REVIEW ARTICLE The Cytoskeleton of Entamoeba histolytica: Structure, Function, and Regulation by Signaling Pathways Isaura Meza, a Patricia Talama ´s-Rohana, b and Miguel A. Vargas a a Departamentos de Biomedicina Molecular and b Patologı ´a Experimental, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Me ´xico D.F., Me ´xico Received for publication September 23, 2005; accepted September 26, 2005 (ARCMED-D-05-00387). Pathogenesis in the parasite Entamoeba histolytica has been related to motility of the trophozoites. Motility is an important feature in amebas as they perform multiple motile functions during invasion of host tissues. As motility depends on the organization and regulation of the cytoskeleton elements, in particular of the actin cytoskeleton, the study of the molecular components of the machinery responsible for movement has been a key aspect to study in this parasite. Although many of the components have high homology in amino acid sequence and function to those characterized in higher eukaryotic cells, there are important differences to suggest that parasitic organisms may have developed adap- tative differences that could be useful as targets to stop invasion. The purpose of this review is to evaluate current knowledge about the cytoskeleton of E. histolytica and the ways in which the parasite controls motility. Ó 2006 IMSS. Published by Elsevier Inc. Key Words: Entamoeba histolytica, Cytoskeleton, Cell signaling, Motility, Invasion. Introduction A striking feature of Entamoeba histolytica trophozoites is their high motility manifested by fast locomotion, pleio- morphism, and continuous movement of intracellular com- ponents. In culture, amebas form surface projections that allow pinocytosis, phagocytosis, adhesion, and displace- ment. During infection of the human host, motility is thought to be a main determinant for the invasive behavior of trophozoites, as they become avid phagocytes ingesting damaged cells, extracellular matrix, and erythrocytes. Re- sponses to chemotactic cues are thought to induce directed migration and tissue penetration. The intense motility of amebas would require a very dynamic cytoskeleton making difficult the visualization of cytoskeletal polymers. It was only after introduction of modified microscopic techniques and specific markers, such as fluorescent compounds and antibodies directed to cytoskeleton proteins, that identification of filaments corre- sponding to actin polymers and nuclear microtubules in cul- tured trophozoites was achieved. Molecular approaches allowed identification of genes encoding several cytoskele- tal proteins and the inference of their amino acid sequences and functional domains. These advances, together with elu- cidation of signaling pathways that regulate actin turnover and interactions and the use of specific drugs to block cyto- skeleton functions, have provided important but still incom- plete information about amebic functions in which the cytoskeleton could play a role. Monitoring cytoskeletal re- arrangements occurring during cellular activities still repre- sents a technical challenge and there is much to explore about the mechanisms that induce and regulate motility in the parasite. As motility has been implicated in pathogenic- ity, it will be very important to discern how the machinery for cell movement supports survival of the parasite as com- mensal in the human host but, at the same time, permits cell and tissue damage during invasion. Actin Actin was first identified in trophozoites by immunostain- ing utilizing heterologous antibodies prepared against hu- man and rabbit muscle actins (1,2). Expression of the protein was confirmed when a protein with similar molec- ular and functional characteristics to typical actins was Address reprint requests to: Dr. Isaura Meza, Departamento de Biome- dicina Molecular, Cinvestav-IPN, Apartado 14-740, Me ´xico, D.F., 07360, Mexico; E-mail: [email protected]0188-4409/06 $–see front matter. Copyright Ó 2006 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2005.09.008 Archives of Medical Research 37 (2006) 234–243
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Archives of Medical Research 37 (2006) 234–243
REVIEWARTICLE
The Cytoskeleton of Entamoeba histolytica: Structure,Function, and Regulation by Signaling Pathways
Isaura Meza,a Patricia Talamas-Rohana,b and Miguel A. Vargasa
aDepartamentos de Biomedicina Molecular and bPatologıa Experimental, Centro de Investigacion y de Estudios
Avanzados del IPN, Mexico D.F., Mexico
Received for publication September 23, 2005; accepted September 26, 2005 (ARCMED-D-05-00387).
Pathogenesis in the parasite Entamoeba histolytica has been related to motility of thetrophozoites. Motility is an important feature in amebas as they perform multiple motilefunctions during invasion of host tissues. As motility depends on the organization andregulation of the cytoskeleton elements, in particular of the actin cytoskeleton, the studyof the molecular components of the machinery responsible for movement has been a keyaspect to study in this parasite. Although many of the components have high homology inamino acid sequence and function to those characterized in higher eukaryotic cells, thereare important differences to suggest that parasitic organisms may have developed adap-tative differences that could be useful as targets to stop invasion. The purpose of thisreview is to evaluate current knowledge about the cytoskeleton of E. histolytica and theways in which the parasite controls motility. � 2006 IMSS. Published by Elsevier Inc.
