Characterization of Entamoeba histolytica α-actinin

Molecular & Biochemical Parasitology 145 (2006) 11–17

Characterization of Entamoeba histolytica ␣-actinin Ana Virel, Lars Backman ∗ Biochemistry, Ume˚a University, SE-901 87 Ume˚a, Sweden Received 27 June 2005; received in revised form 18 August 2005; accepted 7 September 2005 Available online 27 September 2005

Abstract We have cloned, expressed and characterized a ␣-actinin-like protein of Entamoeba histolytica. Analysis of the primary structure reveals that the essential domains of the ␣-actinin protein family are conserved: an N-terminus actin-binding domain, a C-terminus calcium-binding domain and a central helical rod domain. However, the rod domain of this Entamoeba protein is considerably shorter than the rod domain in ␣-actinins of higher organisms. The cloned Entamoeba 63 kDa protein is recognized by conventional ␣-actinin antibodies as well as binds and cross-links filamentous actin and calcium ions in the same manner as ␣-actinins. Despite the shorter rod domain this protein has conserved the most important functions of ␣-actinins. Therefore, it is suggested that this 63 kDa protein is an atypical and ancestral ␣-actinin. © 2005 Elsevier B.V. All rights reserved. Keywords: ␣-Actinin; Entamoeba histolytica; Actin

1. Introduction Entamoeba histolytica infection is estimated to cause between 50,000 and 100,000 deaths every year primarily in certain tropical and subtropical areas. Thereby is E. histolytica induced amoebiasis the third most common parasitic disease in humans after malaria and schistosomiasis [1–3]. Only one of the Entamoeba species humans can host is known to cause disease. The clinical manifestation of parasite infection is amoebic colitis as well as amoebic liver abscess and other extraintestinal lesions due to the spread of the parasite via the blood to other organs [4–6]. Due to the high incident of infection the study of the E. histolytica life cycle as well as the strategies and regulation of the invasion is of a great importance. The sequencing of the entire E. histolytica genome has been one of the most important approaches to understand and characterize the mechanism and proteins involved in infection [7]. The attachment and penetration of the parasite into the host cells requires not only a Gal/GalNAc lectin [8] but also reorganisation of cytoskeletal and actin-binding proteins [9–11] as well as extracellular cysteine proteases [12]. Some of these

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Corresponding author. Tel.: +46 90 786 5847; fax: +46 90 786 7661. E-mail address: [email protected] (L. Backman).

0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2005.09.003

E. histolytica proteins have been characterized, such as the calmodulin-like protein [13], myosin [14–16] and actin-binding proteins EhABPH and ABP-120 [17,18]. Although these proteins share some of the structural and functional properties with isoforms of higher eukaryotes they also have some atypical features. Indicating that some of its proteins may have remained unique or invariable during evolution. Previously we have found a possible actin-binding protein in the genome of E. histolytica, with high similarity to ␣-actinins. Phylogenetic analysis placed this ␣-actinin-like protein at the bottom of the evolution and identifying it as a possible ancestor to modern ␣-actinins [19]. ␣-Actinins is a ubiquitous actinbinding protein, present in most eukaryotic cells with the exception of plants and baker’s yeast (Saccharomyces cervisiae) [19]. The hallmark of ␣-actinin is the presence of three distinct structural domains: an N-terminus actin-binding domain composed of two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminus calcium-binding domain with EF-hand motifs [20,21]. The actin-binding domain, and to a slightly lesser extent, the calcium-binding domain have been conserved during evolution. The rod domain is the most variable part; the amino acid sequence varies not only among the different isoforms but also among the different spectrin repeats. In some primitive organisms like protozoa and yeast (Schizosaccharomyces pombe) the rod domain contains only one or two spectrin repeats instead of the usual four repeats [19].

