Evidence for a Functional Interaction between Cingulin and ZO-1 in Cultured Cells

Abstract

Cingulin, a protein component of the submembrane plaque of tight junctions (TJ), contains globular and coiled-coil domains and interacts in vitro with several TJ and cytoskeletal proteins, including the PDZ protein ZO-1. Overexpression of Xenopus cingulin in transfected Xenopus A6 cells resulted in the disruption of endogenous ZO-1 localization, suggesting that cingulin functionally interacts with ZO-1. Glutathione S-transferase pull-down experiments showed that a conserved ZO-1 interaction motif (ZIM) at the NH 2 terminus of cingulin is required for cingulin-ZO-1 interaction in vitro. An NH 2 -terminal region of cingulin, containing the ZIM, was sufficient, when fused to coiled-coil sequences, to target transfected cingulin to junctions. However, deletion of the ZIM did not abolish junctional localization of transfected cingulin in A6 cells, suggesting that cingulin can be recruited to TJ through multiple protein interactions. Interestingly, the ZIM was required for cingulin recruitment into ZO-1-containing adherens junctions of Rat-1 fibroblasts, indicating that cingulin junctional recruitment does not require the molecular context of TJ. Cingulin coiled-coil sequences enhanced the junctional accumulation of expressed cingulin head region in A6 cells, but purified recombinant cingulin did not form filaments under physiological conditions in vitro, suggesting that the cingulin coiled-coil domain acts primarily by promoting dimerization.The ability of vertebrate organisms to develop a complex body organization depends critically on the creation and maintenance of separate body, organ, and tissue compartments. This, in turn, depends on the ability of epithelia and endothelia lining different body surfaces to form semipermeable barriers that prevent the free diffusion of molecules and cells between extracellular compartments. The structure that allows epithelia and endothelia to form a semipermeable barrier is the tight junction (TJ), 1 a gasket-like seal surrounding the apicolateral region of polarized cells. TJ also define the separation between apical and basolateral domains of the plasma membrane that show different composition and function. Thus, TJ are essential for separation of tissue compartments and maintenance of epithelial cell polarity. The structure and function of TJ vary depending on type of tissue and physiological and pathological states, suggesting that changes in the expression and/or function of different TJ components underlie such modulation.Much progress has been made in recent decades in elucidating the molecular organization of TJ. TJ comprise membrane and submembrane (cytoplasmic plaque) domains. The membrane domain of TJ contains transmembrane Ig-like proteins and four-membrane pass proteins important for cell-cell adhesion and selective paracellular permeability (see Refs. 1 and 2 for reviews). The cytoplasmic plaque of TJ contains a large number of proteins, including PDZ-containing, signaling, and cytoskeletal proteins (2, 3). TJ plaque proteins probably play an important role in epi...

A Role for ZO-1 and PLEKHA7 in Recruiting Paracingulin to Tight and Adherens Junctions of Epithelial Cells

“…Paracingulin shares with cingulin a similar dynamic behavior and domain organization, with globular head and tail domains, and a coiled-coil rod domain, but unlike cingulin, it is localized not only at TJ, but also at AJ (26,27,32). Although the TJ protein ZO-1 is required for the efficient recruitment of cingulin to TJ (13,33,34), it is not known whether paracingulin interacts with ZO-1 and whether this interaction is important for its recruitment to TJ. Furthermore, the question remains open about any potential molecular mechanism of association of paracingulin with AJ.…”

“…25), and removal of the ZIM-containing N-terminal region abolished this interaction (Fig. 1B) (33). A 110-residue N-terminal fragment of the paracingulin head domain, which contains the ZIM sequence, was sufficient to interact with ZO-1 (Fig.…”

“…The ZIM sequence, which is involved in the junctional recruitment and interaction of cingulin with ZO-1 (33), is present in the N-terminal region of the head domain of paracingulin and shows 75% identity to the homologous sequence of cingulin (Fig. 1B).…”

“…To this purpose, we first expressed fluorescently tagged paracingulin in Rat1 fibroblasts. Rat1 fibroblasts do not have TJ and TJ proteins, but form spot-like AJ at sites of cell-cell contact which contain ZO-1, allowing one to examine the interaction of exogenous proteins with ZO-1, independently of TJ (33,40,41). Importantly, spot-like AJ of Rat-1 fibroblasts do not contain the AJ protein PLEKHA7, which in these cells is only detectable at centrosomes (data not shown and Ref.…”

Cingulin is dispensable for epithelial barrier function and tight junction structure, and plays a role in the control of claudin-2 expression and response to duodenal mucosa injury

“…CGN exists as a dimer, each subunit comprising globular domains (head and tail), and a central a-helical coiled-coil domain (rod) (Cordenonsi et al, 1999). CGN interacts with several cytoplasmic TJ proteins, including ZO-1, ZO-2 and ZO-3, as well as with actin and myosin (Cordenonsi et al, 1999;D'Atri et al, 2002). The head domain of CGN is required for CGN recruitment to junctions, primarily via its interaction with ZO-1 (Cordenonsi et al, 1999;D'Atri et al, 2002).…”