RON2mCherry is properly targeted to rhoptries and released to the junction during invasion and RON2mC tachyzoites have normal pre-invasive and intracellular growth properties. (A) Confocal imaging of two RON2mC tachyzoites in the course of entering into HeLa cells; (top panel). Cells were labeled for the surface-exposed P30 T. gondii protein (blue) prior to cell permeabilization and the host cell F-actin (red) after TX-100 cell permeabilization, (bottom panel) for the total RON4 protein a subset of which being localized at the junction (green) and serving as a marker (bottom panel); the pink arrowheads point to the junction; note the overlap between RON4 and RON2mC in the rhoptry compartments and the junction as well as the recruitment of host cell F-actin beneath the RON-formed junction. (B) Histograms comparing tachyzoites expressing untagged RON2 (RON2) or RON2-mCherry (RON2mC) tagged for: (left panel), the frequency of each pre-invasive behavior and (right panel) the frequency of PV containing 4, 8, 16 or 32 tachyzoites 28 hours post infection. (C, D) Time lapses showing RON2 trafficking to the tip of the extruded conoid, followed by RON2 assembly into (C) the HFF cell PM (DIC and the RON2mC (pseudo-colored in green) merged signals, (D) the HeLa Myr-Palm-GFP- (red, top panel) and HeLa GFP-PH-PLCδ-labeled PMs (red, bottom panel) when indicated by a white arrowhead, the green line points to the position of the junction at the early and ending (right frame) times of cell penetration; all scale bars: 5 μm. DIC, HeLa, human epithelial cervical cancer cells; HFF, human foreskin fibroblasts; Myr-Palm, myristoylated and palmitoylated; PM, plasma membrane; PV, parasitophorous vacuole; RON, RhOptry Neck.