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Due to the ability to form antiparallel dimers, ␣-actinin is able to cross-link actin filaments in a wide number of structures such as Z-disc, adhesion plaques, stress fibers and at the leading edge in motile cells [21]. In protozoan such as Acantamoeba and Trichomonas vaginalis, ␣-actinin is located mainly in the cortical areas mediating morphological changes during the infection [22,23] whereas in yeast (S. pombe), ␣-actinin appears to play an important role in the formation of the medial ring in cytokinesis [24]. Recently it has been shown that an Entamoeba ␣-actinin is attached to the Gal/GalNAc lectin, which is known to be important for the pathogenesis of the parasite [8]. Structure analysis of the E. histolytica ␣-actinin-like protein have predicted a protein with a conserved actin-binding domain with two calponin homology domains and a calcium-binding domain with three putative EF-hands. The predicted rod domain connecting these two domains is considerably shorter (ca. 145 residues) than in typical ␣-actinins. The phylogeny placed this ␣-actinin-like protein as one of the most ancestral ␣-actinins and suggested that the four spectrin repeats present in modern ␣-actinins arose after two consecutive intragenic duplications from a single repeat ␣-actinin. In addition, the phylogenetic analysis suggested that the rod domain is most similar to the first spectrin repeat of chicken ␣-actinin [19]. Here we report the cloning, expression and characterization of this ␣-actinin-like protein of E. histolytica. Biochemical analysis showed that the cloned 63 kDa protein is recognized by conventional ␣-actinin antibodies as well as binds filamentous actin and calcium ions in the same manner as ␣-actinins. Thus, despite the shorter rod domain this protein has conserved the most important functions of ␣-actinins.

0.5 mM and grown overnight (15 h) at 23 ◦ C. Cells were harvested by centrifugation, resuspended in 25 mM sodium phosphate, 300 mM NaCl, pH 7.6, and stored at −20 ◦ C until purification. The fusion protein was purified using nickel HiTrapTM affinity columns from Amersham Biosciences. Briefly, after sonication, incubation with detergent and centrifugation, the clear supernatant was loaded on the affinity column. Unbound material was eluted by 25 mM sodium phosphate, 300 mM NaCl, pH 7.6, containing 5 mM imidazole. Bound material was eluted by an imidazole gradient in the same buffer and dialyzed against 25 mM sodium phosphate, 300 mM NaCl, pH 7.6. Tobacco etch virus (TEV) protease (generously provided by Dr. David S. Waugh) was used to remove the His-tag. Affinity chromatography was used again to separate the His-tag. The final ␣actinin-like protein was concentrated using an Amicon ultra-15 centrifuge filter device. Protein concentration was determined by absorbance measurements at 280 nm, using a computed extinction coefficient of 1.3 ml g−1 for a 0.1% solution.

2. Material and methods

2.3. Actin co-sedimentation assays

2.1. Cloning, expression and isolation of a E. histolytica α-actinin

Actin was purified according to [26] from rabbit skeletal muscle and stored in G buffer (5 mM Tris–HCl, pH 8.0, 0.2 mM ATP, 0.2 mM CaCl2 , 0.5 mM ␤-mercaptoethanol). Actin was polymerized by adding KCl and MgCl2 to 100 and 1 mM, respectively, and incubated at room temperature for 45 min. The actin-binding properties of the ␣-actinin-like protein were analysed by a co-sedimentation assay. In a final volume of 150 ␮l, actin (4.5 ␮M) and varying amounts of ␣-actinin-like protein were mixed with EDTA or CaCl2 (1 mM final concentration) before polymerization by KC1 and MgCl2 at room temperature for 45 min. Samples were centrifuged at low-speed (13,000 rpm) in an Eppendorf centrifuge for 15 min. Fifteen percent SDS-PAGE was used to analyse protein in pellet and supernatant. Coomassie blue stained gels were scanned and quantified using ScionImage software.

The gene encoding the E. histolytica ␣-actinin-like protein gene was amplified using PCR Master Mix (Promega) from an E. histolytica genomic DNA library (generously provide by Dr. E. Tannich) using the following primers: 5 -tttggatccaacatgactggaaataaagaatggg (forward) and 5 -tttctcgagtcaaataagtccaagaactaagtt (reverse) containing BamHI and XhoI restriction sites, respectively. The resulting fragment was purified with QIAquick PCR purification kit (Qiagen) and digested with BamHI (Fermentas) and XhoI (Takara). The fragment was ligated into the pET-TEV, created by introducing a TEV protease cleavage site in pET-19b, producing the plasmid pET-TEVehabp. The sequence was verified using the ABI PRISM BigDye terminator cycle sequencing kit (Applied Biosystems). This plasmid was used to transform E. coli BL21(DE3) cells by heat shock. Cells were grown at 37 ◦ C in Luria-Bertani media containing 100 ␮g/ml carbencillin until mid-log phase (OD 600 ≈ 0.5). His-tagged protein expression was induced by addition of isopropyl thio-␤-d-galactoside to a final concentrations of

