FIGURE 8:

Ectopic expression of fluorescently tagged TLAP2 from a tubulin promoter induces the formation of microtubule bundles in T. gondii. (A) 3D-SIM images of a single eGFP–Tgβ2-tubulin (pseudocolored red) transgenic parasite transiently expressing mAppleFP-TLAP2 (pseudocolored green). Top, projection; bottom, single surface section. Microtubules extend from the apical to the basal end of the parasite instead of terminating at approximately two-thirds as seen in the wild-type parasite. The arrowhead indicates the conoid in the apical complex (see Figure 1). Arrows indicate a microtubule decorated by mAppleFP-TLAP2 prominently at its distal section. Green, mAppleFP-TLAP2. Red, eGFP-Tgβ2-tubulin. Scale bars, 2 μm. (B) Projections of 3D-SIM images of a single eGFP–Tgβ2-tubulin transgenic parasite transiently expressing mAppleFP-TLAP2. As seen in A, some microtubules extend from the apical to the basal end of the parasite and are decorated by mAppleFP-TLAP2 on the distal section (arrows). Some TLAP2-decorated microtubules coalesce to form bundles (insets). The arrowhead indicates the conoid. Insets: 2×. (C) Projections of 3D-SIM images of a mass of eGFP–Tgβ2-tubulin transgenic parasite transiently expressing mAppleFP-TLAP2, in which the organization of the microtubule cytoskeleton and the morphology of the parasite are dramatically altered. The arrowhead indicates the conoid. (D) Projections of deconvolved wide-field images of parasites transiently expressing mEmeraldFP-TLAP2 (green) and labeled with a rabbit anti-IMC1 antibody (red), which marks the cortex of the parasite. Multiple fibers containing mEmeraldFP-TLAP2 form in the cytoplasm, and the shape of the parasite mass (∼14 μm × 16 μm) is severely distorted. The anti-IMC1 labeling highlights irregular extensions close to the periphery of the parasite. The inset (1×) shows a surface section in the region indicated by the frame.