A striking feature of Entamoeba histolytica trophozoites istheir high motility manifested by fast locomotion, pleio-morphism, and continuous movement of intracellular com-ponents. In culture, amebas form surface projections thatallow pinocytosis, phagocytosis, adhesion, and displace-ment. During infection of the human host, motility isthought to be a main determinant for the invasive behaviorof trophozoites, as they become avid phagocytes ingestingdamaged cells, extracellular matrix, and erythrocytes. Re-sponses to chemotactic cues are thought to induce directedmigration and tissue penetration.
The intense motility of amebas would require a verydynamic cytoskeleton making difficult the visualization ofcytoskeletal polymers. It was only after introduction ofmodified microscopic techniques and specific markers,such as fluorescent compounds and antibodies directed tocytoskeleton proteins, that identification of filaments corre-sponding to actin polymers and nuclear microtubules in cul-tured trophozoites was achieved. Molecular approaches
Address reprint requests to: Dr. Isaura Meza, Departamento de Biome-
8-4409/06 $–see front matter. Copyright � 2006 IMSS. Published by Else: 10.1016/j.arcmed.2005.09.008
allowed identification of genes encoding several cytoskele-tal proteins and the inference of their amino acid sequencesand functional domains. These advances, together with elu-cidation of signaling pathways that regulate actin turnoverand interactions and the use of specific drugs to block cyto-skeleton functions, have provided important but still incom-plete information about amebic functions in which thecytoskeleton could play a role. Monitoring cytoskeletal re-arrangements occurring during cellular activities still repre-sents a technical challenge and there is much to exploreabout the mechanisms that induce and regulate motility inthe parasite. As motility has been implicated in pathogenic-ity, it will be very important to discern how the machineryfor cell movement supports survival of the parasite as com-mensal in the human host but, at the same time, permits celland tissue damage during invasion.
Actin
Actin was first identified in trophozoites by immunostain-ing utilizing heterologous antibodies prepared against hu-man and rabbit muscle actins (1,2). Expression of theprotein was confirmed when a protein with similar molec-ular and functional characteristics to typical actins was
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purified from trophozoites. Amebic actin showed the capac-ity to form filaments in vitro that could be decorated withheavy meromyosin and to induce myosin ATPase activitywith similar kinetics to that induced by rabbit skeletal mus-cle actin (3). Although this actin did not bind to DNase I orpolymerized at low temperature as other actins do (3,4),antibodies to the purified protein recognized actin fromseveral eukaryotes, including other protozoa, suggesting acertain degree of homology between species and its possi-ble involvement in cell motility (5,6). Availability of a spe-cific marker for polymerized actin (rhodamine phalloidin)allowed localization and quantification of polymerized ac-tin in phagocytic cups of trophozoites formed during theprocess of ingestion of complete erythrocytes and afterstimulation with erythrocyte-derived liposomes (7,8). Atthe same time, polymerized actin was identified in pinocyt-ic invaginations, the uroid in the posterior part of the cell,and in the cortical region of cultured trophozoites (9,10).A possible correlation between actin polymerization andphagocytosis, endocytosis and virulence was drawn fromobservations where the specific inhibitor of actin polymer-ization, cytochalasin D, blocked these processes and frommutants in phagocytosis that were hindered in their abilityto cause cellular damage (11,12).
Later experiments showed that actin could be induced toorganize transient but complex structures such as adhesionplates and focal adhesions that can function as signal trans-duction organelles when trophozoites interacted with anddegraded extracellular matrix proteins (13,14). It was foundthat interaction with fibronectin activates trimeric G-proteinreceptor-coupled signaling pathways that promote actin po-lymerization and interaction with other proteins at specificsites to carry processes such as adhesion, secretion of pro-teases and directed locomotion. This cytoskeleton organiza-tion was stable as long as the inductor was present buteasily reversed to the unorganized state once the stimuluswas removed or the signaling pathways were blocked(15–18). Description of other signaling pathways recentlyidentified in E. histolytica indicates that actin organizationis regulated by a complex network of signals respondingto different cues that could be promoting pathogenic behav-ior (18–21).
The first nucleotide sequence analyses of actin cDNAclones and a genomic clone in Entamoeba histolytica re-vealed multiple genomic copies of the actin gene and theabsence of intervening sequences (22,23). The inferred ac-tin sequence showed 89 and 86% amino acid homology tohuman cytoplasmic and skeletal muscle actins, respectively,but the nucleotide sequence homologies were significantlylower at 69 and 67%. Moreover, distribution of variable nu-cleotide residues throughout the sequence showed strongpreference for the placement of an adenine or thymine res-idue in the third position of a codon (22). Discovery of thisunusual codon usage for amebic actin that displays a greaterdegree of similarity to lower eukaryotic than to higher
eukaryotic codon usage was successfully utilized to pursuethe identification and characterization of other genes ofE. histolytica. It was also reported that actin mRNA tran-scription was controlled by the trophozoite growth condi-tions and presence of inductors of actin polymerizationand its direct effect on actin organization (24).