2.2. Calcium binding assay The binding of calcium was determined using 45 Ca autoradiography as described [25]. Different amounts of purified ␣actinin-like protein were blotted onto a PDVF membrane, previously equilibrated with buffer (60 mM KCl, 5 mM MgCl2 , 10 mM imidazole–HCl, pH 6.8). The membrane was incubated in the same solution containing ∼50 ␮M 45 CaCl2 (20.87 mCi/mg, Perkin-Elmer Life and Analytical Sciences) for 1 h, rinsed with buffer and dried. Calcium-binding proteins were detected by autoradiography.

2.4. Inmunoblotting analysis Protein samples were separated by 15% SDS-PAGE and electroblotted onto PVDF membrane. The membrane was blocked overnight with 5% non-fat milk in PBS–Tween (10 mM

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Na2 HPO4 , 3 mM KH2 PO4 , 140 mM NaCl, 0.05% Tween 20) to block non-specific binding. After three washes in PBS–Tween, membranes were incubated for 1 h with two different polyclonal chicken ␣-actinin antibodies (generously provide by Ana Sarasa and from Sigma). After three washes with PBS–Tween, the membrane was incubated for 1 h with horseradish peroxidaseconjugate secondary antibodies (Sigma) and washed three times with PBS–Tween. The secondary antibody was detected using 9-etyl-3amino-carbazol as chromogen. 2.5. Electron microscopy Filamentous actin (4 ␮M) was mixed with His-tagged ␣actinin and the solution was incubated for at least 30 min before staining. The protein suspension was allowed to adhere to Formvar-coated Cu-grids, stained with 1% sodium silicotungstate and examined with a JEOL 1230 electron microscope. 2.6. Circular dichroism spectroscopy Circular dichroism spectroscopy was used to examine the secondary structure of the purified His-tagged recombinant protein. Far-UV spectra were collected between 190 and 260 nm using Jasco J-810 spectrophotometers at 20 ◦ C. Ten accumulated spectra were collected using a 0.1 cm cuvette. 2.7. MALDI-TOF mass spectrometry and N-terminal analysis MALDI-TOF mass spectrometry was used to analyse degradation of the ␣-actinin-like protein. After separation by SDS-PAGE and staining, major bands at 63 and 40 kDa were digested by trypsin and analysed in a Voyager DESTR mass spectrometer (Applied Biosystems) as described [27]. Edman degradation was used to determine the N-terminal sequence of the 23 kDa fragment after separation by 15% SDSPAGE, electroblotting onto PVDF membrane and Coomassie blue staining.

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gene was also nearly identical to another determined Entamoeba sequence (accession number: AF208390); the difference being a stretch of TTTT that is AAAA at nucleotide 222 in the gene we isolated. We also found an entry (accession number: XM 649337) annotated also as grainin2 that is identical to the sequence we obtained except for a region of 192 nucleotides that it is repeated in the sequence. When searching directly in the Entamoeba database, we only find reads that matched the sequence we isolated, confirming that XM 643283 is the correct ␣-actinin gene sequence. Since it is unlikely that exactly the same nucleotide sequence appears twice in the same gene, we believe, that the repeated stretch in XM 649337 is the result of an assembly error in the automated annotation. There is also another grainin2 in the databases (accession number: AF082530) that show a weak similarity to the other grainin2 sequences. This grainin2 has been suggested to be a calcium-binding protein [31]. After expression, affinity purification and proteolytic removal by TEV protease of the His-tag, bands around 63 and 40 kDa as well as a weak band around 23 kDa appeared in the Coomassie stained SDS-PAGE gel. With time the lower molecular weight bands became more pronounced (Fig. 1), suggesting a continuous degradation of the purified protein. The 63 and 40 kDa bands were analyzed by MALDI-TOF fingerprinting and both were identified as the ␣-actinin-like protein. The matched fragments of the 63 kDa band covered almost the whole length of the sequence whereas the 40 kDa band did not give rise to any fragments located in the C-terminus of the complete sequence. The N-terminal sequence of the 23 kDa fragment was AAADAWLLQ, localizing the proteolytic cleavage site to the peptide bond between residues Q332 and A333. Therefore, the 43 kDa band corresponds to an N-terminal fragment of the 63 kDa full length protein, spanning the first 332 residues (Fig. 2). In addition, the molecular weight of the His-tagged full length protein was 66.9 kDa according to MALDI-TOF analysis, which corresponds favourable with the theoretical size of 66.7 kDa.

