In this study, we have identified raft-like microdomains in the plasma membrane of E. histolytica by staining with two different fluorescent lipid analogs, FAST-DiI and DiIC16. We have also shown that pinocytosis and adhesion to host cell monolayers are raft-dependent cellular events in this pathogen. Interestingly, constitutive secretion of cysteine proteases was not inhibited by raft disruption. Finally, we determined that the heavy, intermediate, and light subunits of the Gal/GalNAc adherence lectin partially purify with the detergent-insoluble portion of the membrane and are enriched in cholesterol-containing regions of the membrane.
In other systems, it is established that cholesterol-rich membrane domains play a role in endocytosis. Reduction of cholesterol by inhibiting its synthesis with oxygenated cholesterol derivatives was shown to decrease pinocytic rates in L cells (23
). More recently, the uptake of an apoptotic agent alkyl-lysophospholipid or the transferrin receptor was blocked by treatment with MBCD or filipin (63
). In another example, depletion of cholesterol was coupled with an increased residency of clathrin in the plasma membrane and a decrease in the proportion of deeply invaginated clathrin-coated pits, suggesting that raft-like domains participate in the early formation of endocytic vesicles (59
). While clathrin has been identified in Entamoeba
), its role in endocytosis in this organism has not been established. Here, we demonstrate the importance of intact raft-like domains to fluid-phase pinocytosis in Entamoeba
cells, suggesting that molecular mechanisms, similar to those in higher eukaryotes, may govern this process in this pathogen.
Secretion is a vital component to the virulence of Entamoeba
cells, as several released toxic molecules, such as cysteine proteases and the pore-forming peptide amoebapore, participate in the destruction of the colonic epithelium, gut-resident bacteria, and erythrocytes. Lipid rafts have been implicated in the secretory capabilities of several cell lines. For example, the neuroendocrine tumor cell line, AtT-20, was defective in the formation of both constitutive and regulated secretory vesicles from the trans
-Golgi network upon cholesterol depletion (70
). Notably, after cholesterol depletion of rat pancreatic cells, amylase was secreted constitutively rather than in a regulated fashion (50
). These results suggest that, at least for rat pancreatic cells, regulated secretory events are lipid raft dependent, whereas constitutive secretion is lipid raft independent. Similarly, the release of cysteine protease from Entamoeba
cells, which is a constitutive secretory event (32
), was not affected by the disruption of raft-like domains. However, we cannot rule out the possibility that regulated secretory events, such as the release of the amoebapore, are sensitive to raft disruption in Entamoeba
Amoebic infection is dependent on the ability of the trophozoite to adhere to the colonic epithelium. Here, adhesion of Entamoeba
cells to a CHO monolayer was blocked upon treatment with raft-disrupting agents MBCD and filipin (Fig. ). Our results are consistent with those of other reports that have described the necessity of microdomains for cell-cell adhesion. In D. discoideum
, cell-cell adhesion was also shown to be sensitive to raft-disrupting agents, such as filipin or digitonin (21
). Furthermore, the Dictyostelium
cell adhesion molecule gp80 was isolated in the low-density, raft-like fractions of a sucrose gradient (21
Adhesion complexes in higher eukaryotes have also been shown to be raft associated. In RBL-2H3 mast cells, the immunoglobulin E receptor, Fc
RI, was evenly distributed in the plasma membrane but was observed to colocalize with the lipid raft marker, fluorescent cholera toxin B subunit, upon ligand binding (reviewed in reference 28
). Connexin 43, an adhesion molecule important in gap junctions, has been determined to reside in raft-like microdomains at the junctional membrane regions of NIH 3T3 fibroblasts and human embryonic kidney 293T cells (51
). In leukocytes, lipid rafts are critical in adhesion as well as in the cellular response to a presented antigen (30
). For example, binding of the T-cell receptor to antigen initiates its association with a number of proteins, including cytoplasmic protein tyrosine kinases, membrane-associated Src-family kinases, and several receptor-associated proteins in raft-like microdomains (27
). Once this activation and signaling machinery assembles in the raft, downstream signaling events mediated by the small GTPase, Rap1A, focal adhesion kinase, proline-rich tyrosine kinase-2, and mitogen-activated protein kinase and actin reorganization occur (27
). Disruption of microdomains in these cells results in an inability to propagate this antigen-dependent signal (27
The precise molecular mechanism by which Entamoeba
adheres to mammalian cells has not been discerned. However, it is known that the Gal/GalNAc-inhibitible lectin is necessary for both adhesion to host cells and virulence (14
). To date, the heavy and light subunits have always been detected jointly in amoebae or by Western blots of native proteins (35
). Likewise, in this study, the heavy and light subunits exhibit similar distribution in the sucrose gradient. Studies in which the heavy subunit was mutagenized identified β2 and β7 integrin-like sequences in the cytoplasmic tail of the heavy subunit (68
). Interestingly, the integrin family of adhesion proteins, which consists of allosteric signaling molecules that mediate intracellular (inside out) or extracellular (outside in) signals, are often associated with raft-like microdomains and with the cytoskeleton through several actin-binding proteins (29
). The results of this study, which demonstrate the enrichment of the Gal/GalNAc lectin heavy subunit in both the cholesterol- and actin-rich domains supports the notion (68
) that this adhesion molecule may be functionally similar to integrins.
