Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the −6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 β2-β3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.     

Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: Physiological rearrangement of tight junctions

Summary All epithelia slough dying cells but the consequences of this physiological process to epithelial barrier functions is unknown. In mammalian small intestine absorptive cells are known to migrate from the villus base to the villus tip from which they slough. These villus tip extrusion zones are often envisioned as sites at which macromolecules could leak across the epithelium. However, only trace amounts of macromolecules cross this epithelium even though, based on known epithelial turnover rates, extrusion events occur millions of times daily. Here, we examine the characteristics of the epithelial barrier to macromolecular permeation at villus tip extrusion zones in rats and hamsters. Freeze-fracture, light and electron microscope studies reveal that extruding cells do not leave transient holes behind as they lift from the epithelium. Rather, as cells extrude, processes of adjacent cells extend under them. Moreover, tight junction elements proliferate between extruding cells and their neighbors and appear to move down the lateral margin of the extruding cell as it extends into the lumen. These observations suggest that newly formed junctional elements zipper the epithelium closed as extrusion proceeds thus preventing epithelial discontinuities from occurring. Correlative in vivo perfusion experiments using horseradish peroxidase as a macromolecular-tracershow that the above described dynamic alterations in tight junctions at extrusion sites are generally sufficient to prevent transepithelial leaks of macromolecules.     

Intercellular junctions in the hypodermis,salivary gland and gené's organ of the cattle tick,Boophilus microplus

Summary The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.The authors would like to thank Mr. R. Lamb for preparing the plates. M.W.J. Megaw was supported by an S.R.C. Studentship     

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.     

Maps, books and other metaphors for systems biology

We briefly review the use of metaphors in science and progressively focus on fields from biology and molecular biology to genomics and bioinformatics. We discuss how metaphors are both a tool for scientific exploration and a medium for public communication of complex subjects, by various short examples. Finally, we propose a metaphor for systems biology that provides an illuminating perspective for the ambitious goals of this field and delimits its current agenda.     

Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium

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Biology and regulation of ectoplasmic specialization, an atypical adherens junction type, in the testis

Anchoring junctions are cell adhesion apparatus present in all epithelia and endothelia. They are found at the cell-cell interface (adherens junction (AJ) and desmosome) and cell-matrix interface (focal contact and hemidesmosome). In this review, we focus our discussion on AJ in particular the dynamic changes and regulation of this junction type in normal epithelia using testis as a model. There are extensive restructuring of AJ (e.g., ectoplasmic specialization, ES, a testis-specific AJ) at the Sertoli-Sertoli cell interface (basal ES) and Sertoli-elongating spermatid interface (apical ES) during the seminiferous epithelial cycle of spermatogenesis to facilitate the migration of developing germ cells across the seminiferous epithelium. Furthermore, recent findings have shown that ES also confers cell orientation and polarity in the seminiferous epithelium, illustrating that some of the functions initially ascribed to tight junctions (TJ), such as conferring cell polarity, are also part of the inherent properties of the AJ (e.g., apical ES) in the testis. The biology and regulation based on recent studies in the testis are of interest to cell biologists in the field, in particular their regulation, which perhaps is applicable to tumorigenesis.     

Protective role of p120-catenin in maintaining the integrity of adherens and tight junctions in ventilator-induced lung injury

Background

Ventilator-induced lung injury (VILI) is one of the most common complications for patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Although p120 is an important protein in the regulation of cell junctions, further mechanisms should be explored for prevention and treatment of VILI.

Methods

Mouse lung epithelial cells (MLE-12), which were transfected with p120 small interfering (si)RNA, p120 cDNA, wild-type E-cadherin juxtamembrane domain or a K83R mutant juxtamembrane domain (K83R-JMD), were subjected to 20 % cyclic stretches for 2 or 4 h. Furthermore, MLE-12 cells and mice, which were pretreated with the c-Src inhibitor PP₂ or RhoA inhibitor Y27632, underwent 20 % cyclic stretches or mechanical stretching, respectively. Moreover, wild-type C57BL/6 mice were transfected with p120 siRNA-liposome complexes before mechanical ventilation. Cell lysates and lung tissues were then analyzed to detect lung injury.