Actin needs the concourse of other proteins to form thecomplex structures responsible for cellular movements.Organization of these structures is accomplished by interac-tion between actin and multiple actin-binding proteins ofwhich some connect actin filaments into extensive networksthat can bind to other proteins in the cytoplasm or to pro-teins and lipids in plasmatic and vesicular membranes,while others have a function in nucleation and stabiliza-tion/destabilization cycles of the actin polymers (25). InE. histolytica trophozoites several actin-binding proteinshave been identified by their association with actin-contain-ing structures or by their direct binding to monomeric andpolymerized actin. In adhesion plates formed during inter-action to extracellular matrix proteins actin filament bun-dles were found associated with the cell membrane. Inaddition to actin, actin-binding proteins such as vinculin,a actinin, and myosins I and II were identified as compo-nents of the plates (14). These actin-binding proteins areknown to participate in the organization of adhesive struc-tures in higher eukaryotic cells (26). Two kinases were alsoidentified as constituents of the adhesion plates, one wasPKC, a serine/threonine kinase, and the other the specifickinase of adhesion plates called FAK125 that phosphorylatestyrosines in several of the proteins associated with theplates (26). These two kinases were later identified as im-portant elements in phosphorylation cascades that regulatereorganization of actin (15–17,27).
Cell locomotion requires dynamic turnover of actin forthe extension of the cell leading edge, attachment to the sur-face, and pulling forward the cell body. Profilin is a proteinthat sequesters actin monomers and could then inhibit orpromote actin polymerization in a precise site within thecell. Profilin, together with other proteins like cofilin andactin depolymerizing factor (ADF), both of which facilitateactin filament depolymerization, regulate actin turnover.Only profilin has been identified so far in E. histolyticatrophozoites where two isoforms are expressed. In the basicisoform, only 4 of the 21 residues that make contact withactin are present. However, the recombinant protein wascapable of binding to plant and mammalian actin (28).ABP-120, another actin-binding protein present in tropho-zoites, could be an important factor in the stabilization/destabilization of actin filaments necessary for assembly/disassembly of transient structures. EhABP-120 has beenlocalized at the sites where actin is concentrated during pro-cesses such as pseudopod extension and cap formation (29).The cloned EhABP-120 gene is a single copy and codes fora protein of approximate molecular weight of 93.6 kDa. Ithas an actin-binding site and similarities to the family of
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actin-binding proteins called filamins, which are regulatedby phosphoinositides. The interaction of membrane lipidsand actin-binding proteins forms a molecular bridge be-tween the plasma membrane and the actin cytoskeletonand favors the recruitment of special types of lipids to formmembrane domains or lipid rafts involved in functions suchas intracellular traffic, apical sorting, regulation of mem-brane proteases, and signal transduction (30). EhABP-120can interact with phosphatidic acid and with Gal-ceramide(31). Therefore, this protein could be a key element in func-tions relevant to the pathogenic behavior of trophozoites.One of the regulatory factors in the function of cytoskeletalproteins is the complex Ca/calmodulin. Calmodulin isa Ca21 regulator in eukaryotic cells where it activates a di-versity of proteins. Calmodulin is present in E. histolyticatrophozoites where it has a role in several of the signalingpathways conducive to actin organization and promotionof motility functions, in particular secretion of proteinases(32–34). Two other Ca-binding proteins have recently beencharacterized, one of which interacts with actin filaments(35). Although these results suggest an important role ofCa-binding proteins in ameba motility, blocking expressionof EhCaBP1 was shown to have a negative effect in unrelat-ed functions such as cellular proliferation, endocytosis andphagocytosis that could result from secondary effects due tothe wide range of proteins that depend on Ca21 activation.
