Coactosin Phosphorylation Controls Entamoeba histolytica Cell Membrane Protrusions and Cell Motility

Differential phosphoproteomes of EhMSP-1-silenced and control E. histolytica trophozoites. (A) Identification and quantification of phosphopeptides. After phosphopeptide enrichment (IMAC and TiO₂), 60 dimethyl-labeled phosphopeptides were identified and quantified in both independent biological replicates (Bio Rep 1 and 2; indicated in red and black, respectively). The points indicating log₂ ratios [EhMSP-1 (−)/WT] corresponding to the EhCoactosin (EHI_168340A) S147-containing phosphopeptide of interest is circled for each biological replicate. (B) Heat map showing the log₂ ratios [EhMSP-1 (−)/WT] for 6 unique phosphopeptides with a change of either >1.5-fold or <0.67-fold and coefficient of variation of <30% in both biological replicates, listed from high to low according to their average log₂ fold change. Note that the EhCoactosin S147-containing phosphopeptide (black dots; EHI_168340A) was detected as both a fully trypsin-digested peptide and a miscleaved variant. See also Table S2 in the supplemental material.

Phosphorylation of EhCoactosin at S147. (A) Identification of EhCoactosin S147-containing phosphopeptide. The coactosin S147 phosphopeptide (EHI_168340A) KAGGADYSFNTTS(phospho)N was identified via SEQUEST with XCorr = 3.31, and precursor ΔM (experimental m/z and theoretical m/z) = −0.23 ppm. The MS/MS spectrum is annotated by Scaffold. The primary amines at the N terminus and on the lysine residue are dimethyl labeled. (B) Increased abundance of EhCoactosin S147-containing phosphopeptide in EhMSP-1-silenced Entamoeba histolytica. The MS1 spectrum of the light- (dimethyl-d0) and heavy-labeled (dimethyl-d6) EhCoactosin S147-containing phosphopeptides and the extracted ion chromatograms showing the light and heavy isotopologues eluting symmetrically at the same retention time are shown on the left and right, respectively. Heavy/light (H/L) was quantified by precursor ion elution profiles to be an average of 1.93 from multiple measurements from both biological replicates (one representative chromatograph [H/L = 1.98] is shown).

Expression and localization of nonphosphorylatable (S147A) and phosphomimetic (S147D) EhCoactosin variants. HM-1:IMSS strain trophozoites were transfected with expression vectors designed for tetracycline-regulated EhCoactosin expression with an N-terminal hemagglutinin (HA) epitope tag fused to either wild type (Coac-WT), S147A (Coac-A), or S147D (Coac-D). (A) Western blot. Protein expression was induced with tetracycline followed by immunoprecipitation (IP) and blotting with an anti-HA antibody. The expected protein size was 17 kDa. (B) Localization of EhCoactosin and S147A and S147D variants during erythrophagocytosis. Trophozoites expressing each EhCoactosin variant protein were incubated with human erythrocytes and then prepared for fluorescence microscopy by staining F-actin with phalloidin (green) and the EhCoactosin proteins with a mouse anti-HA monoclonal antibody. The Coac-WT, Coac-A, and Coac-D proteins all localized to the phagocytic cup. Bars, 10 μm.

Phosphomimetic (S147D) EhCoactosin expression increased the formation of E. histolytica cell membrane protrusions. Qualitative differences in appearance of the empty vector control-, wild-type (Coac-WT)-, S147A (Coac-A)-, and S147D (Coac-D)-expressing trophozoites in culture were quantified using the NIH ImageJ plugin QuimP3 and time-lapse light microscopy. (A) Representative time-lapse images for coactosin-overexpressing and vector control trophozoites. An increased rate of pseudopod formation in the Coac-D-expressing trophozoites was immediately evident in tissue culture (see also Movies S1 and S2). Time is set as 0 s for the first image of the presented series. Yellow-green lines denote membrane protrusion sites identified by QuimP3. Regions of cell membrane scored by QuimP3 are highlighted by program-generated lines color-coded according to the rate of expansion (as in panel B). For clarity, these areas are indicated with arrows at the time of first appearance (i.e., one arrow per pseudopod scored by QuimP3). (B) Motility maps generated by QuimP3 for video microscopy of representative empty vector, Coac-WT, Coac-A, and Coac-D trophozoites. The x axis denotes the normalized position of 1,200 defined nodes along the cell perimeter, and the y axis denotes the video frame number (i.e., time). The color scale corresponds to membrane velocity at each point along the cell perimeter at a given time. Cell protrusions are identified as sites of maximum local velocity. (C) Effect of EhCoactosin S147A and S147D expression on the number of cell membrane protrusions versus time. Data for the empty vector, Coac-WT, Coac-A, and Coac-D strains are shown. Cells (n = 40 per cell line) were imaged for 3 min with 0.4-s intervals, and cell protrusions were identified with QuimP3. Data shown are the number of cell protrusions formed/minute by each analyzed amoeba, along with the mean and standard deviation (SD) for each cell line. ***, P < 0.0001 for ordinary one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test.

EhCoactosin S147A expression increases the range of E. histolytica movement within a model extracellular matrix. Trophozoites trapped within Matrigel matrix were imaged for 2 h at 2-min intervals, and movement of individual trophozoites was tracked using the manual tracking plugin of NIH ImageJ. Six independent experiments were conducted with the empty vector-, wild type (Coac-WT)-, EhCoactosin S147A (Coac-A)-, and EhCoactosin S147D (Coac-D)-expressing trophozoites, with 18 trophozoites imaged/cell line/experiment. (A) Superimposed motility tracks for the vector control and EhCoactosin WT-, S147A (Coac-A)-, and S147D (Coac-D)-overexpressing trophozoites. Tracks from one experiment are shown (n = 18 per cell line). Pixel coordinates for each trophozoite from the original images were corrected to the midpoints of the extreme x and y axis values for display on a single graph. Pixel length = 0.32 μM. (B) Effect of EhCoactosin S147A and S147D expression on amoebic movement. The distance between the two farthest points was determined for each trophozoite and used to determine the mean range of movement (i.e., maximum distance moved) for each cell line during a given experiment. Each data point represents the mean maximum distance moved for the 18 trophozoites of that cell line measured in one experiment, with data for six biological replicates shown. *, P < 0.03 by one-way ANOVA with Tukey’s multiple-comparison test.