Figure 1.
Distribution of EhCaBP3, EhCaBP1 and actin in E. histolytica during erythrophagocytosis.
Localization of endogenous EhCaBP3 and actin (A) and EhCaBP3 and EhCaBP1 (B). Cells were grown for 48 h and incubated with RBC for 10 min at 37°C. The cells were then fixed and immunostained with rabbit anti- EhCaBP3 antibody followed by Alexa-488 (green) secondary antibody, and F-actin was stained with TRITC-phalloidin (red) and viewed using confocal microscope. The secondary antibody used for EhCaBP1 was Alexa-556 (red). Arrow heads represent phagocytic cups and an asterisk mark showing the enrichment of EhCaBP3 in phagosome. Bar represents 10 µm. (DIC, differential interference contrast). (C) Schematic representation of GFP-EhCaBP3 constructs. (D) Western blot of amoebic cell lysates expressing GFP-EhCaBP3 construct at different G418 concentrations (10, 20, 30 µg ml−1). Thirty microgram of the lysate was loaded in each lane and the blot was probed with anti-GFP antibody. The blots were stripped and re-probed with anti-EhCaBP3 antibody. The EhCaBP3 antibody stains both endogenous EhCaBP3 band at 17 kDa and GFP-fused EhCaBP3 at 43 kDa. (E–G) Immunolocalization of GFP-EhCaBP3 in E.histolytica during erythrophagocytosis. Cells expressing GFP alone (E) or GFP-EhCaBP3 (F, G) were incubated with RBC, followed by fixation and immunostaining. GFP− tagged protein were labeled using anti-GFP antibody and F-actin by phalloidin (red), followed by Alexa 488 (green) secondary antibody. (Scale bar, 10 µm; DIC, differential interference contrast).
Figure 2.
Time lapse imaging of GFP-EhCaBP3.
Micrograph showing the de novo formation of a phagocytic cup during phagocytosis of RBC by an amoeba expressing GFP-EhCaBP3. The montage shows a time series of a representative cell showing the formation of phagocytic cup (arrow head), the closure of cup (star) and finally a complete phagosome (asterisk). Bar represents 20 µm.
Figure 3.
(A) Co-sedimentation assay of EhCaBP3 with F-actin. Polymerized F-actin (5 µM) was incubated with purified recombinant proteins EhCaBP3 (5 µM, lanes 7, 8), EhCaBP1 (5 µM, lanes 3, 4) and EhCaBP2 (5 µM, lanes 5, 6) as indicated. EhCaBP3 and EhCaBP1 were also incubated with actin (lanes 11, 12). EhCaBP3 alone was incubated without actin (lanes 9, 10). Actin alone without CaBP3 (lanes 1, 2). Ultra-centrifugation (100,000×g) was performed to separate supernatant (S) and pellet (P) fractions, followed by SDS-PAGE. (B) EhCaBP3 binding to G-actin. 5 µM Actin was coated overnight on 96well plate at 4°C, followed by blocking with BSA. EhCaBP3 was added in increasing concentrations as indicated in the presence (5 mM CaCl2) or the absence of Ca2+ (2 mM EGTA) and EhCaBP1 (5 µM) was used as a control. This was followed by incubation with anti-EhCaBP3 or anti-EhCaBP1 antibodies. The amount of bound protein was detected using anti-rabbit IgG-HRPO. Absorbance was determined at 405 nm. The histogram shows relative mean intensity ± SD of three independent experiments. (C) Effect of EhCaBP3 on actin polymerization. Pyrene-labeled actin was polymerized without and with indicated concentrations of EhCaBP3 as described in “Materials and methods”. The fluorescence of pyrene was observed at 407 nm. (D) F-actin bundling in presence of EhCaBP3. Polymerized F-actin (3 µM) was incubated with EhCaBP3 (5 µM) in presence of 5 mM CaCl2 and 2 mM EGTA for 2 h. The samples were then centrifuged at (10,000×g) for 15 min. Under this condition, unbundled F-actin remains in supernatant and bundled F-actin sediments. Both supernatant (S) and pellet (P) were analyzed by SDS-PAGE.
Figure 4.
Immunolocalization of EhCaBP3 and myosin IB in erythrophagocytosing E. histolytica.
Cells were first grown for 48 hours and then incubated with RBCs for indicated time points at 37°C. These were then fixed, permeabilized followed by immunostaining with anti-GFP and anti-myosin IB antibodies, followed by Alexa 488 (green, EhCaBP3) and Alexa 556 (red, myosin IB) secondary antibodies. Scale bar: 10 µm. Solid arrow indicates the region of enriched co-localization of these two proteins at phagocytic cups, open arrow head denotes attached RBC and star shows their co-localization during closure of phagocytic cups.
Figure 5.
Interaction of EhCaBP3 with myosin 1B.
Total E. histolytica lysate (800 µg) was incubated with Sepharose-anti-CaBP3 antibody conjugate for 6 h at 4°C with shaking. The beads were then washed and the bound material was then eluted and analysed by western blotting and immunostained with anti-myosin 1B antibody raised in rabbit. The blot was reprobed with anti-CaBP3 antibody raised in mice. The total input lysate was also probed for the presence of EhCaBP3 and myosin 1B by their respective antibodies.
Figure 6.
Generation and functional characterization of EhCaBP3mEF.
