The fluid mosaic model of the plasma membrane has evolved considerably since its original formulation 30 years ago. Membrane lipids do not form a homogeneous phase consisting of glycerophospholipids (GPLs) and cholesterol, but a mosaic of domains with unique biochemical compositions. Among these domains, those containing sphingolipids and cholesterol, referred to as membrane or lipid rafts, have received much attention in the past few years. Lipid rafts have unique physicochemical properties that direct their organisation into liquid-ordered phases floating in a liquid-crystalline ocean of GPLs. These domains are resistant to detergent solubilisation at 4°C and are destabilised by cholesterol- and sphingolipid-depleting agents. Lipid rafts have been morphologically characterised as small membrane patches that are tens of nanometres in diameter. Cellular and/or exogenous proteins that interact with lipid rafts can use them as transport shuttles on the cell surface. Thus, rafts act as molecular sorting machines capable of co-ordinating the spatiotemporal organisation of signal transduction pathways within selected areas (‘signalosomes’) of the plasma membrane. In addition, rafts serve as a portal of entry for various pathogens and toxins, such as human immunodeficiency virus 1 (HIV-1). In the case of HIV-1, raft microdomains mediate the lateral assemblies and the conformational changes required for fusion of HIV-1 with the host cell. Lipid rafts are also preferential sites of formation for pathological forms of the prion protein (PrPSc) and of the β-amyloid peptide associated with Alzheimer's disease. The possibility of modulating raft homeostasis, using statins and synthetic sphingolipid analogues, offers new approaches for therapeutic interventions in raft-associated diseases.
Deoxynivalenol (DON) is a mycotoxin belonging to the tricothecene family that has many toxic effects in animals, including diarrhea and weight loss. Using the human epithelial intestinal cell line HT-29-D4 as an in vitro model, we studied the effect of DON on the uptake of different classes of nutrients, including sugars, amino acids and lipids. At low concentrations (below 10 micro mol/L), DON selectively modulated the activities of intestinal transporters: the D-glucose/D-galactose sodium-dependent transporter (SGLT1) was strongly inhibited by the mycotoxin (50% inhibition at 10 micro mol DON, P < 0.05), followed by the D-fructose transporter GLUT5 (42% inhibition at 10 micro mol/L, P < 0.001), active and passive L-serine transporters (30 and 38% inhibition, respectively, at 10 micro mol/L, P < 0.05). The passive transporters of D-glucose (GLUT) were slightly inhibited by DON (15% inhibition at 1 micro mol/L, P < 0.01), whereas the transport of palmitate was increased by 35% at 10 micro mol/L DON (P < 0.001). In contrast, the uptake of cholesterol was not affected by the mycotoxin. At high concentrations (100 micro mol/L), SGLT1 activity was inhibited by 76% (P < 0.01), whereas the activities of all other transporters were increased. The selective effects of DON on intestinal transporters were mimicked by cycloheximide and deoxycholate, suggesting that inhibition of protein synthesis and induction of apoptosis are the main mechanisms of DON toxicity in intestinal cells.
Although verotoxin-1 (VT1) and verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb(3)), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb(3) assemblies. At 4 degrees C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb(3) microdomains. After 10 min at 37 degrees C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3-6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential 'raft' association. >90% (125)I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb(3)-containing sucrose gradient 'insoluble' fraction, whereas only 30% (125)I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb(3)/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb(3) ratios. VT2 competed less effectively for (125)I-VT1/Gb(3) DRM-binding but only VT2-Gb(3)/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb(3) raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology.
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