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Blood, Vol. 110, Issue 6, 2132-2139, September 15, 2007
Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum
Blood Murphy et al.
110: 2132
Supplemental materials for: Murphy et al, Vol 110, Issue 6, 2132-2139
Files in this Data Supplement:
- Table S1. Distribution of PE, PG, PA, PS, and PI phospholipid species in erythrocyte ghost and drug-induced vesicle membranes as determined by negative mode ESI-MS/MS (PDF, 16.1 KB) -
Footnote for Table S1: Numbers in parentheses indicate the carbon chain length of the two associated fatty acids followed by the degree of unsaturation. m/z, mass-to-charge ratio; DIV, drug-induced vesicles. ‘sm’ indicates presence of a small amount of the particular species.
- Table S2. Distribution of PC phospholipid species in erythrocyte ghost and drug-induced vesicle membranes as determined by positive and negative mode ESI-MS/MS (PDF, 10.5 KB) -
Footnote for Table S2: “+” indicates detection by positive mode MS; “-“ by negative mode MS. Numbers indicate combined carbon chain length of both associated fatty acids followed by the total degree of unsaturation. m/z, mass-to-charge ratio. DIV, drug-induced vesicles.
- Figure S1. Raw data from lipid mass spectrometry of erythrocyte ghost and DIV membranes (JPG, 94.3 KB)
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(A) Representative negative mode MS data (670-980 m/z) of ghost membranes and drug-induced vesicles (DIVs). PE (red *), PG (+), and PI lipids (arrows) are marked to highlight their inclusion or exclusion from control and endovesicle preparations. (B) Representative positive mode MS data (full range m/z 400-1200) of ghost membranes and DIVs. (C) Representative negative mode MS (full range m/z 400-1200) of ghost membranes and DIVs. PI species are indicated with arrows.
- Figure S2. Loading ghosts with the PLCδ1 PH domain does not significantly alter the extent of drug-induced endovesiculation or malarial invasion (JPG, 71.5 KB)
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(A) Mean FITC dextran uptake of mock-treated (−) or primaquine-treated (+) empty and PLC 1 PH domain (5 or 15 µM)–containing erythrocyte ghosts. Loaded ghosts were treated with 4 mM primaquine in the presence of 70 kDa fluoroscein isothiocyanate (FITC)–dextran to induce endovesiculation. To determine whether loading of the PLC1 PH domain altered the frequency or degree of endovesiculation, treated cells were analyzed for FITC fluorescence by flow cytometry in the FL1 channel. 50 000 erythrocyte-gated events were collected per sample; error bars represent the standard deviation of triplicate samples within one experiment. The mean fluorescence per cell is displayed. (B) Parasitemias of empty and PLC1 PH domain-loaded ghosts following standard overnight invasion assays as described in “Materials and methods, Purification of recombinant PLC1 PH domain proteins and loading into erythrocyte ghosts.” Error bars represent the standard deviation of triplicate samples within one experiment. Representative experiment; n equals 2 experiments with non-fluorescent protein; greater than 4 experiments with GFP-PLC1 PH domain (assayed by Giemsa and not shown). (C) Measurement of erythrocyte membrane mechanical stability by ecktacytometry of hemoblobin-free erythrocyte ghosts loaded with GFP-PLC1 PH domain (5, 10 or 25 µM). The T50 (time at half maximal deformability index DI) indicates that there is no difference in the mechanical stability of GFP-PLC1 PH domain-loaded ghosts versus controls. (D) Effect of annexin V loading on malarial invasion normalized to control infection. Ghosts were loaded and resealed with 2.5 µM AlexaFluor 594-labeled annexin and invasion assays were carried out as described for panel B.
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