Tale of the two domains in twinfilin: Deciphering phagocytosis through actin dynamics

Abstract

Phagocytosis is an indispensable cellular function in phagocytic cells or protists. It is choreographed by actin and its surfeit of regulatory proteins. This process is critical in Entamoeba histolytica’s lifecycle as it relies primarily on phagocytosis for its nutritional uptake as well as its pathogenesis. In our studies we have tried to puzzle out the role of Twinfilin‐ an actin binding protein in E. histolytica's pathogenesis. Twinfilin is an Actin Depolymerizing Factor (ADF)/cofilin family member, containing two ADF‐homology domains, one each as N‐terminal (NTD) and C‐terminal (CTD).We have solved a 1.5 Å resolution crystal structure of EhTwinfilin NTD and determined that it contains typical ADF homology fold i.e., central β sheets surrounded by α helices. With the help of fluorimetric and SPR studies we have shown the actin binding activities of both the domains and interestingly we found that NTD stabilized F‐actin whereas CTD depolymerized it. NTD has stronger G‐actin binding while CTD binds to F‐actin with much higher affinity; this is rather role reversal of the studies published on twinfilin from other organisms. Structural and actin binding studies of EhTwinfilin provides explanation for some of the functional differences observed with respect to the human and Plasmodium falciparum's ADF such as the ability to bind G‐actin. Cellular localization studies established its significant role during phagocytosis. GFP tagged EhTwinfilin was visualized using confocal imaging, it was found to colocalize with F‐actin at phagocytic cups. Down‐regulation of EhTwinfilin expression leads to an increased rate of phagocytosis. This is the second protein so far that was seen to be present throughout the phagocytosis, up till phagosome maturation and first protein to perform actin severing activity in E. histolytica.Support or Funding InformationSERB, Department of Science and Technology, IndiaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.