PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica

Abstract

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.

Deciphering the liquid–liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein β-lactoglobulin

“…The fluorescence recovery was analyzed using Cell Sens software (Olympus), where the half-lifetime ( τ 1/2 ) was determined as the time at which 50% fluorescence recovery occurred. The following double exponential fit equation was used for analyzing the recovery curves: 34 …”

“…For further analysis of droplet images, ImageJ soware was used. 34 2.2.7 Fluorescence recovery aer photobleaching (FRAP). FRAP experiments were carried out on an Olympus FV3000 confocal microscope using a FRAP module.…”

“…Fluorescence lifetime imaging microscopy (FLIM) and uorescence intensity-based images for b-LG droplets were recorded, respectively, on an inverted confocal microscope setup (PicoQuant, MicroTime 200, Olympus IX-71) and Olympus FV3000 confocal microscope as described elsewhere. [32][33][34] For FLIM/uorescence imaging, the liquid droplets were obtained by mixing 5% CPM-labeled proteins with unlabeled proteins to avoid any possible effect of CPM on the LLPS processes. For sample preparation, 5 mL of the phase-separated sample was drop cast on a clean microscope glass slide and covered with a commercially available coverslip.…”

The autotaxin-LPA axis promotes membrane trafficking and secretion in yolk sac visceral endoderm cells

“…Given that LPA signaling remodels actin dynamics ( Mir et al, 2020 ) and that the actin cytoskeleton is involved in membrane trafficking ( Lanzetti, 2007 ), the defects in intracellular membrane trafficking in Enpp2 −/− VE cells are likely related to and caused by dysregulation of the Rho-ROCK-LIM kinase pathway. Association between LPA and membrane trafficking was reported in other cell systems: LPA enhanced internalization of LPA 1 and transferrin receptors via a clathrin- and cholesterol-dependent manner ( Urs et al, 2005 ); LPA stimulated micropinocytosis via Rho GTPases and subsequent actin modification in Entamoeba histolytica , an anaerobic parasitic amoebozoan ( Apte et al, 2022 ).…”

EhRacM differentially regulates macropinocytosis and motility in the enteric protozoan parasiteEntamoeba histolytica

“…By this screening, we successfully identified EhRacM as a negative regulator of macropinocytosis (Figs 2A). To date, several studies have demonstrated specific roles of E. histolytica Rho proteins in endocytic pathways, such as EhRacA (21), EhRho1(20), EhRacG(22), and EhRho5 (EhRacD1 (19)) (37). This study is the first to identify EhRacM as a macropinocytosis-specific regulator, independent from other endocytic pathways.…”