A RhoGEF activates RacM to selectively promote motility without affecting macropinocytosis in <i>Entamoeba histolytica</i>

Rho guanine nucleotide-exchange factors (RhoGEFs) activate Rho and Rac small GTPases, orchestrating cellular processes, such as actin remodeling, endocytosis and migration. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, 22 Rho GTPases and ∼62 Dbl-homology (DH) domain-containing RhoGEFs have been identified, yet most remain uncharacterized. Our previous work revealed that E. histolytica (Eh)RacM (also known as EhRho13) negatively regulates macropinocytosis while promoting directional migration. Here, we characterize a previously uncharacterized RhoGEF, EHI_158230 (designated EhGEFM), identified as a potential EhRacM interactor through interactome analysis. Co-immunoprecipitation and in vitro GEF assays confirmed that EhGEFM directly binds and specifically activates EhRacM. Although EhgefM silencing did not affect macropinocytosis, it impaired directional migration, partially phenocopying EhracM silencing. Fixed and live-cell imaging revealed colocalization of EhGEFM and EhRacM at the cell periphery; notably, only EhRacM was recruited to maturing macropinosomes following actin coat disassembly. These findings indicate that although EhRacM is involved in both macropinocytosis and directional migration, its activator EhGEFM is required only for directional migration. This study provides the first direct evidence of distinct regulatory pathways governing Rho small GTPase function in E. histolytica.