Immunostaining of F-actin with phalloidin in both EhP3 upregulated and down regulated cells further revealed differential actin dynamics in these cells

Immunostaining of F-actin with phalloidin in both EhP3 upregulated and down regulated cells further revealed differential actin dynamics in these cells. cell collection in absence of tetracycline and reddish arrowheads indicate RBC attachment site in tetracycline induced cells. (Level pub, 5 m; DIC, differential interference contrast).(TIF) ppat.1007789.s004.tif (2.1M) GUID:?36D045B1-A746-4870-BCB2-1A21FF2506F5 S5 Fig: EhP3 is essential for motility. (A) Immunofluorescence analysis of EhP3 at pseudopods in cells transporting antisense construct of EhP3. AG-494 White colored arrowheads show pseudopods. (Level pub, 5 m; DIC, differential interference contrast). (B) Migrated cell count with indicated constructs in presence AG-494 and absence of tetracycline. The number of migrated cells towards serum comprising press were counted using haemocytometer. Experiment was performed twice in duplicates.(TIF) ppat.1007789.s005.tif (1.2M) GUID:?E71DCD85-B2E8-41D6-BF35-0E2466D4A23A S6 Fig: Immunolocalization of EhP3 during phagocytosis of live CHO cells. trophozoites phagocytosing CHO cells stained with cell tracker blue CMAC dye, were fixed and stained for GFP antibody. Localization of EhP3 are demonstrated at different methods of phagocytosis.(TIF) ppat.1007789.s006.tif (794K) GUID:?2951545D-353C-4829-A7E4-2BD113A757CD AG-494 S7 Fig: Immunolocalization of EhP2 in GFP-EhP2 expressing cells. trophozoites actively AG-494 phagocytosing RBCs were fixed and stained for GFP antibody and TRITC phalloidin (for visualisation of F-actin).(TIF) ppat.1007789.s007.tif (912K) GUID:?330B1441-8CF6-4CC4-AC69-15094A6F85E7 S1 Movie: Live cell imaging of GFP-EhP3 during pseudopod formation. The movie represents pseudopod formation in GFP-EhP3 expressing trophozoites. The enrichment of EhP3 is definitely visualized in the leading edge of a moving amoebae. (Level pub, 10 m).(AVI) ppat.1007789.s008.avi (9.1M) GUID:?927DC241-8655-40D8-AA5D-E32E17C48A29 S2 Movie: Live cell imaging of GFP-EhP3 during erythrophagocytosis. The movie represents the process of erythrophagocytosis in presence of fluorescent labelled RBCs. GFP-EhP3 CDC25L accumulated rapidly at the site of attachment of RBC and remained till the formation of phagosome. (Level pub, 10 m).(AVI) ppat.1007789.s009.avi (4.8M) GUID:?E4A286F6-7897-4756-BB87-F897DD5DB657 S1 Table: Summary of 14-3-3 isoform sequences. Summary of 14-3-3 gene family members present in lower eukaryotic pathogens which are retrieved from multiple data bases for sequence alignment.(TIFF) ppat.1007789.s010.tiff (875K) GUID:?241A4869-47E5-497D-8BF1-A7842A4E94D2 S2 Table: Selected list of EhP3-connected proteins. EhP3-connected proteins recognized by LC-MS after immuno-pull down from whole cell lysate using anti-EhP3 antibody.(TIFF) ppat.1007789.s011.tiff (489K) GUID:?8DE66F1A-DEE1-49C8-B253-7BF9C7227726 Data Availability StatementAll relevant data are within the manuscript and its Supporting Info files. Abstract The highly conserved proteins of the 14-3-3 family are common adaptors known to regulate an enormous range of cellular processes in eukaryotes. However, their biological functions remain mainly uncharacterized in pathogenic protists comprising of several 14-3-3 protein isoforms. In this study, we statement the part of 14-3-3 in coordinating cytoskeletal dynamics during phagocytosis in a professional phagocytic protist AG-494 is the etiological agent of human being amebiasis and a major cause of morbidity and mortality particularly in developing countries [26C28]. The majority of infected individuals are asymptomatic and only a small fraction of the people displays medical symptoms with invasions in the intestinal cells or in extra intestinal sites, such as liver [29C30]. Though we understand some of the cellular processes such as motility and phagocytosis, involved in the promotion of invasiveness and pathogenesis of the parasite, detailed mechanisms are not obvious. Understanding these mechanisms would help develop better treatments against amebiasis. Motility and phagocytosis are both essential processes for the survival and invasion of sponsor cells from the parasite, and are mainly dependent on a highly dynamic actin cytoskeleton [30C32]. The parasite offers evolved several homologs of mammalian cytoskeletal proteins as well as some of the novel molecules such as EhCaBP1, EhCaBP3, EhAK1 and EhC2PK to fulfil the need for high rate of actin dynamics during phagocytosis [33C36]. EhCaBP1, EhCoactosin, EhC2PK and EhAK1 have been shown to be involved in the methods including initial particle attachment, progression of phagocytic cups and channeling of actin dynamics for progression of cups [33, 35C37]. EhAK1 has been.