EhRacM differentially regulates macropinocytosis and motility in the enteric protozoan parasite Entamoeba histolytica

Abstract

Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica, the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E. histolytica. Through gene silencing of highly transcribed Ehrho/Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM's multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E. histolytica, highlighting the significance of EhRacM in both macropinocytosis and cellular migration.

Fig 1. Gene silencing of Rho small GTPases in E. histolytica trophozoites.

(A) Confirmation of gene silencing by RT-PCR analysis of Ehrho/Ehrac gene silenced (Ehrho gs) strains. Transcripts of indicated Ehrho/Ehrac and RNA polymerase II (RNA pol II, EHI_056690) genes were amplified by RT-PCR from cDNA isolated from the transformants and examined by agarose gel electrophoresis. Mock control generated by transfection with empty psAP2-Gunma vector was used as a reference (psAP2 (mock)). (B) qRT-PCR analysis of the relative expression level of the targeted Ehrho/Ehrac gene in each Ehrho/Ehrac gs strain and psAP2 mock strain. The steady state mRNA levels of Ehrho/Ehrac genes are indicated relative to that of RNA pol II. Statistical significance was examined with unpaired t-test, and p-values are shown in the graph (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant). Error bars indicate standard deviations of three replicates.

Fig 2. EhracM gene silencing enhanced macropinocytosis.

(A) Macropinocytosis of EhracM and EhracJ gene silenced (gs) and psAP2 mock control strains. Trophozoites were incubated in RITC dextran-containing BIS medium to evaluate macropinocytosis. The fluorescence intensity of incorporated RITC-dextran by each strain was measured by FACS as described in Materials and Methods. RITC-dextran incorporation of each strain was estimated by calculating the geometric mean of the fluorescence intensity after the background signal from unlabeled parasites was subtracted from the fluorescence intensity in PE-A channel of each strain, and is shown relative to the value of mock strain at 120 min. Statistical significance was examined with Two-Way ANOVA (*p<0.05, ***p<0.001, ****p<0.0001, ns: not significant). Error bars indicate standard deviations of three biological replicates. (B) Cell volume of EhracM, EhracJ gs, and control strains. Cell volume was estimated from the cell diameter measured by CASY system as described in Materials and Methods. Statistical significance was examined with One-Way ANOVA (**p<0.01, ns: not significant). Error bars indicate standard deviations of three biological replicates.

Fig 3. Gene silencing of EhracM caused defects in linear and directed movement.

(A) Representative micrographs showing trajectories of live trophozoites (magenta) migrating on a glass surface. Left panel, EhracM gs strain; right panel, psAP2 mock control strain. The images were acquired with CQ1 (Yokogawa). Tracking of the cell trajectory was shown in lines. Bars, 100 μm. (B) A dot in five panels indicates the total distance, net distance, maximum distance, linearity of forward progression, and confinement ratio of each trophozoite during 300 seconds of incubation. 121 trophozoites of EhracM gs strain and 154 trophozoites of psAP2 mock strain were included in the analysis. Average values are shown as bars. Statistical significance was examined with an unpaired t-test (ns: not significant, ****p<0.0001).

Fig 4. Expression and cellular localization of HA—and GFP-fused EhRacM in E. histolytica trophozoite.

