Fig. 4. T. cruzi associates with host cell plasma membrane markers in an actin-independent entry process. (A,B) CHO cells transfected with myr-GFP were infected with T. cruzi trypomastigotes for 15 minutes. The extracellular region of partially internalized parasites was stained with a T. cruzi-specific antibody followed by TR-conjugated secondary antibody (arrowheads) highlighting the ability of T. cruzi to enter cells either (A) posterior end (DAPI-stained, blue) or (B) anterior end first. (C) Fully internalized trypomastigote in CHO cells expressing PLC-PH-GFP 15 minutes after infection. (D) Akt-PH-GFP-expressing L6E9 infected with T. cruzi for 15 minutes were stained with TR-phalloidin (E; arrowhead). (F) Partial co-localization of T. cruzi-associated Akt-PHGFP and F-actin is observed (merged image). (G) Phagocytic uptake of IgG-coated 3 µm latex beads by FCRIII-CHO cells stained with TR-phalloidin (arrowheads). (H,I) CHO cells transfected with PLC-PH-GFP were pre-treated with 10 µM cytochalasin D or vehicle control for 10 minutes, infected with trypomastigotes for 15 minutes and the number of intracellular parasites (H) and the percentage of intracellular parasites associating with PLC-PH-GFP (I) was determined. Data are represented as means ± s.d. for 3 coverslips. Scale bars: 5 µm.