Myosins
Myosins are proteins that function as molecular motors be-ing essential for cellular movement. Myosins move on actinfilaments converting chemical energy into mechanical workby ATP hydrolysis, producing contraction of the filaments.In eukaryotic cells there are at least 18 different classes ofmyosins and specific functions have been assigned to onlya few of them. In Entamoeba histolytica, only two myosinshave been identified. Characterization of a myosin II heavychain gene (Myo II) showed that the globular head domaincontains the specific amino acid regions involved in ATPand actin binding, as well as the sites for interaction withmyosin light chains. The tail domain is organized as ana-helical coiled coil structure interrupted by 2 prolines, in-dicating that the tail folds twice on itself as occurs in othermyosins. In addition, this myosin amino acid sequence pre-sented similarities in its phosphorylation sites with thosefound in the heavy chains of smooth and non-muscle verte-brate myosin II (36). Immunolocalization of myosin II introphozoites indicated the highest density of the proteinin the uroid and low density along the cell membranesand the frontal part of the cell, suggesting its participationin cell locomotion (37). Later, myosin II together with actinwere reported as components of ligand-receptor complexesor caps formed in the surface of trophozoites (38). The useof cytochalasin D and of specific drugs to block kinases
such as PKC and caseine kinase II, found as componentsof the caps, suggested that phosphorylated myosin II wasinvolved in the capping and that this process could dependon actomyosin contraction. Overexpression of the lightmeromyosin domain of myosin II rendered a dominat neg-ative mutant characterized by abnormal movement, failureto form the uroid and failure to induce capping in the pres-ence of concanavalin A. These defective trophozoites wereunable to achieve proper contact with target cells and didnot show the subsequent cellular damage inflicted by con-trol cells (39). Very recently, identification of Rho proteinsin trophozoites and the use of inhibitors of Rho signaling toblock actin–myosin interactions have provided additionalevidence to support that contractility could be the drivingforce in several motile activities of trophozoites (40).
The other form of myosin partially characterized introphozoites is myosin IB. This myosin form shows severalsimilarities to other non-conventional myosins in higher eu-karyotic cells (41). It was found that this myosin interactswith actin and a actinin and that it has the ATP or GTPbinding site, a putative domain for interaction with calmod-ulin and a SH3 domain to mediate interaction with cellmembranes. Overexpression of MyoIB indicated that thisprotein could be involved in phagocytosis, as a strain ex-pressing three-fold more myosin IB than the wild-typestrain was deficient in erythrocyte uptake, a characteristicfeature of invasive trophozoites (42).
The availability of the E. histolytica genome data (TIGRInstitute database at http://www.tigr.org/tdb/e2k/eha1) per-mitted identification of several putative genes for possiblemyosin light chains, important elements for myosin activi-ty, but none of these has been identified in the parasite. Sev-eral kinases and phosphatases that in other cellular systemsare known to activate or inhibit proteins that participate inthe assembly of the complex machinery required for move-ment are also waiting for identification. At the moment,only through putative gene identification can we infer thepossible ways in which actin and myosin functions areregulated. Experimental data are necessary to identify allthe components and characterize their associations andrequirements.
Tubulins
Cytoplasmic microtubules have not been observed in troph-ozoites of E. histolytica, in contrast with microtubulesinside dividing nuclei that could be identified utilizing sev-eral microscopy techniques. The intranuclear mitotic spin-dle is formed by a bundle of microtubules that do notconverge at the poles in well-defined asters, although whatcould be a single microtubule organizing center (MTOC)has been described in one of the poles in the nucleus ofdividing cells (43). Gicquaud described for the first timein 1979 what could be considered the phases of mitosis in
237Entamoeba histolytica: Cytoskeleton and Signaling
E. histolytica trophozoites (44). Mitosis started with a pro-phase where several dense bodies, possibly chromosomes,and microtubules interacted, followed by a very fast move-ment of the chromosomes to the center and then to the polesof the nucleus that became elongated, followed by the for-mation of two separate nuclei. The latter, which are not typ-ical phases of mitosis, could correspond to metaphase andanaphase. The nuclei formed remained together for sometime and slowly became separated. In all stages the nuclearmembrane was conserved intact. Phase contrast microscopyimages have shown the high refringence of microtubulebundles inside dividing nuclei, and recent results obtainedwith antibodies to amebic b-tubulin depict microtubule-likestructures in one of the two nuclei undergoing mitosis ina trophozoite (Figure 1). Furthermore, microtubules thatcould be present in the cytoplasm have not been revealedusing antibodies to different types of tubulins, which onlydepict fluorescence in the nucleus (45,46).
By genetic approaches a, b, and g tubulin encodinggenes have been identified. E. histolytica a tubulin-deducedamino acid sequence showed approximately 50% identityto a tubulins from other protozoa and to human a tubulin(47). Alpha tubulin in amebas lacks a polyacidic motif inthe C terminus that is involved in polymerization and bind-ing to microtubule-associated (MAPS) proteins and a Tyrresidue that participates in microtubule stabilization. Thesedifferences with other eukaryotic tubulins could explainvariations in assembly of amebic microtubules and their ap-parent lability.
Beta tubulin encoding genes showed 54 and 58% identi-ty to b tubulins from several organisms and apparently a sin-gle gene copy is present in E. histolytica and otherEntamoeba species in contrast with a-tubulin genes thatare present as several copies (47,48).
