Cingulin and paracingulin show similar dynamic behaviour, but are recruited independently to junctions.

Cingulin (CGN) and paracingulin (CGNL1) are structurally related proteins that regulate Rho family GTPases by recruiting guanine nucleotide exchange factors to epithelial junctions. Although the subcellular localization of cingulin and paracingulin is likely to be essential for their role as adaptor proteins, nothing is known on their in vivo localization, and their dynamics of exchange with the junctional membrane. To address these questions, we generated stable clones of MDCK cells expressing fluorescently tagged cingulin and paracingulin. By FRAP analysis, cingulin and paracingulin show a very similar dynamic behaviour, with recovery curves and mobile fractions that are distinct from ZO-1, and indicate a rapid exchange with a cytosolic pool. Interestingly, only paracingulin, but not cingulin, is peripherally localized in isolated cells, requires the integrity of the microtubule cytoskeleton to be stably anchored to junctions, and associates with E-cadherin. In contrast, both proteins require the integrity of the actin cytoskeleton to maintain their junctional localization. Although cingulin and paracingulin form a complex and can interact in vitro, the junctional recruitment and the dynamics of membrane exchange of paracingulin is independent of cingulin, and vice-versa. In summary, cingulin and paracingulin show a similar dynamic behaviour, but partially distinct localizations and functional interactions with the cytoskeleton, and are recruited independently to junctions.