1 School of Life Sciences, Jawaharlal Nehru University, Aruna Asaf Ali Marg, New Delhi, 110067 India
2 School of Environmental Sciences, Jawaharlal Nehru University, Aruna Asaf Ali Marg, New Delhi, 110067 India
3 School of Physical Sciences, Jawaharlal Nehru University, Aruna Asaf Ali Marg, New Delhi, 110067 India
4 Unité de Biologie Cellulaire du Parasitisme, INSERM U389, Institut Pasteur, 25-28 rue du Dr Roux, 75015 Paris, France
* Author for correspondence (e-mail: alok0200{at}mail.jnu.ac.in)
Accepted 8 March 2004
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Summary |
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Key words: Phagocytosis, Antisense, Actin, Atomic force microscopy, Cytoskeleton
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Introduction |
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Calcium activation events have been less studied in E. histolytica. Fibronectin-mediated adhesion in E. histolytica can modify cytosolic calcium concentration. This induces the formation of actin adhesion plates and focal contacts, which is a link between calcium signaling and cytoskeletal structures (Carbajal et al., 1996). Also it has been shown that protein kinase C relocates from the cytosol to the membrane fraction in actively phagocytosing trophozoites (De Meester et al., 1990
). The ubiquitous calcium binding protein calmodulin has been shown biochemically to be present in E. histolytica (Munoz et al., 1991
). CaM has been implicated in many functions, such as channel activation, electron dense granule release, cell proliferation and pathogenic activity of E. histolytica (Ravdin et al., 1982
; Makioka et al., 2001
). However, the mechanism of CaM action is not known, as the corresponding gene has not yet been characterized. A number of other calcium binding proteins have been identified in E. histolytica. Two EF-hand calcium binding proteins, grainin 1 and grainin 2 were purified from the granules of E. histolytica and are thought to be involved in endocytosis (Nickel et al., 2000
). E. histolytica also has a functionally diverse calmodulin-like calcium binding protein (EhCaBP1) with no known homologue in the database (Prasad et al., 1992
). Though this protein is similar to calmodulins in many structural properties, it has been shown to be functionally different (Yadava et al., 1997
). Inhibition of the expression of the EhCaBP1 gene by regulatable antisense RNA expression results in loss of cell growth, suggesting that the gene is essential for E. histolytica (Sahoo et al., 2003
). In this paper, we show the involvement of EhCaBP1 in actin cytoskeleton-dependent events such as erythrophagocytosis and endocytosis. A detailed analysis suggests that EhCaBP1 may participate in cytoskeleton dynamics through direct interaction with actin.
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Materials and Methods |
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Immunoprecipitation
The cell lysate was prepared in 1 mM Tris-HCl, pH 7.5, 1% SDS containing 2 mM p-hydroxymercuribenzoic acid (PHMB), 1 mM phenylmethylsulfonyl fluoride (PMSF), 6 mM leupeptin and 1 mM N-ethyl-maleimide, and was centrifuged at 15,000 rpm to remove the cellular debris. The lysate (500 µg) pre-absorbed on protein A-Sepharose beads, was incubated with the EhCaBP1 antibody (Prasad et al., 1993) at 1:20 dilution for 2 hours at 4°C in a reaction volume of 200 µl. Immune complexes were separated by using protein A beads (50 µl suspension; Sigma, USA) followed by three washes with buffer 1 [10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% ovalbumin (w/v), 0.1% Triton X-100 (w/v), 0.05% sodium azide (w/v)] followed by buffer 2 [10 mM Tris-HCl, pH 7.5, 150 mM NaCl] and buffer 3 [0.06 M Tris-HCl, pH 6.8]. The pellet was resuspended in 50 µl of SDS-PAGE buffer (125 mM Tris-HCl 6.8, 2% SDS, 0.1 M DTT, 30% glycerol, 5% ß-mercaptoethanol, and Bromophenol Blue) and boiled for 5 minutes. The bound proteins were separated from beads by brief centrifugation and the supernatant was analyzed by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes by semi-dry electrophoretic transfer in Tris-glycine buffer. Western blotting was performed as described previously (Vargas et al., 1997
). Western blots were developed with monoclonal anti-actin (ICN Biomedicals) at 1:1000 dilution.