Gamma tubulin is the third type of major tubulins pres-ent in eukaryotic cells and it is exclusively associated withMTOC. Microscopy studies with trophozoites detected as-sociation of dense DNA-containing material with clustersof microtubules that could correspond to MTOC (43).The presence of a gene encoding for g tubulin and the stain-ing of a region in the central part of the nucleus with anti-bodies to recombinant g tubulin strongly suggested thatthese organelles could be present in trophozoites. Addition-ally, it was shown that the maximum g-tubulin expressionwas reached during the S/G2 phase of the parasite’s cellularcycle (49,50). Reduction of expression of g tubulin introphozoites by the use of RNAi techniques has shown dis-array of the nuclear fluorescence detected by antibodies toamebic g tubulin, supporting the possible disorganizationof organelles required for chromosome separation (45).
Experiments with flow cytometry utilizing E. invadensand E. histolytica trophozoites have shown that DNA syn-thesis occurs in defined phases of the nuclear division cycleand that several rounds of genome duplication occur beforecytokinesis takes place so it is plausible that well-defined
mechanisms control the cell cycle and nuclear division(51). However, trophozoites in culture, which can containseveral nuclei, show asynchrony in the stages of mitosis,and it is also a well-known fact that cytokinesis, even whenwell advanced, can be halted, originating multinucleatedcells. The low identity of amino acid sequences of amebictubulins with other tubulins could explain the low responseof microtubules to different drugs known to alter microtu-bule integrity in other eukaryotic cells (52,53). Differencesin the amino acid sequences required for polymerizationand stabilization of formed microtubules between amebasand other eukaryotic cells could be a factor in the high sen-sitivity of amebic microtubules to temperature and fixa-tives, making their visualization so difficult. A structuralmodel of tubulin dimers proposed that the carboxyl termi-nals of amebic tubulins interfere with the linkage of themolecules and their polymerization (54). It will be impor-tant to perform biochemical studies focused on polymeriza-tion of the tubulins to validate these possibilities.
From the above data it is easy to conclude that microtu-bule functions are also poorly defined in E. histolyticatrophozoites, in addition to their participation in mitosis.This is an area that needs to be explored, particularly whenmicrotubules are described nowadays as important ele-ments in adhesive, secretory and endocytic activities inmany cells.
The third major element in the cytoskeleton of eukary-otic cells, intermediate filaments, has not been describedin trophozoites. As these types of filaments are more stableand contribute to cell rigidity, it is possible that in a cell ashighly motile as E. histolytica these organelles do not formand, therefore, the proteins that constitute them are not ex-pressed. This possibility will have to be proven not onlysearching for the corresponding genes but also by detectingthe proteins.
Cell Signaling
E. histolytica, either as a commensal or as an invasive cell,requires constant interaction with its extracellular environ-ment and with other cells to survive. These interactions leadto changes in cell physiology, cellular architecture, andgene expression. External signals are perceived by mem-brane-bound receptors, resulting in changes in their bio-chemical or physical states that initiate a cascade ofsignaling events within the cell (55). One of the main func-tions of the cytoskeleton is to mediate cell motility and cellshape in response to extracellular stimuli. Organization andfunction of the cytoskeleton are regulated by signaling cas-cades (56).
Activation by different stimuli induce trophozoites toperform specialized functions that include adhesion, pino-cytosis, phagocytosis, chemotaxis, capping, secretion, andcytokinesis (18,57). For all these biological functions,
238 Meza et al./ Archives of Medical Research 37 (2006) 234–243
Figure 1. Proteins in E. histolytica identified as possible participants in motility-related functions. Photographs on the left side show rhodamine-phalloidin
staining of trophozoite actin adhesion plates (adhesion), polymerized actin during chemotaxis, and actin filaments during cytokinesis. Anti-b tubulin staining
depicts microtubules in dividing nuclei (mitosis). Anti-ConA staining reveals a well-formed cap (capping). Endocytosis is illustrated in a trophozoite ingest-
ing two-color fluorescently labeled fluid-phase markers. Secretion is shown in a transmission electron microscopy image of secreted collagenase-containing
granules, seen outside the ameba surface on the collagen substrate. Micrography was kindly donated by Dr. J.L. Rosales-Encina. On the right side, proteins
identified as possible participants in the corresponding function are indicated.
239Entamoeba histolytica: Cytoskeleton and Signaling
organization of the actin cytoskeleton must be tightly regu-lated both temporally and spatially.
