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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01662

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Band 3 modifications in Plasmodium falciparum-infected AA and CC erythrocytes assayed by autocorrelation analysis using quantum dots

¹ Biochemical and Biophysical Parasitology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8132, USA
² Malaria Genetics Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8132, USA
³ Optical Technology Division, Physics Laboratory, National Institute of Standards and Technology, Stop 8443, 100 Bureau Drive, Gaithersburg, MD 20899, USA

View larger version (26K): [in a new window]   Fig. 1. Analysis of band 3 clustering in uninfected and parasitized erythrocytes. (A) Immunoblot of membrane extracts from both uninfected and parasitized erythrocytes using a mAb against the band 3 cytoplasmic domain (cdb3). All lanes, except for parasitized CC erythrocytes, were loaded with 0.1 µg of total protein. 0.3 µg of total protein from parasitized CC erythrocytes was used to obtain approximately the same amount of signal from band 3 protein. The arrows point out the signal from band 3 oligomers found in parasitized AA and CC erythrocytes. Much greater oligomerization is apparent in the lane of parasitized CC erythrocytes. (B) Quantification of band 3 in SE-HPLC fractions of AA and CC erythrocyte membrane extracts. The amount of protein used in CC extracts (0.63 µg/µl) was approximately double that in AA extracts (0.33 µg/µl). Band 3 in the SE-HPLC fractions was semi-quantified by ELISA using mAb against cdb3 at 1/5000 and 1/2000 dilutions. The anti-band 3 signal obtained for AA extracts is markedly higher than for CC extracts. In a competition ELISA, increasing the amount of mAb caused only a minor increase in anti-band 3 signals.

View larger version (70K): [in a new window]   Fig. 2. Immunocytochemistry of infected erythrocytes. Membranes were immunolabeled with quantum dots for anti-band 3 mAb (red) and parasite nuclei were labeled with Hoechst 33258 (blue). These images are the result of 3D reconstruction and deconvolution. (A) Parasitized AA erythrocytes surrounded by uninfected erythrocytes. (B) Parasitized CC erythrocytes surrounded by uninfected erythrocytes. Note the abnormal membrane appearance of uninfected CC erythrocytes. Comparisons of erythrocyte shapes at early, mid, and late stages of parasitized AA (C-E) and CC erythrocytes (F-H). Bars, 5 µm (A,B); bars, 1 µm (C-H).

View larger version (31K): [in a new window]   Fig. 3. Fluorescence intensity measurements from uninfected and parasitized erythrocytes. (A) Representation example of fluorescence symmetry through a z-stack series of a horizontally oriented, healthy AA erythrocyte. Although the z-stack consisted of 70 frames, only those frames containing the erythrocyte are shown. Each fluorescence intensity profile was centered along the vertical axis for representational purposes. Owing to the high photostability of quantum dots, no measurable reduction in fluorescence intensity occurred throughout the z-stack sequence. (B) Cell-by-cell comparison of mean total fluorescence intensity occurring during the development of malaria parasites in AA and CC erythrocytes. The total fluorescence was measured using 3D-reconstructed, sum projection images. Data were normalized against uninfected erythrocytes (U) for each genotype. Error bars represent the standard error of the mean (s.e.m.). n=3-11 (see Table 1).

View larger version (48K): [in a new window]   Fig. 4. Analysis of band 3 clustering. (A) Example of a line profile of fluorescence intensity drawn at the periphery of the center section of a deconvolved erythrocyte (arrow). Bar, 10 µm. (B) A graphical representation of the profile shown in A. 1 pixel=45 nm. (C) Power spectrum of the frequency profile of measured fluorescence fluctuations shown in B plotted as the inverse of pixel distance (f). (D) Double logarithmic plots of power spectra show linear relationship between frequency and normalized power. Multiple data sets for a developmental stage in AA erythrocytes and their linear regression lines are shown. (E) A comparison of regression slope values between AA and CC erythrocytes as a function of parasite developmental stage. These slopes demonstrate that CC erythrocytes have higher degrees and rates of clustering than AA erythrocytes throughout the intracellular parasite cycle. (F) Autocorrelation analyses of measured fluorescence intensity profiles of representative uninfected and parasitized AA and CC erythrocytes. Only the first 1 µm of the intensity profile is shown for clarity. Autocorrelation values were normalized against the value at C0. The first decay function of the data was fitted (solid lines) using a first-order exponential function to estimate characteristic decay lengths. The slower decay values shown by both uninfected and parasitized CC cells and parasitized AA erythrocytes represent higher degrees of band 3 clustering.

View larger version (57K): [in a new window]   Fig. 5. Proposed model for the mechanism of fluorescence reduction due to band 3 aggregation in AA and CC erythrocytes. Anti-band 3 mAb against cdb3 has easy access to band 3 epitopes in uninfected AA erythrocytes. During the course of P. falciparum infection, however, hemichromes form and bind with high affinity to the cdb3, hindering the anti-band 3 mAb binding. Band 3 also clusters to form oligomeric forms which may provide better recognition site for autologous antibody. Uninfected CC erythrocytes have increased band 3 oligomerization compared to uninfected AA erythrocytes, and a considerable amount of hemichromes already bound to the cdb3 that make those epitopes less accessible to anti-band 3 mAb, as shown in Fig. 1B. When these CC erythrocytes become infected, hemichromes form even larger deposits that further reduce antibody accessibility to band 3 epitopes, and band 3 forms higher levels of clustering, as shown Fig. 1A, Fig. 3B, Fig. 4E and 4F.

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