Immunofluorescence labeling
E. histolytica cells resuspended in incomplete TYI-S-33 medium at 37°C were transferred onto acetone-cleaned coverslips placed in a Petri dish and allowed to adhere for 10 minutes at 37°C. The culture medium was removed and cells were fixed with 3.7% pre-warmed paraformaldehyde (PFA) for 30 minutes. After fixation, the cells were permeabilized with 0.1% Triton X-100/PBS for 1 minute. Additional permeabilization for 3 minutes with 20°C methanol was needed for myosin II and myosin IB staining. Cells were then washed with PBS and quenched for 30 minutes in PBS containing 50 mM NH4Cl. The coverslips were blocked with 1% BSA/PBS for 30 minutes, followed by incubation with primary antibody at 37°C for 1 hour. The coverslips were washed with PBS followed by 1% BSA/PBS before incubation with secondary antibody of 30 minutes at 37°C. When F-actin was labeled with phalloidin, the methanol step was omitted. Antibody dilutions used were: EhCaBP1 at 1:50 (Prasad et al., 1993), phalloidin (Sigma; 1 mg/ml) at 1:500, myosin IB at 1:30 (Voigt et al., 1999
), PAK at 1:30 (Labruyère et al., 2003
), anti-rabbit Alexa 488 (Molecular Probes) at 1:200, anti-rabbit Alexa 594 (Molecular Probes) at 1:300. The preparations were further washed with PBS and mounted on a glass slide using DABCO [1,4-diazbicyclo (2,2,2) octane (Sigma) 10 mg/ml in 80% glycerol]. The edges of the coverslip were sealed with nail-paint to avoid drying.
Confocal laser scanning microscopy
Fluorescent samples were examined on an LSM 510 confocal laser scanning microscope (Zeiss, Germany) equipped with a 63x objective. Alexa-red-labeled samples were visualized after excitation at 543 nm using a He/Ne laser, and Alexa-green-labeled samples after excitation at 488 nm using an argon laser. Focal sections of 0.8 µm with a shift of objective by 1 µm, were captured for 20-30 planes from the bottom to the top of each cell. Images were processed using LSM 510 software, Zeiss, Germany.
FITC-dextran uptake analysis
The endocytosis of E. histolytica was studied by observing the uptake of FITC-dextran. Mid-log phase cells were harvested, washed and resuspended in fresh medium. Cells were incubated with FITC-dextran (2 mg/ml, FD-40; Sigma) for 30 minutes at 36°C followed by harvesting and washing with PBS. The slides were prepared in the presence of 70% glycerol in PBS containing 0.1% 2,5-diphenyl-1,3,4-oxadiazole (PPD). The uptake was observed in the presence or absence of tetracycline (5 µg/ml) for the EhCaBP1-S and EhCaBP1-AS cell lines under a microscope with a fluorescence attachment (Axiovert 25, Zeiss). The total number of fluorescent vesicles engulfed by a cell was counted for 10 cells randomly from each slide at 100x magnification, and for each sample five such slides were counted. The amount of endocytosed material was also determined by measurement of total fluorescence in a cell using a fluorescence microscope (Varion, Cary).
Phagocytosis of red blood cells by trophozoites
To quantify the red blood cells (RBC) ingested by amoebae, the colorimetric method of estimation with some modifications was followed (Rabinovitch and Stefano, 1971). Briefly, 1x108 RBCs were washed with PBS followed by TYI-S-33 and then incubated with 1x106 amoebae for 10 minutes or, as indicated, at 37°C in 0.2 ml culture medium. The amoebae and erythrocytes were pelleted and non-engulfed RBCs were lysed with cold distilled water and centrifuged at 1000 g for 2 minutes. This step was repeated twice, followed by resuspension in 1 ml formic acid to burst amoebae containing engulfed RBCs. The optical density of the samples was determined by a spectrophotometry at 400 nm using formic acid as the blank.
Actin and EhCaBP1 co-sedimentation assay
A co-sedimentation assay was carried out following essentially the published conditions (Vargas et al., 1997). Briefly, 5 µM rabbit muscle G-actin (Sigma) per reaction was polymerized for 60 minutes in polymerization buffer containing 100 mM KCl and 2 mM MgCl2 at room temperature. After polymerization, actin was mixed with 0.2 mM ATP and the appropriate target protein (5 µM) in a total volume of 150 µl of G buffer (10 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 2.5 mM ß-mercaptoethanol, 0.5 M KCl, 10 mM MgCl2) and incubated for 2 hours at room temperature. The samples were centrifuged at 100,000 g for 45 minutes at 4°C. The supernatant (one fourth of total) and pellet fractions (total) were analyzed by 15% SDS-PAGE followed by Coomassie Blue staining. To calculate the binding affinity, increasing concentrations of EhCaBP1 (1.6 µM, 6 µM and 17 µM) were incubated with 2.5 µM actin in polymerization buffer. The supernatant and pellet fractions were collected after ultracentrifugation and analyzed by SDS-PAGE, followed by western blotting for EhCaBP1 at 1:2000 dilution. The band intensity was determined by densitometry to estimate the ratio of bound to free EhCaBP1 and calculate the binding affinity.
Solid phase assay
Different wells of a 96-well plate were coated with 5 µM G-actin in PBS overnight at 4°C and were blocked with 3% BSA in PBS for an additional 24 hours. After washing with PBS-T (Tween 20, 0.05% w/v), EhCaBP1 was added to the wells, in duplicate, at concentrations ranging from 1.7 µM to 10 µM. Bound EhCaBP1 was detected with anti-EhCaBP1 antibody followed by Alkaline phosphatase-linked anti-rabbit IgG using the colorimetric substrate p-nitro-phenylphosphate (PNPP; Sigma). The absorbance was monitored at 405 nm with a microplate reader (Bio-Rad, USA). The concentrations of EhCaBP1 were calibrated to allow reading of the absorbance under a linear range of detection.
Actin polymerization assay
Polymerization of actin was monitored by the increase in fluorescence of pyrene-labeled actin with excitation at 366 nm and emission at 407 nm. The assays were carried out at 20°C in a Safas flx spectrofluorimeter. Samples (80 µl) contained typically 2.5 µM MgATP-G-actin (10% pyrene-labeled), 6 µM EhCaBP1 and other reagents as indicated in polymerization buffer (5 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol, 0.2 mM ATP, 0.1 mM CaCl2, 0.01% NaN3, 0.1 M KCl and 1 mM MgCl2).
Atomic force microscopy (AFM)
The AFM data was collected employing contact mode AFM using the CP-Research model of Thermomicroscopes, USA. The cantilevers employed for this purpose had a force constant of 0.2 nN/m. Images were obtained at a scan rate of 1.5 Hz. The set force in contact mode applied by the cantilever was kept below 5 nN. Topographical dimensions of actin strands were analyzed using the IP 2.1 software supplied with the instrument by the manufacturer. Before each experiment fresh F-actin were prepared by polymerization in presence or absence of EhCaBP1 as described above with some modifications. Briefly, 5 µM rabbit muscle G-actin (Sigma) per reaction was polymerized for 1 hour in polymerization buffer containing 100 mM KCl and 2 mM MgCl2 at room temperature. After polymerization, actin was mixed with 0.2 mM ATP and appropriate target protein (5 µM) in a total volume of 150 µl of buffer (10 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 2.5 mM ß-mercaptoethanol, 0.5 M KCl, 10 mM MgCl2) and incubated for 3 hours at room temperature. The sample was diluted 1:5000 in double distilled water before laying on freshly cleaved mica and air dried for 30 minutes at room temperature. The specimen was then mounted on a metal disc for imaging by AFM.
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Results |
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Trophozoites in culture or in the presence of red blood cells (RBCs) were fixed, immunostained with anti-EhCaBP1-purified antibody and analyzed by laser scanning confocal microscopy (LSCM). The cellular compartments found to contain EhCaBP1 included cytoplasm, vacuoles and membrane extensions. EhCaBP1 was particularly enriched within the phagocytic cup (Fig. 1). To examine whether EhCaBP1 co-localizes with proteins that are known to be associated with the E. histolytica cytoskeleton during phagocytosis, we performed double staining of trophozoites with antibody against EhCaBP1 and one of the following: phalloidin, an F-actin cross-linker (Fig. 1A); an antibody against myosin IB (Fig. 1B), or an antibody against PAK (Fig. 1C). Actin molecules present in the RBCs were also decorated and revealed with phalloidin during these experiments. The cells undergoing erythrophagocytosis showed enrichment of F-actin (Fig. 1A), myosin IB (Fig. 1B) and PAK (Fig. 1C) around phagocytic cups and early phagosomes. EhCaBP1 was found to co-localize with F-actin and myosin IB around the phagocytic cups (Fig. 1A,B) and within membrane extensions with PAK (Fig. 1C).
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Depletion of EhCaBP1 in E. histolytica inhibits cytoskeleton-related functions such as endocytosis and phagocytosis
Membrane deformation leading to pseudopod protrusion is important for phagocytosis and cell motility in E. histolytica. The fact that EhCaBP1 localizes in protruding pseudopods prompted us to investigate the role of EhCaBP1 in these two phenomena. We took advantage of parasite strains depleted of EhCaBP1 by antisense RNA technology. In E. histolytica cells expressing EhCaBP1 antisense RNA, the level of EhCaBP1 protein is about 40% that of control parasites (Sahoo et al., 2003). The tetracycline-inducible antisense expression system was used and the cell lines carrying the two chimeric plasmids, pEhCaBP1-S (sense) and pEhCaBP1-AS (antisense) were referred to as EhCaBP1-S and EhCaBP1-AS, respectively. The EhCaBP1-AS cells showed a defect in cellular proliferation (Sahoo et al., 2003
). Endocytosis constitutes an important physiological property of E. histolytica (Batista et al., 2000
). Since amoebae ingest as much as 30% of their volume per hour and endocytosis is the major source of food and nutrients, a defect in the endocytic pathway would affect the growth of these organisms.
Fluid-phase endocytosis was inferred by determining the level of uptake of the fluorescent marker FITC-dextran in EhCaBP1-S and EhCaBP1-AS cells in the presence and absence of tetracycline. The level of endocytosis was determined by the number of vesicles containing the fluorescent marker. In EhCaBP1-expression-blocked cells uptake was reduced by 70% compared to un-induced cells (Fig. 2A). There was no significant difference in the uptake of FITC-dextran in EhCaBP1-S cells in presence or absence of tetracycline.
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Phagocytosis of human cells is a major indicator of E. histolytica pathogenesis. Erythrophagocytosis was measured by a spectrophotometric assay where the quantity of haemoglobin contained in the amoebae as a result of engulfment of RBCs was determined. The amount of erythrophagocytosis by EhCaBP1-AS cells in absence of tetracycline and EhCaBP1-S cells in presence and absence of tetracycline was found to be comparable (Fig. 2B). In contrast, EhCaBP1-AS cells displayed a reduction of 60% in phagocytosis of RBC. The decline in erythrophagocytosis activity was visible within ten minutes of addition of RBCs. The number of RBCs taken up in 10 minutes was estimated as described in Materials and Methods. EhCaBP1-AS cells engulfed about 5 RBCs in 10 minutes in comparison to 10-15 RBCs for control cells (Fig. 2C). The results show that both endocytosis and phagocytosis are inhibited in E. histolytica cells where expression of EhCaBP1 is blocked, suggesting the involvement of EhCaBP1 in these processes.
Altered actin organization in EhCaBP1-AS cells
We examined whether the reduction in EhCaBP1 could affect actin localization as phagocytosis of RBCs was inhibited in EhCaBP1 antisense-blocked cells and EhCaBP1 and F-actin co-localized around phagocytic cups. EhCaBP1-AS and EhCaBP1-AS+tet cells incubated with RBCs were fixed, stained with phalloidin and anti-EhCaBP1-purified antibody and analyzed by LCSM. The distribution of EhCaBP1 and actin in EhCaBP1-AS cells was equivalent to the distribution observed in the wild-type cells (Fig. 3A). EhCaBP1 along with actin localized around the phagocytic cups. In contrast, in tetracycline-treated EhCaBP1-AS cells, where the level of EhCaBP1 was reduced, the recruitment of actin to phagocytic cups was largely impaired (Fig. 3B), indicating that EhCaBP1 is necessary for changes in the actin-rich cytoskeleton during phagocytosis.
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Localization of EhCaBP1 and actin in the presence of inhibitors of actin dynamics
To investigate the dynamic of actin and EhCaBP1 interaction, we incubated parasites with agents that affect actin dynamics and then stained them for EhCaBP1 and F-actin. We utilized jasplakinolide, which inhibits depolymerization of F-actin, and cytochalasin D (CD), which prevents addition of G actin by capping barbed end of F-actin. In cells treated with jasplakinolide, staining of EhCaBP1 and F-actin showed colocalization of both EhCaBP1 and F-actin (Fig. 4). In CD-treated parasites, bright patches containing actin were seen and there was no co-localization of EhCaBP1 with these actin patches (Fig. 4). Both these inhibitors did not affect the cytoplasmic distribution of EhCaBP1 in E. histolytica cells. The data suggests that co-localization may involve interaction of EhCaBP1 with F-actin.
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EhCaBP1 interacts with F-actin
The cellular analysis by confocal microscopy indicated that EhCaBP1 co-localizes with F-actin in cell protrusions and this may have a role in endocytosis and phagocytosis. We addressed the question of direct interaction of EhCaBP1 with actin filaments using an in vitro test involving immunoprecipitation with specific EhCaBP1 antibody. The proteins collected along with the immune complex were analyzed by electrophoresis and immunoblotting. Antibodies against proteins related to actin cytoskeleton dynamics were used to identify the proteins co-immunoprecipitated with EhCaBP1. These included antibodies against actin, myosin IB, PAK and profilin. Of these, only actin was detected in the western blot. Thus EhCaBP1 may interact directly with actin despite the absence of any bonafide actin-binding site in this protein (Fig. 5A).
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The ability of EhCaBP1 to bind F-actin was also examined by a co-sedimentation assay. Actin filaments were incubated with purified EhCaBP1 and assessed for co-sedimentation at high speed. A known actin-binding protein -actinin, used as a positive control, co-sedimented with actin in the pellet fraction as expected (Fig. 5B, lane 8). EhCaBP1 by itself was not observed in the pellet fraction (Fig. 5B, lane 4). However, upon incubation with actin, EhCaBP1 was readily detected in the pellet fraction (Fig. 5B, lane 6). In contrast, BSA, a negative control could not be pelleted down with actin (Fig. 5B, lanes 1 and 2). This result suggests a direct interaction between F-actin and EhCaBP1. By using the co-sedimentation assay, the affinity constant of EhCaBP1 for actin was determined (Fig. 5C). The apparent Kd for EhCaBP1 binding to F-actin was 2±0.1 µM, indicating that EhCaBP1 is a potential binder of F-actin. The binding affinity was also determined by a solid phase method where binding of EhCaBP1 to G-actin was determined by an ELISA-based assay. From a standard curve of known values of EhCaBP1, the bound and free values were calculated (Fig. 5D). The Kd value of 2.6±0.3 µM was found to be similar to that obtained by sedimentation assay. The role of calcium ions in this binding was investigated by carrying out the solid phase binding reaction in presence of EGTA. There was 40-50% reduction in the amount of EhCaBP1 bound in presence of EGTA (Fig. 5F) suggesting that calcium ions play an important role, as expected.
The specificity of binding of EhCaBP1 was further tested by studying binding of EhCaBP2 to actin by co-sedimentation (Fig. 5E). The major difference between EhCaBP1 and EhCaBP2 proteins is in the central linker (Chakrabarty et al., 2004). The results showed that there was no significant binding of EhCaBP2 with actin. Since the central linker region showed maximum diversity between the two proteins the role of the central linker region in actin binding was further checked by using delcenEhCaBP1, a central-linker deletion mutant of EhCaBP1. This mutant did not bind to actin as seen from absence of the molecule in the pellet. Moreover, no significant binding was observed when EhCaBP2 was used instead of EhCaBP1 in the solid phase assay (data not shown here). The results suggested that EhCaBP1 specifically binds actin and that the interaction is probably through the central linker region.
EhCaBP1 does not affect the rate of polymerization of G-actin
It is probable that changes seen in the organization of F-actin in EhCaBP1-expression-blocked cells may be due to changes in the kinetics of actin polymerization. In order to check this hypothesis, actin polymerization assay in the presence of EhCaBP1 was carried out in vitro. The result is shown in Fig. 6. There was no significant difference in rates of nucleation and polymerization of actin in the presence or absence of EhCaBP1, suggesting that filament treadmilling is not affected by EhCaBP1.