(A) Schematic representation of EhCaBP3 indicating the three EF hand domains. The sequence of all the EF hands is shown and the first aspartate (D) of each EF hand and the last (E) amino acid residue was mutated to alanine (A) to generate CaBP3mEF mutant. (B) Cosedimentation assay of EhCaBP3 mutant with F-actin. Briefly 5 µM of F-actin was incubated with target proteins for 2 h. This was followed by ultracentrifugation and SDS-PAGE. (C) Solid-phase assay showing that CaBP3mEF was not able to bind G-actin efficiently as compared to wild type (WT). EhCaBP3mEF was added as indicated in the presence of 5 mM CaCl2 and 2 mM EGTA. EhCaBP3 (10 µM) was used as a control. (D) Cells expressing GFP-CaBP3mEF were maintained at different G418 concentrations (5, 10, 30 µg ml−1). Thirty microgram of the lysate was used for western blotting and protein was detected using anti-GFP antibody or anti-CaBP3 antibody. (E) Erythrophagocytosis of cells expressing GFP-CaBP3mEF. The slides were prepared as described in Experimental procedures. GFP-CaBP3mEF was stained with anti-GFP antibody (green) and TRITC-phalloidin (red) and viewed using confocal microscope. Upper panel: Confocal section of a representative cell showing enrichment of mutant in phagocytic cups and its co-localization with F-actin. Lower panel: Lack of EhCaBP3 mutant in phagosome. Bar represents 10 µm. Note the accumulation of GFP− CaBP3mEF in the phagocytic cup (arrow) and absent in phagosome (asterisk). (F) EhCaBP3mEF undergoing erythrophagocytosis followed by fixation. The cells were stained for EhCaBP3 or mutant (green) and TMK-9(red). Phagocytic cup represented by arrow heads and a phagosome by asterisk. Bar represents 10 µm. (G) Immunostaining of myosin 1B in the cells expressing GFP-EhCaBP3mEF. Cells were stained with anti-GFP antibody (green) and myosin 1B antibody (red). Arrow heads indicate the presence of EhCaBP3mEF and the absence of myosin 1B in the phagocytic cups. (H) Graph represents the number of phagocytic cups and phagosomes in over-expressed cell lines of EhCaBP3 and EhCaBP3mEF. Fifty cells were randomly selected for each experiment and counted the number of phagocytic cups and phagosomes present in all cells (blue, EhCaBP3 and red, EhCaBP3mEF).
Figure 7.
Downregulation of EhCaBP3 reduces the rate of phagocytosis.
(A) Schematic representation of sense and anti-sense constructs. EhCaBP3 was cloned in the sense and the anti-sense orientation in BamH1 and Kpn1 sites of pEhHyg-TetR-O-CAT vector. (B, C) Western blot analysis of cell lines carrying the anti-Sense (B) and Sense (C) constructs. Thirty microgram of lysate from indicated cells in the presence and the absence of tetracycline were analyzed. Cells carrying without any construct were used as positive control and EhCaBP1 as loading control. (D) RBC uptake assay performed in cells expressing the sense and the anti-sense constructs in the presence and the absence of tetracycline. The experiment was repeated three times independently in triplicates. ANOVA test was used for statistical comparisons. P-values for * and ** are P<0.01 and P<0.001, respectively. #P>0.05. (E) Amoebic cells with and without indicated constructs were incubated with red blood cells (RBCs). These cells were then fixed and stained with TRITC-Phalloidin. The accumulation of actin at phagocytic cups were marked by a solid arrow heads and phagosomes were marked by asterisk. Open arrow heads mark attached RBCs at the site of phagocytosis. (F) Seventy five cells were randomly selected for each experiment and the number of phagocytic cups present in all cells were counted (blue, normal HM1: MSS; red, EhCaBP3S; green, EhCaBP3AS).
Figure 8.
Distribution of myosin IB and actin in cells expressing EhCaBP3AS and EhCaBP3S.
(A) Immunolocalization of myosin 1B and actin in EhCaBP3AS cell line. Cells were incubated with RBCs at indicated time points and then were fixed and stained with anti-myosin IB antibody (red) and FITC-phalloidin (green). (B) Immunolocalization of myosin 1B and actin in EhCaBP3S cell line. B1- Cells overexpressing EhCaBP3S were incubated with erythrocytes and were labeled with anti-EhCaBP3 antibody (Green) or TRITC-phalloidin (red). B2- Cells overexpressing EhCaBP3S were incubated with erythrocytes and were labeled with anti-myosin antibody (red) or FITC-phalloidin (green). Solid arrow heads indicate phagocytic cups and star shows the presence of CaBP3 and myosin 1B; and absence of actin at the site of cup closure.
Figure 9.
Localization of EhC2PK and EhCaBP1 in the anti-sense cell line.
Cells were grown in the presence of tetracycline for 24 h and then incubated with RBCs at different time points. The cells were then fixed and immunostained with specific antibodies as described before. The antibodies used were anti-CaBP3 (green) and anti-CaBP1 (red) antibodies or anti-C2PK antibody (red) and anti-CaBP1 antibody (green) and viewed using Confocal Scanning Laser Microscope. Arrow head depicts the mild accumulation of EhCaBP1 and EhC2PK at the site of phagocytosis. Scale bar represents 10 µM.