In higher eukaryotes, actin cytoskeleton assembly is reg-ulated at multiple levels. One level downstream of surfacereceptors is the activation of complex networks of interac-tive intracellular signals that in turn activate several path-ways that induce actin monomers to form actin polymersand the superorganization of actin polymers into filamen-tous structures. A large number of actin-binding proteinsregulate actin assembly by controlling nucleation of mono-mers, elongation and stabilization/destabilization of thefilaments, and cross-linking of the actin network (58). Fila-ment association with other proteins to form particularstructures is also a regulatory step in actin superorganiza-tion (58).
Many proteins associated with the actin cytoskeleton arelikely targets of signaling pathways that modulate actin as-sembly at different levels. The activities of these proteinsare often regulated by signaling molecules such as Ca21,phosphoinositides, and different types of kinases (59). Asmentioned above, some actin-binding proteins have beenidentified in E. histolytica, of these, profilin is known tointeract with monomeric actin (28). Others like vinculin,a actinin, ABP-120, and myosins I and II interact with po-lymerized or F-actin-containing structures (14,29,37,42).Knowledge about the regulation of trophozoite functionsthrough signaling pathways is incipient, as many of theproteins known to be components of conventional path-ways in other eukaryotic cells have not been characterizedin E. histolytica. Some advances have been achieved forfunctions in which actin and myosin participate, so we willrefer only to these as practically nothing is known aboutregulation of microtubule organization or microtubule-dependent functions.
Integrin-Type Mediated Signaling
During tissue invasion, amebas bind to and destroy differenttypes of cells and extracellular matrix components. In eu-karyotic cells, actin is nucleated and polymerized at multi-molecular complexes formed at the cell membrane at sitesof contact. These complexes facilitate attachment of cells tothe substrate and consist of integrin-type receptors that caninteract with the extracellular matrix and intracellularlyassociate with protein complexes containing actin, talin,vinculin, a actinin, paxillin, tensin, zyxin, and focal adhe-sion kinase (pp125FAK) (56). In E. histolytica, at least twosurface proteins, one of 37 kDa (13) and one of 140 kDa,this last one with antigenic and functional similarities tob1 integrin (60), have been reported to have fibronectin(FN)-binding capacity (61,62). Additionally, a multimolecu-lar complex consisting of phosphorylated paxillin, vinculin,pp125FAK, and the 140-kDa molecule has been described(63). A monoclonal antibody produced against the 140-kDa
molecule was able to inhibit trophozoite adhesion to FNand collagen (COL) but not to laminin (64), suggesting thatthis putative receptor may have the ability to bind not onlyFN but also COL, possibly in addition to 30-kDa collagen-binding proteins also present in trophozoites (65).
The initial demonstration that binding of trophozoites toFN induced formation of actin adhesion plates (13) provid-ed an in vitro model to discern a series of events occurringduring remodeling of the actin cytoskeleton. Analysis of thecomposition of adhesion plates after stabilization of actinfilaments with phalloidin revealed the presence of F-actin,the 37-kDa FN-binding molecule, several actin-bindingproteins, at least one cysteine proteinase, and signaling pro-teins such as PKC and pp125FAK, suggesting that these ad-hesion plates may function as signal transduction organelles(14). Signaling events such as elevation of intracellular pH,Ca21 transients, and protein tyrosine phosphorylation oc-curring during the interaction with FN supported the ideathat signaling events triggered by integrins or other recep-tors could be taking place (19,33). Interaction of tropho-zoites with collagen substrates, which involves celladherence and formation and release of electron-densegranules containing collagenase activity, could also triggersignal transduction (32). The process is Ca21/CaM depen-dent and proteins such as pp125FAK and p42MAPK wereidentified as components of the collagen-induced response(66).
Using the FN interaction model, the search for signalingpathways was started in E. histolytica. A rise in inositol tri-phosphate (IP3), intracellular Ca21 (Cai
21), and cyclic aden-osine monophospate (cAMP), all second messengers in thephosphoinositide and adenylyl cyclase signaling pathwaysthat lead to PKC- and PKA-mediated protein phosphoryla-tion, were described during formation of adhesion plates(15,16,33). In addition, participation of functional trimericG-proteins as important elements in the activation of en-zymes such as phospholipase C that generates IP3 andDAG, and adenylyl cyclase that catalyzes the productionof cAMP from ATP was demonstrated (15–17). Further-more, in the same works increases in PKC and PKAactivities were also reported, as well as increased phosphor-ylation of proteins associated with cytoskeleton-membranefractions.