Results

cyclic stretches of 20 % actived c-Src, which induced degradation of E-cadherin, p120 and occludin. However, loss of p120 increased the degradation and endocytosis of E-cadherin. Immunoprecipitation and Immunofluorescence results showed a decrease in the association between p120 and E-cadherin, while gap formation increased in p120 siRNA and K83R-JMD groups after 20 % cyclic stretches. Loss of p120 also reduced the occludin level and decreased the association of occludin and ZO-1 by enhancing RhoA activity. However, the altered levels of occludin and E-cadherin were reversed by PP₂ or Y27632 treatments compared with the cyclic stretch group. Consistently, the expression, redistribution and disassociation of junction proteins were all restored in the p120 overexpression group after 20 % cyclic stretches. Moreover, the role of p120 in VILI was confirmed by increased wet/dry weigh ratio and enhanced production of cytokines (tumor necrosis factor-α and interleukin-six) in p120-depleted mice under mechanical ventilation.

Conclusions

p120 protected against VILI by regulating both adherens and tight junctions. p120 inhibited E-cadherin endocytosis by increasing the association between p120 and juxtamembrane domain of E-cadherin. Furthermore, p120 reduced the degradation of occludin by inhibiting RhoA activity. These findings illustrated further mechanisms of p120 in the prevention of VILI, especially for patients with ALI or ARDS.     

Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis

The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram‐negative and Gram‐positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell‐to‐cell junction factors including E‐cadherin, occludin, and claudin‐8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development.     

Building momentum for systems and synthetic biology in India

Biological systems are inherently noisy. Predicting the outcome of a perturbation is extremely challenging. Traditional reductionist approach of describing properties of parts, vis-a-vis higher level behaviour has led to enormous understanding of fundamental molecular level biology. This approach typically consists of converting genes into junk (knock-down) and garbage (knock-out) and observe how a system responds. To enable broader understanding of biological dynamics, an integrated computational and experimental strategy was formally proposed in mid 1990s leading to the re-emergence of Systems Biology. However, soon it became clear that natural systems were far more complex than expected. A new strategy to address biological complexity was proposed at MIT (Massachusetts Institute of Technology) in June 2004, when the first meeting of synthetic biology was held. Though the term ‘synthetic biology’ was proposed during 1970s (Szybalski in Control of gene expression, Plenum Press, New York, 1974), the usage of the original concept found an experimental proof in 2000 with the demonstration of a three-gene circuit called repressilator (Elowitz and Leibler in Nature, 403:335–338, 2000). This encouraged people to think of forward engineering biology from a set of well described parts.     

How to be an anti-reductionist about developmental biology: Response to Laubichler and Wagner

Alexander Rosenberg recently claimed (1997) that developmental biology is currently being reduced to molecular biology. cite several concrete biological examples that are intended to impugn Rosenberg's claim. I first argue that although Laubichler and Wagner's examples would refute a very strong reductionism, a more moderate reductionism would escape their attacks. Next, taking my cue from the antireductionist's perennial stress on the importance of spatial organization, I describe one form an empirical finding that refutes this moderate reductionism would take. Finally, I point out an actual example, anterior-posterior axis determination in the chick, that challenges the reductionist's belief that all developmental regularities can be explained by molecular biology. In short, I argue that Rosenberg's position can be saved from Laubichler and Wagner's criticisms and putative counter-examples, but it would not survive a different kind of counter-example.     

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. In this review, we will focus on specific mechanisms of endothelial tight and adherens junctions.     

Reproductive biology of Trichocentrum pumilum: an orchid pollinated by oil-collecting bees

The reproductive biology, reward production and pollination mechanism of Trichocentrum pumilum were studied in a gallery forest in the interior of the State of São Paulo, southeast Brazil. The floral visitors and pollination mechanism were recorded, and experimental pollinations were carried out in order to determine the breeding system of this species. Trichocentrum pumilum blooms in spring. Each paniculate inflorescence bears an average of 85 flowers that present a central yellow callus and finger‐like trichomes on the lateral lobes of the lip. A lipoidal substance is produced and stored among these trichomes. In the studied population, T. pumilum is exclusively visited and pollinated by two bee species (Tetrapedia diversipes and Lophopedia nigrispinis). Pollinaria are deposited on mouthparts of bees during collection of the lipoidal substance from the lateral lobes of the labellum. Trichocentrum pumilum is self‐incompatible and pollinator‐limited. Natural fruit set was low (9%, compared to 45% in experimentally cross‐pollinated flowers). Potentially viable seed exceed 97% in fruits obtained through cross‐pollination and in natural conditions (open pollination).     

Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins

Tight junctions (TJs) and adherens junctions (AJs) are dynamic structures linked to the actin cytoskeleton, which control the paracellular permeability of epithelial and endothelial barriers. TJs and AJs are strictly regulated in a spatio-temporal manner by a complex signaling network, including Rho/Ras-GTPases, which have a pivotal role. Rho preferentially regulates TJs by controlling the contraction of apical acto-myosin filaments, whereas Rac/Cdc42 mainly coordinate the assembly-disassembly of AJ components. However, a subtle balance of Rho/Ras-GTPase activity and interplay between these molecules is required to maintain an optimal organization and function of TJs and AJs. Conversely, integrity of intercellular junctions generates signals through Rho-GTPases, which are involved in the regulation of multiple cellular processes. Rho/Ras-GTPases and the control of intercellular junctions are the target of various bacterial toxins responsible for severe diseases in man and animals, and are part of their mechanism of action. This review focuses on the regulation of TJs and AJs by Rho/Ras-GTPases through molecular approaches and bacterial toxins.     

细胞间隙连接通讯与肿瘤

由连接蛋白构成的细胞间隙连接通讯(GJIC)广泛存在于脊椎动物中,可以直接介导细胞间小分子物质的传递,而不需要通过细胞间质。GJIC与肿瘤密切相关,多数肿瘤GJIC能力降低或丧失,连接蛋白不表达或胞内定位,而恢复GJIC可以抑制肿瘤, GJIC已成为肿瘤预防与治疗研究的潜在靶点之一。     

Multi-scale modeling in biology: how to bridge the gaps between scales?

Human physiological functions are regulated across many orders of magnitude in space and time. Integrating the information and dynamics from one scale to another is critical for the understanding of human physiology and the treatment of diseases. Multi-scale modeling, as a computational approach, has been widely adopted by researchers in computational and systems biology. A key unsolved issue is how to represent appropriately the dynamical behaviors of a high-dimensional model of a lower scale by a low-dimensional model of a higher scale, so that it can be used to investigate complex dynamical behaviors at even higher scales of integration. In the article, we first review the widely-used different modeling methodologies and their applications at different scales. We then discuss the gaps between different modeling methodologies and between scales, and discuss potential methods for bridging the gaps between scales.     

Improved understanding of gene expression regulation using systems biology

This article reviews the current state of systems biology approaches, including the experimental tools used to generate ‘omic’ data and computational frameworks to interpret this data. Through illustrative examples, systems biology approaches to understand gene expression and gene expression regulation are discussed. Some of the challenges facing this field and the future opportunities in the systems biology era are highlighted.     

冷冻电子显微学在结构生物学研究中的现状与展望

冷冻电子显微学近年来在电子显微镜的硬件设备及结构解析的软件算法等方面取得了多个重要的技术突破,正在成为结构生物学研究的重要技术手段,为越来越多的生物学研究者所重视.冷冻电子显微学的技术特点决定了它所具备的一些独特优势和发展方向,同时作为一个正在迅速发展的科学技术领域,需要多学科的交叉促进.本文主要介绍冷冻电子显微学的研究现状及面临的技术挑战,并提出未来可能实现结构生物学与细胞生物学不同尺度的研究在冷冻电子显微学技术上融合的新方法.     

进化细胞生物学的提出及其任务

作者提出应创建一门源于进化生物学与细胞生物学两者的交叉学科一进化细胞生物学(细胞的进化生物学)。其根本任务在于用进化的观点考察真核细胞的一切方面,从它们的起源和演化来认识它们的现在。文中列举了其具体的研究内容,并分析了其研究方法上的特点,指出在这里需要把进化生物学的综合性分析与细胞生物学的实验研究最紧密地结合起来。文中还论述了真核细胞的细胞器的“不进化”现象,指出其根本原因在于进化焦点的转移。     

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway

Large‐scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi‐dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein–protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA‐mediated gene knockdown on Wnt signaling. Meta‐analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates β‐catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.