THE NEW ROLE OF AP-2 ADAPTOR PROTEIN IN Giardia lamblia ENCYSTATION

FELIZIANI, CONSTANZA; RIVERO MR; QUASSOLLO, GONZALO; ROPOLO AS; TOUZ MC

Congreso; SAIB - SAMIGE Joint meeting 2021 on line; 2021

Cells can exchange material and information with their environment through receptors on the cell surface that are involved inprocesses that range from nutrient uptake to signaling responses. Consequently, endocytosis constitutes a powerful mechanismto regulate both events. G. lamblia is unable to synthesize cholesterol de novo and acquires cholesterol through receptormediated endocytosis (RME) of lipoproteins and is vital for in vitro growth. On the other hand, during encystation, the decreasein available cholesterol triggers the differentiation of trophozoites to cysts. In this work, we addressed the participation of theendocytic machinery in the process of encystation via clathrin-RME by analyzing the µ (gµ2) subunit of the adaptor protein 2(AP2). IFA and confocal microscopy results showed that the relative number of Encystation-Specific Vesicles (ESVs) andcysts were significantly increased in ds-gµ2 transgenic trophozoites, in which the expression of gµ2 and RME were downregulated, compared to control cells. Also, using Real-Time PCR, we found an increase in the expression of specific encystinggenes (cwp1-3 and myb1-like) in growing conditions. Moreover, an increased number of cysts was observed in ds-gµ2 cellsalthough they denoted characteristics of incomplete cysts unable to survive in water overnight. Interestingly, when we laterinduce trophozoites to encyst, a reduction of cysts was observed in ds-gµ2 cells, suggesting that gµ2 might play another roleduring encystation. Examination of the subcellular localization of gµ2 and the cyst wall protein1 (CWP1) in wild-typetrophozoites, by direct immunofluorescence and confocal microscopy, showed the presence of CWP1 inside the formed ESVsand gµ2 on the endolysosomal peripheral vacuoles (PVs) at the beginning of the encystation process. However, later duringthe encystation, gµ2 and CWP1 partially colocalized, resulting in the formation of small ESVs and CWP1 deposition formingthe cyst wall. When the expression of gµ2 was inhibited, no alterations in the morphology of PVs or ESVs were observed.However, the formation of small vesicles and the formation of the cyst wall was blocked. Here, we show that impairing theRME of cholesterol is sufficient to induce encystation in growing trophozoites. However, a more complex scenario is necessaryto accomplish the entire process of cell differentiation. Also, we show how the clathrin-adaptin subunit GlAP2 participates inthe process of encystation by a unique and unusual mechanism of cyst wall protein sorting to the plasma membrane. Our datareinforce the importance of cholesterol deficiency in the process of encystation and demonstrate the essential role of GlAP2in the production of the infective stage of this parasite.