Cingulin

Cingulin (CGN; from the Latin cingere “to form a belt around”) is a cytosolic protein encoded by the CGN gene in humans  localized at tight junctions (TJs) of vertebrate epithelial and endothelial cells.

Discovery
Cingulin was originally discovered at the MRC Laboratory of Molecular Biology (Cambridge, UK) by Dr. Sandra Citi, as a protein present in chicken intestinal epithelial cells, that co-purified with non-muscle myosin II and was specifically localized at tight junctions (zonulae occludentes).

Structure & interactions
Cingulin is a homodimer, each subunit containing a N-terminal globular "head" domain, a long α-helical coiled-coil "rod" domain and a small globular C-terminal "tail" region. This organization is highly conserved throughout vertebrates. However, cingulin homologs have not been detected in invertebrates.

In vitro, cingulin can bind to and bundle actin filaments, and interact with myosin II and several TJ proteins including ZO-1, ZO-2, ZO-3, paracingulin and occludin. Moreover, cingulin forms a complex with JAM-A, a tight junction membrane protein. Most of cingulin protein interactions are through the globular head domain. Cingulin interacts with ZO-1 through an N-terminal ZO-1 interacting motif (ZIM) in its head region. The rod domain is involved in dimerization and interaction with the RhoA activator, GEF-H1.

Cingulin has also been found to interact with microtubules (MTs) through the N-terminal head region, and these interactions was regulated by phosphorylation by the adenosine monophosphate-activated protein kinase (AMPK).

Function
The function of cingulin has been studied by knockout (KO), knockdown (KD) and over-expression approaches. Embryoid bodies derived from embryonic stem cells where one or both cingulin alleles were targeted by homologous recombination show apparently normal tight junctions, but changes in the expression of a large number of genes, including tight junction protein genes (claudin-2, claudin-6, claudin-7 and occludin) and transcription factors (including GATA4). Changes in the expression of claudin-2 and ZO-3 are also observed in cultured kidney cells (MDCK) depleted of cingulin by shRNA.

In 2012, the phenotype of cingulin-knockout mice was described, proving that functional TJ in vivo can be formed in the absence of cingulin. Together with paracingulin, cingulin also was reported to regulate claudin-2 expression through RhoA-dependent and independent mechanisms. The role of cingulin in development has been studied by morpholino. oligonucleotide-mediated depletion in chicken, indicating that cingulin is involved in neural crest development. In early mouse and frog embryos, maternal cingulin is localized in the cell cortex. Through early mouse development, cytocortical cingulin in present from oogenesis (cumulus-oocyte contact sites) until 16-cells morulae stage (apical microvillous zones) during early embryogenesis; then maternal cingulin is degraded by endocytic turn-over from the 32-cells stage. Regarding the zygotic cingulin, it accumulates at the tight junctions from 16-cells stage, 10 hours after ZO-1 assembly. Furthermore, the synthesis of cingulin in early mouse embryos is tissue-specific and it occurs in blastocyst (up-regulated in trophectoderm and down-regulated in inner-cells). In Xenopus laevis embryos, maternal cingulin is recruited to apical cell-cell junctions from 2-cells stage.

Homologs
In 2004, a protein homologous to cingulin was discovered and named JACOP (also known as paracingulin, or cingulin-like 1 protein; CGNL1).

Human diseases
Although cingulin has been involved in regulation of RhoA signaling and gene expression in cultured cells and KO mice, nothing is known about the specific role of cingulin in human diseases. Cingulin expression has been studied in human carcinomas and shown to be expressed in adenocarcinomas and down-regulated in squamous carcinomas. Furthermore, histone deacetylase inhibitors, such as sodium butyrate, strongly upregulate its expression in some cultured cells. Cingulin, as other junctional proteins could be used as a marker of epithelial differentiation, and as a diagnostic marker to distinguish adenocarcinomas from squamous carcinomas.