Adaptor protein 2 regulates receptor-mediated endocytosis and cyst formation inGiardia lamblia

Abstract

Synopsis The parasite Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation, and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In this study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gμ2) of gAP2 showed localization of this complex to the PVs, cytoplasm, and plasma membrane in the growing trophozoites. gAP2 also colocalized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL. Targeted downregulation of the gene encoding gμ2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

“…In Giardia, low-density lipoprotein (LDL) is taken up via a clathrin-dependent pathway (35,36). As with giActin, inhibition of the Giardia clathrin-mediated endocytosis pathway does not prevent the uptake of fluid-phase markers (36). We analyzed the ability of actin knockdown cells to endocytose fluorescent Bodipy-LDL.…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…Several groups have presented evidence for a role of clathrin in endocytosis in Giardia (Touz et al, 2004;Hernandez et al, 2007;Gaechter et al, 2008;Rivero et al, 2010). However, neither typical membrane-associated clathrin lattices nor emerging clathrin-coated pits have been observed in this parasite.…”

“…However, a recent work showed, by immunofluorescence and 3-D reconstruction, that the ER membranes are found throughout the cytoplasm, but do not permeate the space occupied by PVs (Faso and Hehl, 2011). Recently, it was shown that a mechanism of receptor-mediated endocytosis occurs in this organism, with specific molecules selectively directed to the PVs through a classical endocytic mechanism (Rivero et al, 2010;. These data suggest that uptake of soluble material into PVs is not selective at this step but is still capable of redirecting specific molecules to the TVN (Hernandez et al, 2007;Abodeely et al, 2009b).…”

“…The regulated pathway takes place only during encystation and associates with the appearance of encystation-specific vesicles (ESVs), which transport cyst wall components to the plasma membrane of the encysting cell and release their content to the cell exterior during cyst wall formation (Reiner et al, 1989;McCaffery and Gillin, 1994;Lujan et al, 1995;Sun et al, 2003). A selective pathway sorting proteins to the endosomal/lysosome membrane system has been recently demonstrated by our group, although Giardia do not possess distinctive endosomes or lysosomes Touz et al, 2004;Rivero et al, 2010;Rivero et al, 2011).…”

“…Giardia intestinalis, Giardia duodenalis) is an early branching eukaryote that possesses a reduced membrane network with highly polarized vesicles, denominated peripheral vacuoles (PVs), neighboring the plasma membrane (reviewed by Adam, 2001;Faso and Hehl, 2011;. This is an important zone of interaction between the parasite and its environment (the host intestine in vivo or the culture medium in vitro), and was shown to be the place involved in fluid-phase and receptor-mediated endocytosis (Gaechter et al, 2008;Rivero et al, 2010). Another characteristic of these PVs is that they contain hydrolytic activity resembling the function performed by lysosomes.…”