Fig. 5. Extraction of MTIP and CS with Triton X-100 (TX-100). (A) Sporozoites were permeabilized with saponin and fixed with paraformaldehyde (PFA) (upper panel, —TX100) or treated with 1% TX-100 followed by PFA fixation (lower panel, +TX100). Each preparation was labeled with anti-CS (red) and anti-MTIP (green) antibodies. In untreated sporozoites MTIP and CS were localized to the periphery of sporozoites and showed a relatively homogenous distribution. TX-100-treated sporozoites showed no significant change in MTIP distribution or fluorescent intensity. However, CS fluorescence was lost from the periphery of sporozoites by TX-100 treatment, indicating a complete removal of the plasma membrane. The inset in each panel shows overview fluorescence micrographs for each sporozoite preparation at lower magnification. Left and right panels show the same sporozoites labeled with MTIP (left panels) and CS (right panels). Scale bars are 0.5 µm for individual sporozoite micrographs and 10 µm for overview micrographs. (B) Quantification of MTIP and CS fluorescence shown in A. Graphs are the mean of relative fluorescent intensities measured on 50 sporozoites for each the TX-100-treated and untreated populations with constant exposure times±s.d. (C) Immunoblot analysis of TX-100-treated sporozoites with anti-MTIP and anti-CS antibodies. Pellet (P) and supernatant (S) were separated by high speed centrifugation and analyzed by SDS-PAGE followed by blotting and probing with anti-MTIP (upper panel) or anti-CS (lower panel). Most CS was detected in the supernatant, indicating effective solubilization of the sporozoite plasma membrane and associated proteins. However, most MTIP was detected in the pellet, indicating its retention in the IMC. Total parasite extracts are shown for comparison (C). Note that CS was detected as 40/60 kDa species.

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