Fig 1.
14-3-3 family members and their phylogenetic analysis.
(A) Sequence alignment of Eh14-3-3 proteins. The full-length Eh14-3-3 protein sequences (EhP1, EhP2, EhP3) has been aligned with the seven human 14-3-3 isoforms (h14-3-3beta/alpha, h14-3-3theta, h14-3-3gamma, h14-3-3zeta/delta, h14-3-3eta, h14-3-3sigma, h14-3-3epsilon) using Clustal X (V2.1) and CLC sequence viewer (v6.3). Conserved residues are indicated as % conservation bar (0–100%) below the aligned sequences. Residues representing 14-3-3 protein signatures 1 and 2 are indicated by dark black lines. (B) Phylogenetic analysis of EhP3 proteins. Phylogenetic tree was constructed using an iterative neighbor-joining algorithm of MEGA 7.0 software. Bootstrap values (as percentages) are shown at each node.
Fig 2.
Live imaging montage showing dynamics of GFP-EhP3 in motile trophozoites.
(A) The montage depicts a time series of selected frames of fluorescent images of motile trophozoites expressing GFP-EhP3. A number of pseudopods showing increased fluorescent intensity of GFP-EhP3 marked by white arrow head can be visualized in different directions. (Scale bar, 10 μm). (B) The graph represents time course of intensity of GFP-EhP3 at selected ROI in pseudopods vs. cytoplasm. Fluorescent intensity gradually increased at pseudopods in comparison with cytoplasm of the cells. (C) Western blot analysis of GFP-EhP3 in the parasite lysate. 50 μg of the lysate prepared from E. histolytica cells expressing GFP-EhP3 and GFP alone, grown in presence of 30 μg/ml G418 was loaded in each lane and probed with anti-GFP antibody. Anti-GFP antibody detected GFP fused 53 kDa protein (EhP3 + GFP) band in GFP-EhP3 expressing cells as compared to 26 kDa band of GFP alone in control cells. Anti-EhCaBP1 antibody was taken as equal loading control.
Fig 3.
Time-lapse imaging of GFP-EhP3 during phagocytosis of red blood cells (RBCs).
(A) The montage shows time-lapse images of an amoeba cell expressing GFP-EhP3 undergoing phagocytosis of RBCs. RBCs were stained with DiD dye. Represented images are from two different phagocytic events showing de novo formation of a phagocytic cup, closure of cup and formation of phagosome, marked by white arrow heads. GFP-EhP3 accumulated rapidly at the site of attachment of RBC and remained till the formation of phagosome. (Scale bar, 10 μm). (B) The graph represents intensity profile of GFP-EhP3 at selected ROI in phagocytic cup vs. cytoplasm during phagocytosis.
Fig 4.
Immunolocalization of EhP3 in fixed trophozoites during phagocytosis of RBCs and CHO cells.
(A) Localization of EhP3 during different stages of phagocytosis of RBCs. E. histolytica trophozoites actively phagocytosing RBCs were fixed and stained with EhP3 antibody and TRITC phalloidin (for visualisation of F-actin). Localization of EhP3 during different stages of phagocytosis are indicated as arrowheads (nucleation and progression of phagocytic cups), asterisks (closure of cup before scission) and stars (phagosomes). (B) Localization of EhP3 during phagocytosis of CHO cells. E. histolytica trophozoites phagocytosing CHO cells stained with cell tracker blue CMAC dye, were fixed and stained for GFP antibody. Localization of EhP3 are shown at phagocytic cups and phagosomes. (C and D) Co-localization of EhP3 with respect to phagocytic markers. (C) Co-localization of EhP3 with EhCaBP3 in phagocytic cups, just closed cups before scission and phagosomes. (Scale bar, 10 μm; DIC, differential interference contrast). (D) Graph showing the Pearson’s correlation coefficient (r) values of EhP3 with each phagocytic marker protein (EhCaBP1, EhCaBP3, EhAK1, Ehactin) from phagocytic cups. Analysis were done from 10 cells for each of the respective markers, by using Olympus Fluoview FV1000 software.
Fig 5.
EhP3 is essential for phagocytosis.
