Infections with the human malaria parasite Plasmodium falciparum are characterized by sequestration of erythrocytes infected with mature forms of the parasite. Sequestration of infected erythrocytes appears to be critical for survival of the parasite and to mediate immunopathological abnormalities in severe malaria. A leukocyte differentiation antigen (CD36) was previously suggested to have a role in sequestration of malaria-infected erythrocytes. CD36 was purified from platelets, where it is known as GPIV, and was shown to be a receptor for binding of infected erythrocytes. Infected erythrocytes adhered to CD36 immobilized on plastic; purified CD36 exhibited saturable, specific binding to infected erythrocytes; and purified CD36 or antibodies to CD36 inhibited and reversed binding of infected erythrocytes to cultured endothelial cells and melanoma cells in vitro. The portion of the CD36 molecule that reverses cytoadherence may be useful therapeutically for rapid reversal of sequestration in cerebral malaria.
Erythrocytes infected with trophozoites and schizonts ofthe human malaria parasite Plasmodium falciparum develop surface protrusions (knobs) (1) by which the infected erythrocytes (IRBCs)t adhere specifically to venular endothelium in vivo (2, 3) and to human endothelial cells (4) and some lines of melanoma cells (5) in vitro. Cytoadherence between IRBCs and venular endothelium has a critical role in the pathogenesis of falciparum malaria, since it permits the mature parasites to evade spleen-dependent immune mechanisms (6) and since the sequestered parasites may occlude blood flow, as seen in cerebral malaria (7). Antibody in immune serum reacts with a strain-specific parasite-determined antigen on IRBCs and inhibits cytoadherence in vitro (8) and in vivo (9). The inhibition of cytoadherence by antibody may protect the host from the clinical consequences of falciparum malaria.Both cytoadherence and the IRBC surface antigen were shown to be destroyed by incubating IRBCs with proteases (9, 10), suggesting that the two properties are determined by proteins on the IRBC surface. In addition, the cytoadherence phenotype ofP.falciparum parasites and the expression ofthe IRBC surface antigen were modulated together by the spleen in a monkey model of falciparum malaria (9, 10), suggesting that the two properties are linked and perhaps determined by the same protein. A family ofpotential cytoadherence proteins was identified in studies with IRBCs from Aotus monkeys (11) . The members ofthe protein family differed in antigenicity and molecular size among strains ofP.falciparum, but had in common several biochemical properties, including their accessibility to surface radioiodination, detergent solubility, and cleavage by the same concentration of trypsin that inhibited cytoadherence (11) .
Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-function relationships, including elucidation of protein-protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunof luorescence antibody assays. Simultaneous dualcolor excitation and detection with NSOM provided f luorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) f luorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two f luorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA)(parasite)͞protein4.1(host) and P. falciparum histidine rich protein (PfHRP1)(parasite)͞ protein4.1(host) showed good real-space correlation for the MESA͞protein4.1 pair, but relatively poor correlation for the PfHRP1͞protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.One of the crucial aspects of current biological inquiry relates to the organization of cells and how interactions between proteins are involved in important cellular processes, such as signal transduction and receptor-ligand binding. Several experimental systems have been developed in recent years to try and identify such protein-protein interactions including the two hybrid and phage display systems (1, 2). All such attempts to identify interactions in disassembled cellular systems need to be confirmed in vivo, by approaches such as chemical cross-linking in cells or some form of colocalization study using microscopy. A common approach has been the use of confocal microscopy using fluorescent antibody probes. We report here the development of a technique based on dual-color immunofluorescence labeling together with near-field scanning optical microscopy (NSOM) (3, 4), which offers significant advances in the det...
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