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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\n                    <jats:italic>Entamoeba histolytica<\/jats:italic>\n                    , 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\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>histolytica<\/jats:italic>\n                    . Through gene silencing of highly transcribed\n                    <jats:italic>Ehrho<\/jats:italic>\n                    \/\n                    <jats:italic>Ehrac<\/jats:italic>\n                    genes and following flow cytometry analysis, we identified that silencing\n                    <jats:italic>EhracM<\/jats:italic>\n                    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\u2019s multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>histolytica<\/jats:italic>\n                    , highlighting the significance of EhRacM in both macropinocytosis and cellular migration.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1012364","type":"journal-article","created":{"date-parts":[[2024,11,13]],"date-time":"2024-11-13T13:36:07Z","timestamp":1731504967000},"page":"e1012364","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["EhRacM differentially regulates macropinocytosis and motility in the enteric protozoan parasite Entamoeba histolytica"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-2649-5240","authenticated-orcid":true,"given":"Misato","family":"Shimoyama","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9832-916X","authenticated-orcid":true,"given":"Kumiko","family":"Nakada-Tsukui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1354-5133","authenticated-orcid":true,"given":"Tomoyoshi","family":"Nozaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,11,13]]},"reference":[{"key":"ppat.1012364.ref001","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1038\/icb.2011.20","article-title":"Macropinocytosis: An endocytic pathway for internalising large gulps.","volume":"89","author":"JP Lim","year":"2011","journal-title":"Immunology and Cell Biology"},{"key":"ppat.1012364.ref002","doi-asserted-by":"crossref","DOI":"10.1098\/rstb.2018.0146","article-title":"The breadth of macropinocytosis research.","volume":"374","author":"JA Swanson","year":"2019","journal-title":"Philosophical Transactions of the Royal Society B: Biological Sciences"},{"key":"ppat.1012364.ref003","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1038\/nrm2447","article-title":"Shaping cups into phagosomes and macropinosomes.","volume":"9","author":"JA Swanson","year":"2008","journal-title":"Nature Reviews Molecular Cell Biology"},{"key":"ppat.1012364.ref004","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1111\/j.1600-0854.2009.00878.x","article-title":"Defining macropinocytosis.","volume":"10","author":"MC Kerr","year":"2009","journal-title":"Traffic"},{"key":"ppat.1012364.ref005","first-page":"17","article-title":"Pinocytosis","volume":"49","author":"W.H. 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Maniak","year":"2001","journal-title":"Biochimica et Biophysica Acta (BBA)\u2014General Subjects."},{"issue":"17","key":"ppat.1012364.ref008","doi-asserted-by":"crossref","first-page":"2714","DOI":"10.1091\/mbc.e13-02-0092","article-title":"WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion","volume":"24","author":"JS King","year":"2013","journal-title":"Mol Biol Cell"},{"issue":"5","key":"ppat.1012364.ref009","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1083\/jcb.201009119","article-title":"Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis","volume":"193","author":"M Carnell","year":"2011","journal-title":"Journal of Cell Biology"},{"key":"ppat.1012364.ref010","article-title":"The origins and evolution of macropinocytosis.","volume":"374","author":"JS King","year":"2019","journal-title":"Philosophical Transactions of the Royal Society B: Biological Sciences"},{"issue":"7","key":"ppat.1012364.ref011","article-title":"A Review of the Global Burden, New Diagnostics, and Current Therapeutics for Amebiasis.","volume":"5","author":"DAT Shirley","year":"2018","journal-title":"Open Forum Infect Dis."},{"issue":"4","key":"ppat.1012364.ref012","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1002\/cm.20038","article-title":"Dynamics of endocytic traffic of Entamoeba histolytica revealed by confocal microscopy and flow cytometry","volume":"59","author":"I Meza","year":"2004","journal-title":"Cell Motil Cytoskeleton"},{"key":"ppat.1012364.ref013","doi-asserted-by":"crossref","first-page":"179","DOI":"10.3389\/fcimb.2018.00179","article-title":"Morphodynamics of the Actin-Rich Cytoskeleton in Entamoeba histolytica","volume":"1","author":"M Manich","year":"2018","journal-title":"Front Cell Infect Microbiol"},{"key":"ppat.1012364.ref014","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1080\/21541248.2015.1044811","article-title":"Rho GTPases at the crossroad of signaling networks in mammals.","author":"JC Herv\u00e9","year":"2015","journal-title":"6, Small GTPases."},{"key":"ppat.1012364.ref015","article-title":"The Small GTP-Binding Protein rac Regulates Growth Factor-Induced Membrane Ruffling.","volume":"70","author":"AJ Ridley","year":"1992","journal-title":"Cell"},{"key":"ppat.1012364.ref016","doi-asserted-by":"crossref","DOI":"10.1091\/mbc.12.7.2061","article-title":"Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells.","volume":"12","author":"C Fiorentini","year":"2001","journal-title":"Molecular Biology of the Cell"},{"key":"ppat.1012364.ref017","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/nrm.2016.67","article-title":"Regulating Rho GTPases and their regulators.","volume":"17","author":"RG Hodge","year":"2016","journal-title":"Nature Reviews Molecular Cell Biology"},{"key":"ppat.1012364.ref018","first-page":"81","article-title":"Dissecting the Actin Cytoskeleton of Entamoeba histolytica from a Genomic Perspective.","author":"CH Chung","year":"2010","journal-title":"Anaerobic Parasitic Protozoa: Genomics and Molecular Biology."},{"issue":"9","key":"ppat.1012364.ref019","article-title":"Janhawi; Arya R, Bhattacharya S, Bhattacharya A. 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