Several reports demonstrate a direct interaction between lipid rafts and the actin cytoskeleton, which may account for the dual localization of the Gal/GalNAc heavy (and consequently, light) subunit. First, upon cholesterol depletion of hippocampal cells with MBCD, dendritic spines, typical membrane outgrowths of this cell type, were observed to immediately collapse due to F-actin redistribution from the spines to dendritic shafts (26
). Second, actin depolymerization was observed in microvilli and lamellipodia of fibroblasts upon cholesterol depletion (39
). Third, proteomic analysis of the detergent-resistant membrane from neutrophils revealed the association of an F-actin-binding protein, supervillin (41
). Interestingly, the β2-integrin, LFA-1, is excluded from microdomains in unstimulated T cells due to cytoskeletal constraints. Upon activation, the protein moves into the more-ordered domain to presumably mediate a signal either from internal or external sources (33
). Although it is not clear why the subunits of Entamoeba
Gal/GalNAc lectin partition into both raft-like sucrose gradient fractions and actin-rich fractions of higher density, the data support the previously reported notion that mechanisms similar to those for LFA-1 may regulate the Gal/GalNAc lectin (68
). In other words, the Gal/GalNAc heterodimer (which consists of Hgl and Lgl) may be excluded from microdomains through its interaction with the actin cytoskeleton. Upon binding to Gal- or GalNAc-containing ligands, the proteins may be released from the cytoskeleton and become incorporated into raft-like regions of the membrane. This may initiate a signaling cascade that would trigger virulence functions, including the release of the pore-forming peptide amoebapore.
Hgl and Lgl were also found in the pellet that formed at the bottom of the gradient. The pellet (fraction P) may represent incompletely solubilized low-density fractions or large, stable protein complexes that include the lectin. Given the proposed role of the lectin in signaling (68
), it would not be surprising to find this adhesion molecule forming complexes with other proteins. In support of this, it has been shown that integrins form large complexes with transmembrane 4 superfamily proteins that are >20 million Da and remain intact after treatment with Triton (10
The precise function and role of Igl in adhesion and virulence has not been described; however, it is known that amoebic adhesion to CHO cells is blocked upon incubation of Entamoeba
with a monoclonal antibody to the protein (8
). Previously, Igl was shown to have a GPI anchor sequence motif on its carboxy terminus, Gal/GalNAc binding affinity independent of that of Hgl, and a noncovalent association with the Hgl/Lgl heterodimer, and it has recently been postulated to function as a coreceptor for the heterodimer (35
). Our work demonstrates that Igl is associated with lipid raft fractions and actin-rich fractions; however, unlike Hgl and Lgl, it is not found in the densest fractions of the sucrose gradient. This indicates that Igl does not associate with actin as strongly as the Hgl/Lgl heterodimer and/or may not be included in some of the protein complexes containing the other two subunits. In the second scenario, Igl may serve as a regulator for adhesion and/or downstream signaling events associated with virulence.
This analysis is the first to describe raft-like lipid microdomains in Entamoeba cells and to illustrate their potential physiological significance for this human intestinal parasite. Lipidomic and proteomic analyses of the detergent-insoluble regions of the Entamoeba plasma membrane, currently under way, will greatly advance the knowledge of host-pathogen interactions. These future studies will provide greater insight into the role of lipid rafts and the mechanisms governing the virulence of this human pathogen.