Fig. 4

ΔMyoA tachyzoites form a typical ring-shaped RON-positive TJ that coincides with the strong recruitment of F-actin nucleating factors and the assembly of an actin-myosin II network. a–e Laser scan confocal micrographs of ΔMyoA mutant zoites in contact with (a) protein tyrosine kinase 1 (PtK1) epithelial and spontaneously arising retinal pigment epithelial (ARPE-19) cells, (b) human foreskin fibroblasts (HFF) and human brain microvascular endothelial cells (HBMEC), (c–e) U2OS osteosarcoma cells. Samples were fixed and processed for double or triple immunofluorescence staining with (a) anti-RON4 antibodies (red) and phalloidin (green); note the RON-positive TJ (red arrows), (b, c, e) anti-P30 antibodies (blue) prior to permeabilization to selectively label surface-exposed antigens, and (b) phalloidin (green) or (c) anti-Arp2/3 antibodies (green) after cell permeabilization. Note the spectacular co-enrichment of F-actin and the Arp2/3 complex that surrounded the TJ ring or area (green arrows), (d) anti-cortactin antibodies to localize the regulatory Arp2/3-nucleating factor (cortactin, red) and its target (the Arp2/3 complex, green), and (e) anti-Arp 2/3 antibodies (red) and phalloidin (green). Note the co-localization of F-actin and the actin nucleating machinery (Arp2/3 and cortactin) at the TJ (green and red arrows). All scale bars: 5 μm