F., Zahedi, N., Rodionov, V., Yu, C.C., Gross, S.P., 2004. Intracellularactin-based transport: How far you go depends on how often you switch.Proceedings of the National Academy of Sciences United States of America 101,13204–13209.

Swanson, J.A., Baer, S.C., 1995. Phagocytosis by zippers and triggers. Trends in CellBiology 5, 89–93.

Wetzel, D.M., Håkansson, S., Hu, K., Ross, D., Sibley, L.D., 2003. Actin filamentpolymerization regulates gliding motility by apicomplexan parasites. MolecularBiology of the Cell 14, 396–406.

Yarar, D., Waterman-Storer, C.M., Schmid, S.L., 2005. A dynamic actin cytoskeletonfunctions at multiple stages of clathrin-mediated endocytosis. MolecularBiology of the Cell 16, 964–975.