Impaired PKCζ/Tiam1/Rac signaling induces a switch from paracellular to transcellular migration. (A,B) Activated WT and Tiam1^−/− T cells were incubated on top of a monolayer of TNFα-activated bEnd.3 cells overlaid with SDF1α for 30 minutes. Fixed samples were subjected to LFA-1, ICAM-1, and PECAM-1 staining. (A) Representative examples of immunofluorescence images showing paracellular and transcellular migration of a T cell. Scale bars represent 5 μm. (B) Percentage of WT and Tiam1^−/− T cells showing diapedesis via the transcellular route. Three independent experiments were performed. Values are means ± SD; p indicates P value between WT and Tiam1^−/− T cells. (C) Tiam1^−/− T cells migrating on activated bEnd.3 were fixed after 30 minutes and processed for transmission electron microscopy (EM). Image shows typical podosome structures invading the endothelial cell (→). (D,E) Naive T cells were either untreated or treated 2 μm PP2for 1 hour. After accumulation on activated bEnd.3 monolayer, T cells were subjected to shear flow for 15 minutes. Percentages of adherent cells undergoing diapedesis during this period were evaluated. Diapedesis of PP2-treated T cells was expressed as a percentage of control cells (untreated cells = 100%). Two fields for each condition were recorded in 4 independent experiments. Values indicate mean ± SD. (D) Comparison of diapedesis route used by WT and Tiam1^−/− T cells upon PP2 treatment; p indicates P value between percentage WT and Tiam1^−/− T cells undergoing diapedesis. (E) WT T cells were untreated or treated for 1 hour at 37°C with a PKCζ inhibitor (2 μM) before shear flow experiments; p indicates P value between percentage of untreated WT cells and PKCζ inhibitor–treated WT cells undergoing diapedesis. (F) Model illustrating that crawling and the route of transendothelial migration depends on PKCζ/Tiam1-dependent polarization and crawling. Tiam1 is dispensable for T-cell adhesion to endothelial monolayers but is required for polarization and subsequent T-cell crawling on top of endothelial monolayers. T cells with intact PKCζ/Tiam1 signaling preferentially transmigrate through endothelial junctions (paracellular migration). Loss of Tiam1 and disturbed PKCζ signaling in T cells prevents chemokine-induced Rac activation leading to unstable polarization and thereby impaired T-cell crawling on top of endothelial monolayers. As a result, these T cells preferentially cross the endothelial monolayer through individual endothelial cells (transcellular migration).