2.8. Structure prediction The PSIPRED [28] and SMART [29] servers were used to predict secondary structures and structure motifs. MultiCoil was used to predict coiled coil regions [30]. 3. Results 3.1. Cloning, sequencing and expression Previously we have found an E. histolytica gene coding for a ␣-actinin-like protein [19]. PCR primers were designed accordingly and used to amplify the corresponding gene. The isolated gene turned out to be identical to an Entamoeba gene annotated as grainin2 (accession number: XM 643283). The isolated

Fig. 1. SDS-PAGE of purified Entamoeba ␣-actinin-like protein. Entamoeba ␣actinin-like protein was separated by 15% SDS-PAGE directly after purification but before removal of the His-tag (lane 2), after cleavage and removal of the His-tag (lane 3) and after prolonged storage (lane 4). Protein was stained with Coomassie blue. Lane 1: Molecular weight references: 200, 116, 97, 66, 45, 31 and 21 kDa.

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Fig. 2. Schematic structure of the Entamoeba ␣-actinin-like protein. The SMART server (http://smart.embl-heidelberg.de/) was used to predict structural domains in the 537 residues long ␣-actinin-like protein. CH, calponin homology domain; coil, coiled coil region; EF, EF-hand. Numbers represent the predicted borders of domains. The arrow indicate the cleavage site at residue 332 that give rise to the 40 and 23 kDa fragments.

3.2. Immunodetection The identity of the expressed protein was probed by immunodetection. For this purpose, the purified protein was separated by SDS-PAGE, electroblotted onto PVDF membrane and incubated with polyclonal ␣-actinin antibodies. As Fig. 3 shows, one of the antibodies (Sigma) reacted strongly with the 63 kDa band but only weakly with the 43 kDa peptide. On the other hand, the other antibody tested reacted somewhat stronger with the 43 kDa band (not shown). Although the antibodies differed in reactivity, it is evident that epitopes required for staining were present in both the full length protein and in the degraded one. 3.3. Calcium-binding properties Binding of calcium was investigated by an overlay assay. ␣-Actinin was blotted onto a PVDF membrane together with positive (calmodulin) and negative (TEV protease and glutathione-S-transferase) controls. After washing in binding buffer, the membrane was incubated in the presence of 45 Ca, washed to remove unbound radioactive calcium, dried and autoradiographed. The results showed that the ␣-actinin-like protein has affinity for calcium (Fig. 4).

Fig. 3. Immunoidentification of Entamoeba ␣-actinin-like protein. Purified Entamoeba ␣-actinin-like protein was first separated by 15% SDS-PAGE and then transferred to PVDF membranes by semi-dry electroblotting. The gel was stained with Coomassie blue (left) and the membrane probed with ␣-actinin antibodies (right). Lane 1: 7.5 ␮g bovine serum albumin; lanes 2: 5 ␮g ␣-actinin-like protein and 3: 10 ␮g ␣-actinin-like protein (after storage).

Fig. 4. Calcium-binding assay. Calcium-binding properties of Entamoeba ␣actinin-like protein was probed by calcium overlay assay using 45 Ca. Fifty-four micrograms (1A) and 108 ␮g (1B) Entamoeba ␣-actinin-like protein was slotblotted onto a nitrocellulose membrane. Two hundred micrograms calmodulin (2) was used as a positive control and 280 ␮g of glutathione-S-transferase (3) and 200 ␮g TEV-protease (4) were loaded as negative controls.

3.4. Structure analysis and prediction Far-UV CD spectroscopy was used to analyse the helical content and thermal stability of the ␣-actinin-like protein. The obtained CD spectrum (Fig. 5) showed a typical ␣-helix containing protein, with the characteristic minima at 207 and 220 nm. When analysed by DICHROWEB, a ␣-helical content of ca. 40% was obtained. When submitted to the PSIPRED server similarly high ␣-helical content (ca. 52%) was returned. The thermal stability was also determined by far-UV CD spectroscopy (not shown). As expected the secondary structure

Fig. 5. Far-UV CD spectrum. Far-UV CD spectrum of 2 ␮M Entamoeba ␣actinin-like protein in 25 mM sodium phosphate, 300 mM NaCl, pH 7.6. Ten scans between 190 and 260 nm were accumulated at 25 ◦ C.

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was lost upon heating. When the ellipticity at 222 nm versus temperature was analysed, a transition temperature of around 47 ◦ C was obtained. Prediction of the tertiary structure by the SMART or InterProScan servers returned a structure similar to ␣-actinins; an N-terminus actin-binding domain, with two calponin homology domains and two or three EF-hands at the C-terminus (Fig. 2). The central part is much shorter than in the typical ␣-actinins and this region was not recognized by SMART or InterProScan as a typical spectrin repeat, as in other ␣-actinins. However, most of the central region is predicted to form a coiled structure and previously we have found that this region showed a weak similarity to a spectrin repeat [19]. 3.5. Actin-binding properties of Entamoeba α-actinin-like protein To determine the actin-binding properties of this protein, a co-sedimentation assay was used. The rationale being that a lowspeed centrifugation is supposed to pellet only cross-linked actin filaments together with the cross-linker whereas a high-speed centrifugation will pellet actin filaments whether they are bundled or not. After a low-speed centrifugation actin as well as the ␣-actinin-like protein were present in the pellet. Therefore, the results (Fig. 6) implied that the ␣-actinin-like protein not only binds actin filaments but also is able to cross-link actin. Electron micrographs of negatively stained samples also showed that this Entamoeba protein cross-linked actin filaments into bundle-like structures (Fig. 7). In presence of calcium considerably less of the ␣-actinin-like protein was pelleted. However, since calcium also caused precipitation of the 63 kDa protein, we could not investigate the effect of calcium in detail. Anyhow, these results indicated that the ␣-actinin-like protein behaved similar to other calcium-sensitive isoforms of ␣-actinins [32–34]. 4. Discussion We have cloned and expressed a ␣-actinin-like protein from E. histolytica that displays all characteristics of ␣-actinin. This protein binds and cross-links actin filaments in a calciumdependent manner and binds calcium. Structure analysis and predictions indicate that the domain structure is similar to other ␣-actinins; with an N-terminal actin binding domain, containing two calponin homology domains, and a calcium-binding domain in the C-terminal part, comprising at least two EF-hands. In the typical ␣-actinin the rod domain comprises four spectrin repeats that are involved in forming the antiparallel homodimer [35,36] The central region of the Entamoeba ␣-actinin-like protein is much shorter (ca. 145 residues) but still long enough to form a typical 106-residue long spectrin repeat [37]. This region was also predicted to form a coiled structure, though not necessarily a triple-helix. The cross-reactivity with ␣-actinin polyclonal antibodies indicated that some of the recognition epitopes are preserved in the Entamoeba protein. Therefore, we believe that this ␣-actininlike protein of Entamoeba in fact is a genuine ␣-actinin. Thereby lending some support for the previous phylogenic analysis that

Fig. 6. Actin co-sedimentation assay. (a) Low-speed co-sedimentation assay. Actin and Entamoeba ␣-actinin were centrifuged alone or together at different concentrations. After centrifugation (13,000 rpm, 15 min) supernatant (S) and pellet (P) were separated on 15% SDS-PAGE. Lane 1: 4.5 ␮M actin; lane 2: 9.7 ␮M Entamoeba ␣-actinin; lane 3: 4.5 ␮M actin and 1.5 ␮M ␣-actinin; lane 4: 4.5 ␮M actin and 2.3 ␮M ␣-actinin; lane 5: 4.5 ␮M actin and 5.0 ␮M ␣actinin; lane 6: 4.5 ␮M actin and 9.7 ␮M ␣-actinin; lane 7: 4.5 ␮M actin and 9.7 ␮M ␣-actinin plus 1 mM CaCl2 . Arrows indicate the position of actin and ␣-actinin. (b) Quantification of pelleted actin and ␣-actinin. The Coomassie blue stained gel in A was quantified using ScionImage. Actin: black bar; ␣-actinin: grey bars.