Serine/threonine kinases of the PKC family are impor-tant in the intracellular transduction of many receptor-mediated calcium-dependent signals (67). Ravdin et al. (68)and Weikel et al. (69) reported a stimulatory effect of phorbolesters (that directly activate PKC) on the pathogenic capacityof E. histolytica toxicity and suggested a role for Ca21 andPKC in the adhesion and killing of target cells. Additionally,PKC activity, a protein of 68,000 kDa that cross-reacts withanti-PKC heterologous antibodies and gene segments ofPKC, as well as the complete gene sequence for a serine/thre-onine kinase have been described in E. histolytica (70,71).The use of phorbol esters, sphingosine, and a specific
240 Meza et al./ Archives of Medical Research 37 (2006) 234–243
inhibitor of phospholipase C, as probes to modulate the ac-tivity of PKC, indicated the participation of amebic PKCin adhesion to FN, organization of actin into adhesive struc-tures, and the cytolytic activity of E. histolytica trophozoites(15–17).
Additional receptors have been proposed to participatein the host–parasite interphase. Among them, an alcoholdehydrogenase of 97 kDa able to bind to FN, COL, andlaminin (72), the 170-kDa heavy subunit of the Gal/Gal-NAc lectin which shares an epitope with human b2 integrin(73), and an immunodominant variable surface antigen of125 kDa that has a small region of the inferred amino acidsequence that presents similarity to the b chain of the hu-man FN receptor, the band 3 precursor of the chick integrin,and the b chain of the mouse integrin (74), although nonehas been shown to directly induce or regulate actin rear-rangement. An interesting point to highlight is the fact thateven though no integrin-like sequences have yet been iden-tified in the E. histolytica genome (75), several reports haveshown evidence that the FN-binding function is present,i.e., amebas respond to extracellular matrix componentsas if having those type of receptors (13,61,63,66,76).
Integrin activation of signaling pathways also occurs insome cells by direct stimulation of phosphatidyl inositol(PtdIns) phosphate metabolism, activation of Ras proteins,mitogen-activated protein kinases activation, and tyrosinekinases, as signaling pathways communicate among them-selves and activate additional routes (77). Some of thesesignaling routes have been explored in E. histolytica,finding some evidence to support their participation in re-ceptor-mediated responses to extracellular matrix compo-nents. Specific tyrosine phosphorylation activity triggeredby FN through the 140-kDa molecule was demonstratedwhen comparing the activation of tyrosine kinases (PTKs)and the pattern of tyrosine phosphorylated proteins in cellsthat interacted with FN, glass, and concanavalin A (19).
Rho Protein-Mediated Signaling
Signaling pathways involving the participation of Rho fam-ily monomeric GTPases in actin organization have beenextensively studied in higher eukaryotic cells (78). The firstidentified rho gene homologue in E. histolytica was clonedfrom parasite genomic DNA using PCR and degenerate oli-gonulceotide primers to GTP-binding sequences I and IIthat are conserved in ras family proteins. It was postulatedthat EhRho1 protein could participate as a factor control-ling growth and differentiation (79). EhRho1 is not asubstrate for Rho-specific C3 exoenzyme, making it an un-usual member of the Ras family. Since then, another sixgenes encoding proteins belonging to the Rho subfamilyhave been identified in E. histolytica that also lack theamino acid sequences to bind C3 exozyme and share97% amino acid identity among them (40). Antibodies to
recombinant EhRhoA1 indicated that Rho proteins areexpressed in trophozoites and that activation by lysophos-phatidic acid, possibly through a membrane receptor cou-pled to Ga12/13 proteins, induces their participation in Rhosignaling pathways leading to reorganization of actin andits interaction with myosin to form actomyosin structures.It has been suggested that actomyosin structures could havea role in functions requiring contractile force such as cap-ping, phagocytosis of erythrocytes, chemotaxis, and adhe-sion to the substrate (38–40). Rac proteins have also beendescribed in E. histolytica and their participation in actinorganization has been inferred from experiments withmutants constitutively expressing activated Rac that wereaffected in phagocytosis and cap formation (80).
A Rho/Rac guanine nucleotide exchange factor (EhGEF)has been recently characterized as a protein with specificityfor activation of Rac proteins, although it can also activateRho1 to a certain extent (21). Overexpression of this activa-tor causes alterations in functions such as phagocytosisand chemotaxis. A kinase with homology to murine p21-activated kinase and yeast Ste20 was recently identifiedin E. histolytica (EhPAK). This protein can phosphorylatein vitro the regulatory chain of myosin II, myosin light-chain kinase, and myosin 1B, suggesting a link with othersignaling pathways that regulate myosin–actin interactions(81). Presence of monomeric GTPases, their regulator pro-teins and diverse kinases in trophozoites in addition to thetwo main classical signaling routes suggest that regulationof actin organization is a complex process in which partic-ipation and collaboration between diverse signaling cas-cades is necessary.
