The extracellular loop 2 (ECL2) of claudin-5 (Cldn5) is involved in cross-over endocytosis.

Murine Cldn5-mutants with an amino acid substitution in ECL2 fluorescently labelled with yellow fluorescent protein (YFP) or mTurquoise2 (TRQ) were expressed in Madin-Darby canine kidney cells II and grown in cocultures of monotransfected cells. A: Localization of Cldn5_wt, Cldn5_F147A and Cldn5_Q156E. Arrowheads: cross-over endocytosed vesicles (white color). Arrows: vesicles containing only one type of Cldn as indicated by the color of the arrow (not cross-over endocytosed). Purple: ZO-1 used as junctional marker. B: Vesicle numbers of Cldn_wt. Relative number of cross-over endocytosed vesicles vs. total TRQ-Cldn5 vesicles in untreated cocultures; mean±SEM, n>100. C: Vesicle numbers normalized to Cldn5_wt; mean±SEM, for each group n>70. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA.