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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01198

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Calcium binding protein 1 of the protozoan parasite Entamoeba histolytica interacts with actin and is involved in cytoskeleton dynamics

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

View larger version (78K): [in a new window]   Fig. 1. Immunolocalization of EhCaBP1 in erythrophagocytosing E. histolytica. Trophozoites grown for 48 hours were transferred to pre-warmed, acetone-washed cover-slips for 10 minutes at 37°C. The attached cells were further incubated with RBC for 7 minutes. The cells were then fixed with paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS followed by immunostaining with various antibodies mentioned below. Amoebae were double labeled for EhCaBP1 and (A) F-actin (Phalloidin Red), (B) myosin IB and (C) PAK. Myosin IB and PAK were labeled with CY3-conjugated secondary antibody and EhCaBP1 with Alexa-green secondary antibody. Scale bar: 5 µm. Arrow indicates the region of enriched co-localization. Notice co-localization (yellow) of EhCaBP1 with F-actin and myosin IB around the phagocytic cup and with PAK in the pseudopod.

View larger version (25K): [in a new window]   Fig. 2. Phagocytosis and endocytosis are affected by down regulation of EhCaBP1. (A) Fluid-phase endocytosis in EhCaBP1-AS and EhCaBP1-S cells. Transformed cells were grown in 20 µg/ml hygromycin with or without 5 µg/ml tetracycline for 48 hours. For the FITC-dextran uptake assay, 2x105 cells were incubated with 1 mg/ml FITC-dextran in PBS for 30 minutes followed by thorough washing. Quantitative analysis of the fluorescent images was carried out by counting the number of vesicles containing fluorescent FITC-dextran in each cell. The data represent percentage mean (±s.d.) of fluorescent-labeled vesicles per cell. Ten cells were randomly selected per slide and counting of engulfed beads was done for five separate slides for each cell type. (B) Erythrophagocytosis in EhCaBP1-AS and EhCaBP1-S cells. Transformed cells were grown in 20 µg/ml hygromycin with or without 5 µg/ml tetracycline for 48 hours. The cells (2x106) were incubated with red blood cells (RBCs: 2x108) for the indicated times followed by washing with water to remove adhering cells. The amoebae with engulfed RBCs were then lysed with formic acid. The amount of heme was determined by measuring the optical density at 400 nm for the different cell types indicated. Values are mean ± s.d. (C) The number of RBCs internalized for each amoeba cell-type. The data from sector B was used to compute the number of RBCs internalized as described in Materials and Methods.

View larger version (54K): [in a new window]   Fig. 3. Distribution of F-actin is affected in tetracycline-induced EhCaBP1-AS cells during phagocytosis. The attached EhCaBP1-AS (A) and EhCaBP1-AS+tet (B) cells were incubated with RBCs for 7 minutes prior to fixing and immunostaining. Amoebae were double labeled for EhCaBP1 and F-actin (Phalloidin red). EhCaBP1 was labeled with Alexa-green secondary antibody. Notice the actin rich phagocytic cup in the EhCaBP1-AS cell (arrow), absence of a cup around the RBC (arrow) and reduced expression of EhCaBP1 in EhCaBP1-AS+tet cell.

View larger version (48K): [in a new window]   Fig. 4. Distribution of F-actin and EhCaBP1 in jasplakinolide- and cytochalasin D-treated cells. The HM-1: IMSS cells grown for 48 hours were incubated at 37°C with jasplakinolide at 10 µM for 30 minutes or cytochalasin D at 10 µM in a reaction volume of 1 ml for 15 minutes or with DMSO alone. The cells were pelleted and fixed before immunostaining with rhodamine-phalloidin (red) and anti-EhCaBP1 antibody. EhCaBP1 was labeled with Alexa-green secondary antibody. Finally the cell-pellet was diluted in DABCO for laying on glass slides. Notice enrichment of EhCaBP1 in areas where F-actin was fixed by jasplakinolide. Scale bar: 5 µm.

