Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy

Abstract

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 immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence 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) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence 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 MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 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.

Figure 1

Mapping of malarial protein in the erythrocyte membrane. A thin blood smear of the trophozoite infected erythrocytes was fixed, then reacted with antibodies against PfHRP1, and labeled with tetramethylrhodamine. (a) Bright-field image of the blood smear. The NSOM tip was positioned above the three cells in the center of the field-of-view and a scan was taken within the yellow framed area. (Scale bar = 10 μm.) (b) Result of the NSOM imaging in topography (Left) and fluorescence (Right) (128 × 128 pixels, 60 nm/pixel). While all three cells are imaged in topography, only the one in the lower right corner was infected and is visible in the fluorescence image. (c) Zoom around the infected cell, which was seen in the lower right corner of b (128 × 128 pixels, 52 nm/pixel). The fluorescence signal is superimposed as a color value (brightness scale goes from black via red to yellow) to the topography to show the distribution of PfHRP1 in the erythrocyte membrane. (Scale bar for b and c = 2 μm.) Z range for c is 1 μm.

Figure 2

NSOM (a) (256 × 256 pixels, 30 nm/pixel) and confocal (b) (128 × 128 pixels, 70 nm/pixel) imaging of infected erythrocytes labeled with antibodies against PfHRP1. The figures show the fluorescence image (Left) and a cross section through the smallest feature (marked with a line) found in the image (Right). The fluorescence signal is given as a color value (brightness scale goes from black via red to yellow). (a) Individual knobs are resolved by NSOM imaging. The resolution is about 100 nm. (b) Confocal cross section through the cell. The membrane is seen as a bright ring. The resolution is about 310 nm. (Scale bars = 2 μm.)

Figure 3

Colocalization of malarial and host proteins in the erythrocyte membrane. A blood smear of erythrocytes infected with the trophozoite stage parasites was fixed and then reacted with two different antibodies (from rabbit and from mouse) against the proteins under study. Secondary antibodies labeled with FITC (anti-rabbit) and TR (anti-mouse) were used, respectively. The left column shows the two simultaneously acquired fluorescence channels by NSOM dual-color imaging. The center column presents the corresponding overlay of the fluorescence images and the right column is pixel-by-pixel correlation between the red and green images. (a) Control experiment: primary antibodies against PfHRP1 (128 × 128 pixels, 86 nm/pixel). (b) Colocalization of MESA and protein 4.1 (128 × 128 pixels, 48 nm/pixel). (c) Colocalization of PfHRP1 and protein 4.1 (256 × 256 pixels, 30 nm/pixel). (Scale bars = 2 μm.)