scholarly journals Phase separation of zonula occludens proteins drives formation of tight junctions

2019 ◽  
Author(s):  
Oliver Beutel ◽  
Riccardo Maraspini ◽  
Karina Pombo-Garcia ◽  
Cécilie Martin-Lemaitre ◽  
Alf Honigmann
Keyword(s):  
Phase Separation ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Biology ◽  
Active Phase ◽  
Molecular Assembly ◽  
Scaffolding Proteins ◽  
Molecular Binding ◽  
Membrane Bound

AbstractTight junctions are cell adhesion complexes that seal tissues and are involved in cell polarity and signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how ZO proteins organize junction assembly, we performed quantitative cell biology andin vitroreconstitution experiments. We discovered that ZO proteins self-organize membrane attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight junction proteins including adhesion receptors, cytoskeletal adapters and transcription factors. Our results suggest that an active phase transition of ZO proteins into a condensed membrane bound compartment drives claudin polymerization and coalescence of a continuous tight junction belt.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

scholarly journals Direct Current Stimulation Disrupts Endothelial Glycocalyx and Tight Junctions of the Blood-Brain Barrier in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Yifan Xia ◽  
Yunfei Li ◽  
Wasem Khalid ◽  
Marom Bikson ◽  
Bingmei M. Fu
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Blood Brain Barrier ◽  
Direct Current ◽  
Endothelial Glycocalyx ◽  
Microvascular Endothelial Cells ◽  
Bbb Permeability

Transcranial direct current stimulation (tDCS) is a non-invasive physical therapy to treat many psychiatric disorders and to enhance memory and cognition in healthy individuals. Our recent studies showed that tDCS with the proper dosage and duration can transiently enhance the permeability (P) of the blood-brain barrier (BBB) in rat brain to various sized solutes. Based on the in vivo permeability data, a transport model for the paracellular pathway of the BBB also predicted that tDCS can transiently disrupt the endothelial glycocalyx (EG) and the tight junction between endothelial cells. To confirm these predictions and to investigate the structural mechanisms by which tDCS modulates P of the BBB, we directly quantified the EG and tight junctions of in vitro BBB models after DCS treatment. Human cerebral microvascular endothelial cells (hCMECs) and mouse brain microvascular endothelial cells (bEnd3) were cultured on the Transwell filter with 3 μm pores to generate in vitro BBBs. After confluence, 0.1–1 mA/cm2 DCS was applied for 5 and 10 min. TEER and P to dextran-70k of the in vitro BBB were measured, HS (heparan sulfate) and hyaluronic acid (HA) of EG was immuno-stained and quantified, as well as the tight junction ZO-1. We found disrupted EG and ZO-1 when P to dextran-70k was increased and TEER was decreased by the DCS. To further investigate the cellular signaling mechanism of DCS on the BBB permeability, we pretreated the in vitro BBB with a nitric oxide synthase (NOS) inhibitor, L-NMMA. L-NMMA diminished the effect of DCS on the BBB permeability by protecting the EG and reinforcing tight junctions. These in vitro results conform to the in vivo observations and confirm the model prediction that DCS can disrupt the EG and tight junction of the BBB. Nevertheless, the in vivo effects of DCS are transient which backup its safety in the clinical application. In conclusion, our current study directly elucidates the structural and signaling mechanisms by which DCS modulates the BBB permeability.

scholarly journals Enteroaggregative Escherichia coli Disrupts Epithelial Cell Tight Junctions

2010 ◽  
Vol 78 (11) ◽  
pp. 4958-4964 ◽  
Author(s):  
Maura C. Strauman ◽  
Jill M. Harper ◽  
Susan M. Harrington ◽  
Erik Juncker Boll ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Model ◽  
Barrier Dysfunction ◽  
T84 Cells ◽  
E Coli ◽  
Eaec Strain ◽  
Epithelial Barrier Dysfunction

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.

scholarly journals The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

2007 ◽  
Vol 18 (3) ◽  
pp. 721-731 ◽  
Author(s):  
Alan S. Fanning ◽  
Brent P. Little ◽  
Christoph Rahner ◽  
Darkhan Utepbergenov ◽  
Zenta Walther ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Cultured Cells ◽  
Variable Region ◽  
Binding Studies ◽  
Vitro Binding ◽  
Complex Protein ◽  
Regulatory Functions ◽  
Maguk Proteins

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins.

scholarly journals Controlling compartmentalization by non-membrane-bound organelles

2018 ◽  
Vol 373 (1747) ◽  
pp. 20170193 ◽  
Author(s):  
Richard J. Wheeler ◽  
Anthony A. Hyman
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Scaffold Protein ◽  
Theme Issue ◽  
New Class ◽  
Cellular Events ◽  
Condensate Formation ◽  
Membrane Bound ◽  
Two Phases ◽  
In Cells