View larger version (48K): [in a new window]   Fig. 5. Binding of EhCaBP1 to actin. (A) Immunoprecipitation of amoebic proteins with anti-EhCaBP1 antibody and western analysis with anti-actin antibody. E. histolytica cell lysate was used for immunoprecipitation using anti-EhCaBP1 antibody and protein A beads. The immunoprecipitate was eluted from beads in SDS-PAGE buffer and separated on a 10% SDS-PAGE gel. The proteins were immobilized on nitrocellulose by electrophoretic transfer for immunostaining with anti-actin antibody and anti-mouse IgG linked to peroxidase. The bound antibody was detected using the ECL system. Lanes 1: immunoprecipitated amoebic proteins; lane 2: control precipitation with only protein A beads; lane 3: total lysate (10 µg protein). Molecular mass is indicated. (B) Co-sedimentation of EhCaBP1 with F-actin. Purified recombinant EhCaBP1 (5 µM), -actinin (5 µM) or bovine serum albumin (5 µM) were incubated with F-actin (5 µM), in polymerization buffer containing salts, followed by ultracentrifugation to separate the soluble and pellet fractions. Pellet (p) and supernatant (s) were resolved in 15% SDS-PAGE followed by Coomassie Blue staining. Total pellet fraction and one fourth of total supernatant was loaded for gel analysis. (C) Immunoblotting of EhCaBP1 co-sedimented with F-actin. Different amount of EhCaBP1 (1.6 µM, 6 µM and 17 µM) was incubated with F-actin in polymerization buffer containing salt as described in B. The pellet and supernatant fraction after ultracentrifugation were resolved in 15% SDS-PAGE followed by western analysis with anti-EhCaBP1 antibody as described in Materials and Methods. The amount of EhCaBP1 co-sedimented was determined by densitometry. The binding affinity was computed using a Scatchard plot. (D) Scatchard plot for EhCaBP1-actin interaction. Different wells of a microtiter plate were coated with 50 µl of 5 µM actin overnight at 4°C. After blocking with BSA, EhCaBP1 was added at the indicated concentrations ranging from 10 µM to 0.1 µM, followed by incubation with anti-EhCaBP1 antibody. The amount of bound EhCaBP1 was determined using anti-rabbit alkaline phosphatase-linked IgG. The amount of product formed was detected at 405 nm. (E) Co-sedimentation of EhCaBP1, delcenEhCaBP1 and EhCaBP2 with actin. Purified EhCaBP1, delcenEhCaBP1 or EhCaBP2 (5 µM), were incubated with F-actin (5 µM) in sedimentation buffer, followed by ultracentrifugation to separate the soluble and pellet fractions as described in Materials and Methods. Pellet and supernatant fractions were resolved in 15% SDS-PAGE followed by Coomassie Blue staining. Arrow indicates the position of soluble EhCaBP1 (14 kDa). (F) Calcium requirement for binding of EhCaBP1 to actin. Binding was carried out by solid phase assay in the presence of excess calcium (5 mM) or EGTA (2 mM). The plot shows the relative mean intensity (as the percentage ±s.d.) obtained from three independent experiments.

View larger version (12K): [in a new window]   Fig. 6. Effect of EhCaBP1 on actin polymerization. Actin-labeled with pyrene (2.5 µM) with or without EhCaBP1 (6 µM) was polymerized as described in Materials and Methods. The fluorescence of pyrene actin was observed as percentage maximum over a period of 3 hours.

View larger version (65K): [in a new window]   Fig. 7. Atomic force micrographs of F-actin. Actin (5 µM) was incubated in polymerization buffer in absence (A,B) or presence of EhCaBP1 (C,D). The samples were diluted 1:5000 times before mounting on freshly cleaved mica for atomic force microscopy. (A,C) two-dimensional view; 2 µm scan. (B,D) Three-dimensional top view; 0.8 µm scan.