Ca21 Regulation
Intracellular calcium plays a crucial role as a second mes-senger for the control of a variety of cell functions in eukar-yotes, including contraction, secretion, cell division,differentiation, and sodium and potassium permeability(82). The uptake and release of the calcium ion (Ca21)across the plasma membrane and intracellular organellesby the concerted operation of distinct calcium transport-ing systems control intracellular Ca21 concentration. InE. histolytica a partial sequence for a plasma membraneCa21-ATPase, (PMCA)-type Ca21 ATPase has beenreported (reviewed in Reference 82). The presence ofcalmodulin has been documented by biochemical inhibitionassays using CaM antagonists and further purification ofthe molecule (32). Elevation of internal calcium levels isa necessary event associated with cellular functions suchas secretion, contraction, and movement (33). The largeststore for Ca21 in cells is usually found in the endoplasmicreticulum. In amebic trophozoites, InsP3 releases Ca21
from intracellular stores, suggesting the involvement ofan endoplasmic reticulum-like structure (83). Inositol
241Entamoeba histolytica: Cytoskeleton and Signaling
1,3,4,5-tetraphosphate (InsP4) was also shown to releaseCa21 from intracellular stores of E. histolytica (84). Tran-sient increases of intracellular Ca21 and activation of sever-al kinases and Ca21-binding proteins, which in turn modifythe actin cytoskeleton and cell motility, are major eventstriggered by cell binding to ECM proteins. In E. histolytica,external Ca21 influx is an important mechanism in the FN-stimulated signal, and cells depleted of internal calciumcannot respond to FN (33).
Regarding Ca21-binding proteins, a number of CaBPshave been identified in E. histolytica (85), in addition to cal-modulin. Among these, two isoforms of EhCaBP have beendescribed: EhCaBP1, which can bind directly to actin (13)and EhCaBP2, a 15-kDa monomeric protein containingfour canonical EF-hand Ca21-binding loops (85). Compar-ison between the two genes showed an overall identityof 79% and both genes are single copy. EhCaBP1 andEhCaBP2 are functionally different and bind different setsof E. histolytica proteins in a Ca21-dependent manner. Def-inition of the signaling pathways involving these proteinswill help elucidate the role of these two proteins in vivo.
Functions such as phagocytosis, chemotaxis, adhesionand degradation of extracellular matrix proteins, and cap-ping, which are exacerbated during the invasive processof E. histolytica trophozoites, are processes that can alsobe accomplished by trophozoites in culture medium oncethey encounter the adequate stimulus. As seen in this re-view, the present evidence indicates that the machineryfor motility-dependent processes is ready, but its activationobeys different signals. What triggers the activation andfunction of signaling pathways that would result in in-creased motility rendering an aggressive organism is stillan unanswered question related to the pathogenicity of thisparasite.
Despite the impressive progress made with the identifi-cation of several genes and proteins related to cytoskeletonfunctions, there is still scarce knowledge about the proteinstructures, interactions, and mechanisms of regulation.Completion of the E. histolytica genome will make the mo-lecular identification of proteins much easier and faster, butwe still will need to characterize the physiological role ofthe identified proteins. The two most common approachesused to explore the function of cytoskeleton proteins havebeen, on one side, overexpression or its total depletion indominant negative mutants. However, one must keep inmind that an excess of one protein could change homeosta-sis of the cell and the balance between polymerized/unpo-lymerized forms. These situations could perturb thestoichiometry of the polymerization reaction and the inter-action of soluble monomers or polymers with other proteinsto perform a determined function. Depletion, on the otherhand, may require severe reductions in concentration or los-ses in affinity for physiological defects to manifest, so falsenegatives are likely. Approaches such as partial depletion ofa protein by RNAi or inducible expression could be closer
to physiological conditions. The pharmacological approachthat was successful to establish key processes such as thedynamic turnover of cytoskeletal proteins and to infer theirparticipation in several processes has the problem of drugspecificity and unrelated effects. However, if reversibilitytakes place in a biologically relevant time scale, small mol-ecule inhibitors could be of particular value considering thedifferences between human and E. histolytica cytoskeletalstructures. Partial depletion of a particular protein withRNAi or induced expression at a particular stage that wouldcause little perturbation of the physiological conditionscould be excellent tools to study a protein function andits interactions. Microscopy of live cells expressing fluores-cently labeled cytoskeletal proteins could provide direct as-sessment of protein localization and interactions. Finally,biochemical characterization of the proteins, from theiractivity to 3D structure, will also be necessary to unravelthe mystery that still represents the cytoskeleton ofE. histolytica.
AcknowledgmentsWe thank enthusiastic collaborators and members in our laborato-ries for their important contributions at different stages of our re-search with Entamoeba histolytica, as they have made possible thecurrent view on the parasite’s cytoskeleton and its functions.This work was supported by grants from Conacyt, Mexico #42724to IM, #37270 and #28077-M to PTR, and #39511-A1 to MV.
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