Pamela Pulimeno

The cytoplasmic plaque of tight junctions: a scaffolding and signalling center.

The region of cytoplasm underlying the tight junction (TJ) contains several multimolecular protein complexes, which are involved in scaffolding of membrane proteins, regulation of cytoskeletal organization, establishment of polarity, and signalling to and from the nucleus. In this review, we summarize some of the most recent advances in understanding the identity of these proteins, their domain organization, their protein interactions, and their functions in vertebrate organisms. Analysis of knockdown and knockout model systems shows that several TJ proteins are essential for the formation of epithelial tissues and early embryonic development, whereas others appear to have redundant functions.

Regulation of small GTPases at epithelial cell-cell junctions

Abstract Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.

PLEKHA7 Is an Adherens Junction Protein with a Tissue Distribution and Subcellular Localization Distinct from ZO-1 and E-Cadherin

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of Mr ∼135 kDa and ∼145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts (∼5.5 kb and ∼6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, β-catenin and α-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.

Human skeletal myotubes display a cell- autonomous circadian clock implicated in basal myokine secretion

Objective Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. Methods We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. Results Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. Conclusions Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

The Tight Junction Protein Cingulin Regulates Gene Expression and RhoA Signaling

Tight junctions (TJ) regulate the passage of solutes across epithelial sheets, contribute to the establishment and maintenance of epithelial apico‐basal polarity and are involved in the regulation of gene expression and cell proliferation. Cingulin, a Mr 140 kDa protein localized in the cytoplasmic region of TJ, is not directly required for TJ formation and epithelial polarity but regulates RhoA signaling, through its interaction with the RhoA activator GEF‐H1, and gene expression. Here we describe in more detail the effect of cingulin mutation in embryoid bodies (EB) on gene expression, by identifying the genes that show the highest degree of up‐ or downregulation, and the putative canonical pathways that might be affected by cingulin. Furthermore, we show that full‐length canine GEF‐H1, produced in baculovirus‐infected insect cells, interacts with regions both in the cingulin globular head, and in the coiled‐coil rod domain. These results extend our previous studies and provide new perspectives for the mechanistic analysis of cingulin function.

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

Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherin-based cell-cell junctions in Rat1 fibroblasts requires the ZIM-containing region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively.

Cingulin, paracingulin, and PLEKHA7: signaling and cytoskeletal adaptors at the apical junctional complex.

Cingulin, paracingulin, and PLEKHA7 are proteins localized in the cytoplasmic region of the apical junctional complex of vertebrate epithelial cells. Cingulin has been detected at tight junctions (TJs), whereas paracingulin has been detected at both TJs and adherens junctions (AJs) and PLEKHA7 has been detected at AJs. One function of cingulin and paracingulin is to regulate the activity of Rho family GTPases at junctions through their direct interaction with guanidine exchange factors of RhoA and Rac1. Cingulin also contributes to the regulation of transcription of several genes in different types of cultured cells, in part through its ability to modulate RhoA activity. PLEKHA7, together with paracingulin, is part of a protein complex that links E‐cadherin to the microtubule cytoskeleton at AJs. In this paper, we review the current knowledge about these proteins, including their discovery, the characterization of their expression, localization, structure, molecular interactions, and their roles in different developmental and disease model systems.

A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells

To determine the impact of a functional human islet clock on insulin secretion and gene transcription.

The control of gene expression and cell proliferation by the epithelial apical junctional complex

The AJC (apical junctional complex) of vertebrate epithelial cells orchestrates cell-cell adhesion and tissue barrier function. In addition, it plays a pivotal role in signalling. Several protein components of the AJC, e.g. the cytoplasmic proteins β-catenin, p120-catenin and ZO (Zonula Occludens)-2, can shuttle to the nucleus, where they interact with transcription factors to regulate gene expression and cell proliferation. Other junctional proteins, e.g. angiomotin, α-catenin and cingulin, are believed to act by sequestering either transcription factors, such as YAP (Yes-associated protein), or regulators of small GTPases, such as GEF (guanine-nucleotide-exchange factor)-H1, at junctions. The signalling activities of AJC proteins are triggered by different extracellular and intracellular cues, including cell density, and physiological or pathological activation of developmentally regulated pathways, such as the Wnt pathway. The interplay between junctional protein complexes, the actin cytoskeleton and signalling pathways is of crucial importance in the regulation of gene expression and cell proliferation.

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

Abstract 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.