(A and B) Immunoblot detection of HA-EhRacM and GFP-EhRacM in E. histolytica transformants. Approximately 30 μg of total lysates from HA- or GFP-fused EhRacM expressing transformants and mock-transfected control (pEhEx-HA or pEhEx-GFP) were subjected to SDS-PAGE and immunoblot analysis using anti-HA monoclonal antibody (A), anti-GFP monoclonal antibody (B), and anti-CS1 polyclonal antibody (loading control). An arrow indicates HA-EhRacM (A) or GFP-EhRacM (B). (C) The immunofluorescence image of a representative trophozoite of HA-EhRacM strain. HA-EhRacM was visualized with anti-HA antibody (left). A yellow arrow indicates the trajectory of the fluorescence intensity plot shown in the right panel. The arrowheads with alphabets indicate ~10 positions where HA-EhRacM intensity peaked. (D) Immunoblot analysis of HA-EhRacM (left panel) and GFP-EhRacM (right panel) in fractionated cell lysates. Homogenate (Ho) from trophozoites of HA-EhRacM/GFP-EhRacM expressing transformant was fractionated by centrifugation into the low-speed pellet (p13, the pellet fraction of 13,000 × G centrifugation), the high-speed pellet (p100, the pellet fraction of 100,000 × G centrifugation) and the supernatant fractions (s100, the supernatant fraction of 100,000 × G centrifugation). These fractions were subjected to immunoblot analysis using anti-HA/anti-GFP, anti-CS1 (a cytosolic protein), and anti-HgL (a membrane protein) antibodies. The arrows indicate the approximate sizes of HA-EhRacM (left) and GFP-EhRacM (right), respectively.

Fig 5. GFP-EhRacM was recruited to the dextran-containing macropinosomes.

Montage of live imaging of GFP-EhRacM (green) expressing trophozoites that were incubated with RITC dextran (red). The white arrowheads show the site of macropinocytic cup formation and the resultant macropinosome. Three times-magnified images of the regions enclosed by a white square are displayed in the third row in an inverted grayscale. The fourth row shows the fluorescence intensity plots along with the trajectory of yellow arrows depicted in the corresponding third-row panels. Macropinosome areas are highlighted in red, with the GFP signal intensity at the initial macropinocytic cup formation (approximately 18) shown in green. Bars, 5 μm.

Fig 6. GFP-EhRacM persisted on macropinosomes even after 2 hours.

Montage of live imaging of GFP-EhRacM expressing trophozoites incubated with RITC dextran for 15 minutes and then chased in a dextran-free BIS medium for the indicated time. White arrowheads show the macropinosomes decorated by GFP-EhRacM. Bars, 5 μm.

Fig 7. GFP-EhRacM was not recruited to actin-rich macropinocytic cups nor nascent macropinosomes.

Representative images of GFP-EhRacM expressing trophozoites. F-actin was visualized by phalloidin. The white arrows depict the macropinocytic cup (in the upper row) and a semi-closed macropinosome with an F-actin envelope being formed (in the lower row). Note that GFP-EhRacM is not associated with either the macropinocytic cup or the enclosing macropinosome. Bars, 5 μm.

Fig 8. GFP-EhRacM was recruited to macropinosomes following the removal of the F-actin envelope.

Montage of live imaging time series of a representative GFP-EhRacM expressing trophozoite in which macropinocytosis was monitored. F-actin was visualized using SiR-Actin (magenta), while GFP-EhRacM expressing trophozoites were shown in green (second row). The third-row panels are expanded images of the enclosed area by the white squares in the second-row panels. The trophozoites were incubated with RITC dextran (red). The white arrowheads indicate the site of macropinosome formation and the resultant macropinosome. The fourth-row panels show the GFP-EhRacM’s fluorescence intensity plot along with the trajectory of yellow arrows depicted in the corresponding third-row panels. Macropinosome areas are highlighted in red, and the mean GFP-EhRacM’s signal intensity at the initial macropinocytic cup (approximately 30) is shown in green. Bars, 5 μm. The F-actin envelope dissociation was captured at 49.941 [s].

Fig 9. GO enrichment analysis of EhRacM binding protein candidates identified by HA-EhRacM co-IP.

The results of PANTHER GO enrichment analysis on 107 hit proteins obtained from HA-EhRacM co-IP. Proteins were classified by biological process (BP) (A) and molecular function (MF) (B). Twenty GO terms were selected in descending order of fold enrichment for each entry. Each dot size reflects the count size, whereas its color reflects the FDR. The x-axis indicates fold change.

Fig 10. Working model of the role of EhRacM in macropinocytosis.

RacM indicates EhRacM, and colored arrows indicate signals from one compartment or molecule to another. See details in the text.