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EhCaBP1 affects F-actin bundle thickness
Actin interacts with a very wide variety of proteins, including molecular motors such as myosins and other anchoring, sequestering and cross-linking molecules. We decided to explore whether EhCaBP1 could induce changes in the actin filaments. Electron microscopy (negative staining with uranyl acetate and rotary shadowing) of pelleted samples obtained in co-sedimentation experiments was not able to reveal any change in the organization of actin filaments recovered in the presence of EhCaBP1 (data not shown here). We decided to utilize atomic force microscopy (AFM) because of its high signal-to-noise ratio and the fact that AFM allows the observation of macromolecules in hydrated environment. Reproducible images were obtained under different ratios of F-actin to EhCaBP1 (1:1 and 1:2) and incubation periods of polymerization (1-3 hours). F-actin was seen to have varied lengths and branching patterns. Two representative images of actin filaments (polymerized for 3 hours) are shown in Fig. 7A,B. The line plot of the actin strand gives the thickness of the filaments. Images showed that F-actin appeared as a well defined fiber containing several parallel actin filaments. The average full-width of actin strands at half height calculated from ten independent points was found to be 45 nm. In the presence of EhCaBP1, the fiber consistently had thinner strands with an average width of 34 nm. Two representative images of F-actin in presence of EhCaBP1 are shown in Fig. 7C,D. The contribution of EhCaBP1 to the F-actin bundle thickness was ruled out because of its small size and inability to form complexes with self molecules. EhCaBP1 may be affecting the arrangement of F-actin filaments such that in the presence of EhCaBP1 fewer parallel strands of single filaments constitute the actin fiber.
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Discussion |
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Confocal microscopy of cells immunolabeled with EhCaBP1 antibody showed cytoplasmic localization of the protein, excluding the vesicles and the nucleus, but present also on pseudopods and beneath the plasma membrane. The association of EhCaBP1 with different components of the cytoskeleton was also visualized by co-localization studies. Amoebic pseudopods are not only enriched with cytoskeletal proteins, such as myosin IB and actin, but also with EhCaBP1. The enrichment of EhCaBP1 on pseudopods suggests its association with areas rich in actin cytoskeleton and its associated proteins. However, this enrichment pattern was not seen over the portion of the membrane not undergoing any ruffle. Therefore it appears that presence of actin in some of the areas underneath the plasma membrane ensures recruitment of EhCaBP1 or vice versa. When amoebic cells are incubated with RBCs, phagocytic cups can be seen prior to engulfment of the RBCs. EhCaBP1 is observed to be present around the cup along with actin. It is probable that EhCaBP1 is recruited early in the initiation of phagocytosis and is lost after phagocytic vesicles are formed, although association of actin with the vesicles continues. During phagocytosis of RBCs in EhCaBP1-expression-blocked cells, neither actin nor EhCaBP1 were efficiently recruited to form a phagocytic cup and this altered actin dynamics inhibiting membrane deformation and RBC uptake. Jasplakinolide treatment of cells freezes the actin filaments by inhibiting depolymerization. In these cells clear co-localization and enrichment of EhCaBP1 and F-actin was seen. The association of EhCaBP1 with F-actin in jasplakinolide-treated cells may be due to a high affinity of EhCaBP1 for actin bundles. In contrast, when the cells lost these filaments; for instance, in the presence of cytochalasin D, EhCaBP1 dissociates from the remaining array of filaments. This suggests that EhCaBP1 may not play a direct role in actin filament production but rather in the higher organization of these filaments. It is difficult to speculate on the detailed mechanism from these results at present; however, it is clear that EhCaBP1 in association with actin may be involved in dynamic changes of the actin cytoskeleton.
The E. histolytica cells blocked for EhCaBP1 expression using antisense technology were used in many experiments such as FITC-dextran uptake and erythrophagocytosis. Incubation of these expression-blocked cells with human RBCs showed that these amoebae were deficient in erythrophagocytosis, suggesting a role for EhCaBP1 in the pathogenic activity of a human parasite. The level of uptake of RBCs remained low even on prolonged incubation (30 minutes), indicating that attachment and/or intake may be affected in these cells. Fluorescent staining of the erythrophagocytosis-impaired cells failed to show active phagocytic cups around RBCs, as seen for the uninduced, transformed cells and wild-type cells.
The result with EhCaBP1 was found to be similar to the results obtained by overexpression of myosin IB (Voigt et al., 1999) that led to a modification of phagocytosis. A detailed pathway implicating these two molecules at the molecular level needs further exploration.
Whole cell imaging studies have clearly shown an association of EhCaBP1 with actin in the form of co-localization within a specific region of the cell. For in vitro status, interaction of EhCaBP1 with the cytoskeletal fraction was also demonstrated using a co-sedimentation assay and immunodetection of actin in the immunoprecipitated fraction with anti-EhCaBP1 antibody. The presence of EhCaBP1 along with F-actin in the pellet after co-sedimentation suggests their direct interaction in vitro.