(A) (i) Expression analysis of EhP3 in sense and antisense cell lines. 50 μg of the lysate prepared from E. histolytica cells expressing either Tet-O-CAT (TOC) vector alone, antisense EhP3 (AS) or sense EhP3 (S) gene grown in presence and absence of tetracycline (10, 30μg/ml) were loaded in respective lanes. The lanes were probed with anti-EhP3 antibody for expression analysis and anti-EhCaBP1 antibody was taken as equal loading control. (ii) Erythrocyte uptake assay in sense and antisense cell lines. E. histolytica cells carrying the above mentioned constructs were incubated with RBCs for the indicated time points and assessed for RBC uptake by spectrophotometric analysis. The experiments were repeated independently three times in duplicate with error bars indicating the standard error. The statistical comparisons were carried out using oneway AnoVA test (p-value 0.00005). (B) (i) and (ii) Visualization of phagocytic events in sense and antisense cell lines. Cells incubated with RBCs were fixed and stained for actin with TRITC-Phalloidin, or with anti-EhP3 antibody followed by Alexa-488. Image panels in the left indicate TRITC-Phalloidin stained multiple number of EhP3 sense cells (i) and antisense cells (ii), (Scale bar, 5 μm; DIC, differential interference contrast). Image panels in the right indicate cells stained with anti-EhP3 antibody and TRITC-Phalloidin respectively, (Scale bar, 10 μm; DIC, differential interference contrast). (C) (i) and (ii) Quantitative determination of phagocytic events (phagocytic cups and phagosomes) observed in above indicated cell lines. Number of phagocytic cups and phagosomes were counted in randomly selected 50 cells (in triplicates) at denoted time points. One-way ANOVA test was used for statistical comparisons. “*” p-value 0.05, “**” p-value 0.005, “***” p-value 0.0005, “****” p-value 0.00005.
Fig 6.
(A) Graph depicts analysis of G-actin solid phase plate binding assay for EhP3. The histogram represents relative mean intensity of fluorescence observed on binding to actin. CaBP1 was used as binding control. (B) Immunoblot analysis of the F-actin co-sedimentation assay for EhP3. Purified EhP3 protein incubated at different concentration (2.5, 5, 10 μM) with polymerised G-actin (5 μM) or without F-actin were separated into supernatant (S) and pellet (P) fractions by high speed ultracentrifugation and analysed by SDS PAGE. (S-supernatant, P-pellet). (C) Model summarising reported/predicted interactions between 14-3-3 and actin regulatory proteins that controls cell cytoskeleton remodelling in higher systems. The 14-3-3 protein interact indirectly or directly with many Rho regulators, particularly Rac1 binds directly with 14-3-3. The 14-3-3 proteins also control the activity of the ubiquitous F-actin depolymerizing and severing factor cofilin via binding and stabilizing cellular phosphocofilin levels. Interaction of 14-3-3 with other proteins of the ADF/cofilin family (Twinfilin, Coactosin) warrants further investigation. The binding of 14-3-3 with structural proteins (filamin, clathrin), anchoring proteins (spectrin), sequestering proteins (profilin) have been depicted in the proteome studies.
Fig 7.
Interaction of EhP3 with EhCoactosin and its recruitment at the site of phagocytosis.
(A) (i) SPR sensorgram showing the interaction between EhP3 (immobilized) and different set of recombinant actin binding proteins (Coactosin (red), Actophorin (purple), Twinfilin (yellow), Profilin A, B (dark green, blue), Filamin (light green)) at 200 nM concentration as indicated. (ii) SPR sensogram showing the interaction between EhP3 (immobilized) and increasing concentrations of recombinant Coactosin (1, 2 4, 8, 10 μM) with an association constant of 3 μM (B) Validation of EhP3/ EhCoactosin interaction by immunoblot analysis of the coimmunoprecipitated proteins using their respective immune and pre-immune antibodies. A clear band of both EhP3 and EhCoactosin were detected in the pull down with either EhP3 or EhCoactosin antibodies. Total cell lysate was also probed with the respective immune and pre-immune antibodies as control. (C) Immunofluorescence analysis of EhCoactosin in cells carrying antisense construct of EhP3 in the presence and absence of tetracycline. Amoebic cells containing EhP3-AS constructs were incubated with RBCs, fixed and stained with TRITC-Phalloidin, anti-EhP3 or, anti-EhCoactosin antibodies followed by Alexa-488 (EhCoactosin) or, Pacific blue-410 (EhP3). White arrowheads indicate phagocytic cups, asterisks indicate the closure of cups in EhP3-AS cell line in absence of tetracycline and red arrowheads indicate RBC attachment site in tetracycline induced cells. (Scale bar, 5 μm; DIC, differential interference contrast). (D) Quantitative analysis of relative intensity of EhCoactosin (N = 10 cells) at phagocytic cups in EhP3-AS cell line in absence of tetracycline and at RBC attachment site in presence of tetracycline. One-way ANOVA test was used for statistical comparisons. (E) Immunoblot analysis of amoebic cells showing the level of EhCoactosin in tet-inducible vector alone and cells expressing EhP3-AS construct, in the presence and the absence of tetracycline. EhCaBP1 was used as loading control.