placed this protein very early in evolution and identified it as a possible ancestor to modern ␣-actinins. We also suggest that the two sequences of grainin2 (accession numbers: XM 643283 and XM 649337) in fact are the same and that this particular grainin2 is identical to the protein we have cloned. Since it has all hallmarks of a ␣-actinin we suggest that it should be annotated as such. After expression and purification, we noticed a slow degradation of the 63 kDa protein; with time a smaller 40 kDa fragment appeared. Since in-gel trypsination, followed by MALDI analysis and N-terminal sequencing located the cleavage site to residue 332, there seems to be a protease-sensitive region between the rod and the calcium-binding domain. It is tempting to assume that this cleavage site is located to a flexible linker connecting these two modular domains. In humans as well as several other organisms, there are four isoforms of ␣-actinin. Two of these (␣-actinin 1 and 4) are sensitive to calcium whereas the other two (␣-actinin 2 and 3) are insensitive. During evolution these four isoforms arose in the vertebrate-invertebrate divergence [19,38]. Since from an evolutionary aspect a loss-of-function seems more likely than a gain-of-function, any ancestral ␣-actinin should be calcium-

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short length of the rod domain as has been suggested before [41]. Addition of calcium diminished both low-speed pelletable actin filaments and bundle formation. The exact location of Entamoeba ␣-actinin as well as function is unknown. ␣-Actinin-like proteins have previously been identified in the nuclei fraction of Entamoeba [42] as well as in adhesion plates [43] and actin-rich complexes [44]. Recently it has also been found attached to the plasma membrane through its binding to the Gln/GlnNAc lectin. Although ␣-actinin has a wide distribution and function in the cell, ␣-actinins are typically concentrated on membranes and cytoskeletons and involved in membrane-associated events. However, other possible roles have been suggested; in the yeast S. pombe ␣-actinin appears to be more important in cytokinesis than in membrane-associated events [45]. Recently, it has become evident that there are ␣actinin and/or ␣-actinin related proteins also in the nucleus [46–48]. 5. Conclusions Entamoeba ␣-actinin is an atypical and ancestral member of this protein family, containing a single putative spectrin repeat in the rod domain. However, this ␣-actinin still conserves the most essential function in modern ␣-actinins: the ability to crosslink actin filaments in a calcium-sensitive manner. The short rod domain leads to thick and compact actin bundles, supporting the idea that the composition and length of the rod domain is not crucial for actin binding, but it determines the separation between adjacent filaments. This ancestral protein may represent the origin of the ␣-actinin protein family, containing only the indispensable requirements for calcium-sensitive actinbundling. During the evolution more complex and sophisticated isoforms have arisen due to the needs of adaptation to different environments. Acknowledgements

Fig. 7. Electron transmission microscopy. Four micromolars actin was incubated alone (a) or with 2.8 ␮M Entamoeba ␣-actinin in the absence of calcium (b) before electron microscopy as described in Section 2. Samples were added to grids and negatively stained with sodium silicotungstate. Bar: 200 nm.

We thank to Dr. E. Tannich for providing the E. histolytica library, Dr. David S. Waugh for the TEV protease, Ana Sarasa for ␣-actinin antibodies, Katja Petzold for helping us with CD measurements, Leonore Johansson for electron microscopy and Dr. Per-Ingvar Ohlsson for N-terminal sequencing. This work was supported by EU Framework 5 programme (HPRN-CT-2000-00096) and Carl Tryggers stiftelse. References

sensitive. Therefore, the finding that Entamoeba ␣-actinin binds calcium collaborate the previous suggestion that this protein originates at the bottom of evolution [19]. In the dimer, calcium-binding to ␣-actinin 1 or 4 interferes with the actin-binding site on the other monomer and inhibits cross-linking of filaments [32,39,40]. Calcium appeared to interfere also with the ability of Entamoeba ␣-actinin to cross-link actin. In the absence of calcium, it was possible to pellet actin filaments at low-speed centrifugation and actin bundles could be seen in the electron micrographs. The actin bundles we observed were thick and compact. The compactness may depend on the

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