The binding affinities of actin binding proteins were found to vary from 0.1 to about 1.0 µM. EhCaBP1 binds actin with an affinity constant of around 2.0 µM, which is somewhat lower than other actin binding molecules. Therefore EhCaBP1 may be required in higher amounts for cellular activity. Large phenotypic changes observed in expression-blocked cells may be caused by reduction of EhCaBP1 concentration below a threshold. There are however, proteins that bind actin with even lower affinity, such as calponin with 6.0 µM (Lu et al., 1995). The calcium-induced conformational change involves a central linker in target binding. A 50% reduction in binding to actin in the presence of EGTA again confirms calcium-dependent functionality of EhCaBP1. EhCaBP1 is also likely to be involved in different processes/pathways as it is known to bind a number of cytoplasmic proteins (Yadava et al., 1997
). The binding to the cytoskeleton may be through either actin or other molecules that are also associated with it. The binding of EhCaBP1 to actin was also visualized by AFM using an indirect gold-labeled immunological staining. EhCaBP1 appeared to be uniformly distributed on actin with no preferential site, unlike other actin binding proteins, such as N-RAP and nebulin which are found exclusively in sarcomeres and at the end of actin bundles, respectively (data not shown) (Herrera et al., 2000
). Its distribution is similar to actinin, which is found homogenously all over the stress fibers as well as in filaments (Langanger et al., 1984
).
From the data presented here it is clear that in the absence EhCaBP1 there are major changes in actin organization in E. histolytica. This may be the result of EhCaBP1 binding to actin either directly or through other proteins that interact with actin and or EhCaBP1. In general, the changes in F-actin based on activity of binders may be caused by changes in the rates of polymerization and depolymerization of filaments and in bundling of different F-actin molecules. The data presented here showed that in vitro actin polymerization and/or nucleation processes are not altered by EhCaBP1. However, changes were observed in bundling properties as seen from the AFM experiments. This indicates that the association of filaments in a bundle may be regulated by EhCaBP1. Since these experiments were carried out with pure actin and EhCaBP1 the role of other molecules in these processes may have been overlooked.
The detailed mechanism by which EhCaBP1 may be functioning needs further investigation. It may also be involved in the localization of the cytoskeletal complex at the base of the membrane in response to a stimulus signaled by calcium that initiates phagocytosis and/or endocytosis. A number of calcium binding proteins are known to exert influence on the functioning of cytoskeleton. DAP-kinase a calcium-regulated death-promoting kinase is known to bind actin filaments. One of the substrates of DAPk was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing (Shohat et al., 2002). The neuronal calcium sensors are a family of EF-hand-containing Ca2+-binding proteins. Neurocalcin a member of this family is an N-myristoylated calcium-binding protein that directly interacts with actin in a calcium-dependent manner (Mornet and Bonet-Kerrache, 2001
). There is a possibility that neurocalcin delta may be involved in the control of clathrin-coated vesicle traffic (Ivings et al., 2002
). CaM is known to participate in endocytosis and actin cytoskeleton organization (Geli et al., 1998
; Ohya and Botstein, 1994
). Regulation of cytoskeletal organization by CaM is through modulation of PtdIns(4,5)P2 levels and subsequently phospholipase D activity (Desrivieres et al., 2002
). The cytoplasm of E. histolytica has numerous granules. Calcium binding proteins have been identified in these granules that may have important role in granule discharge during cell killing (Nickel et al., 2000
). Therefore it is probable that EhCaBP1 modulates actin-mediated processes through the participation of other proteins in a complex network.
It is also possible that EhCaBP1 may be able to pull the membrane-attached actin filaments away from the membrane or denucleate these into shorter filaments, thereby allowing ingestion of RBC as phagosomes, similar to some of the actin depolymerization factors, such as ADF/cofilins.
This study suggests that EhCaBP1 might be a novel addition to the already known list of essential cytoskeleton effectors. Studies on the correlation between the already characterized factors and this novel protein, which has so far been seen only in the early branching eukaryote E. histolytica, will further help us to understand regulation of some of the fundamental processes such as phagocytosis and endocytosis in this primitive organism.
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Acknowledgments |
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