Compartmentalization is a characterizing feature of complexity in cells, used to organize their biochemistry. Membrane-bound organelles are most widely known, but non-membrane-bound liquid organelles also exist. These have recently been shown to form by phase separation of specific types of proteins known as scaffolds. This forms two phases: a condensate that is enriched in scaffold protein separated by a phase boundary from the cytoplasm or nucleoplasm with a low concentration of the scaffold protein. Phase separation is well known for synthetic polymers, but also appears important in cells. Here, we review the properties of proteins important for forming these non-membrane-bound organelles, focusing on the energetically favourable interactions that drive condensation. On this basis we make qualitative predictions about how cells may control compartmentalization by condensates; the partition of specific molecules to a condensate; the control of condensation and dissolution of condensates; and the regulation of condensate nucleation. There are emerging data supporting many of these predictions, although future results may prove incorrect. It appears that many molecules may have the ability to modulate condensate formation, making condensates a potential target for future therapeutics. The emerging properties of condensates are fundamentally unlike the properties of membrane-bound organelles. They have the capacity to rapidly integrate cellular events and act as a new class of sensors for internal and external environments. This article is part of the theme issue ‘Self-organization in cell biology’.

scholarly journals A novel in vitro platform for the study of SN38-induced mucosal damage and the development of Toll-like receptor 4-targeted therapeutic options

2016 ◽  
Vol 241 (13) ◽  
pp. 1386-1394 ◽  
Author(s):  
Hannah R Wardill ◽  
Rachel J Gibson ◽  
Ysabella ZA Van Sebille ◽  
Kate R Secombe ◽  
Richard M Logan ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Mucosal Damage ◽  
Toll Like Receptor ◽  
Tight Junction Proteins ◽  
T84 Cells ◽  
Toll Like Receptor 4 ◽  
Transmission Electron ◽  
Junction Proteins

Tight junction and epithelial barrier disruption is a common trait of many gastrointestinal pathologies, including chemotherapy-induced gut toxicity. Currently, there are no validated in vitro models suitable for the study of chemotherapy-induced mucosal damage that allow paralleled functional and structural analyses of tight junction integrity. We therefore aimed to determine if a transparent, polyester membrane insert supports a polarized T84 monolayer with the phenotypically normal tight junctions. T84 cells (passage 5–15) were seeded into either 0.6 cm2, 0.4 µm pore mixed-cellulose transwell hanging inserts or 1.12 cm2, 0.4 µm pore polyester transwell inserts at varying densities. Transepithelial electrical resistance was measured daily to assess barrier formation. Immunofluoresence for key tight junction proteins (occludin, zonular occludens-1, claudin-1) and transmission electron microscopy were performed to assess tight junction integrity, organelle distribution, and polarity. Reverse transcription-polymerase chain reaction was performed to determine expression of toll-like receptor 4 (TLR4). Liquid chromatography was also conducted to assess SN38 degradation in this model. Polyester membrane inserts support a polarized T84 phenotype with functional tight junctions in vitro. Transmission electron microscopy indicated polarity, with apico-laterally located tight junctions. Immunofluorescence showed membranous staining for all tight junction proteins. No internalization was evident. T84 cells expressed TLR4, although this was significantly lower than levels seen in HT29 cells ( P = .0377). SN38 underwent more rapid degradation in the presence of cells (−76.04 ± 1.86%) compared to blank membrane (−48.39 ± 4.01%), indicating metabolic processes. Polyester membrane inserts provide a novel platform for paralleled functional and structural analysis of tight junction integrity in T84 monolayers. T84 cells exhibit the unique ability to metabolize SN38 as well as expressing TLR4, making this an excellent platform to study clinically relevant therapeutic interventions for SN38-induced mucosal damage by targeting TLR4.

Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types

1992 ◽  
Vol 262 (2) ◽  
pp. C461-C469 ◽  
Author(s):  
A. G. Howarth ◽  
M. R. Hughes ◽  
B. R. Stevenson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Primary Cultures ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Dermal Fibroblasts ◽  
Cell Contact ◽  
Cell Periphery ◽  
Cell Extracts

ZO-1 is a high molecular mass phosphoprotein peripherally associated with the cytoplasmic surface of tight junctions in epithelial and endothelial cells. We report here that ZO-1 is also present in several nonepithelial cell types in vitro that are not believed to form tight junctions, including primary cultures of astrocytes, Schwann cells, and dermal fibroblasts and the C6 glioma, S-180 (sarcoma), and P3 myeloma cell lines. Immunoblots of cell extracts probed with a ZO-1-specific monoclonal antibody reveal a single band that comigrates with ZO-1 from rodent epithelial cells at 225 kDa. In addition, these cells contain a single mRNA species of identical size to that previously reported for ZO-1 in epithelial tissues, as determined by Northern blots probed with a partial ZO-1 cDNA. Immunofluorescence microscopy demonstrates diverse ZO-1 distributions in these cells. In astrocytes, identified by the presence of glial fibrillary acidic protein, ZO-1 is localized at discrete sites of cell-cell contact as well as within the cell cytoplasm. In contrast, S-180 cells display diffuse staining at the cell periphery and within the cytoplasm. Dermal fibroblasts show no staining above background, although ZO-1 was detected on immunoblots of fibroblast cell extracts. Immunofluorescence staining of frozen sections of mouse brain demonstrates no detectable ZO-1 immunoreactivity outside blood vessels where endothelial cell tight junctions of the blood-brain barrier are located. These studies suggest that, although ZO-1 is found to be associated with the tight junction, it has a broader distribution than previously recognized.

scholarly journals Host–Pathogen Interactions of Chlamydia trachomatis in Porcine Oviduct Epithelial Cells

Pathogens ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1270
Author(s):  
Amanda F. Amaral ◽  
Bryan E. McQueen ◽  
Kimberly Bellingham-Johnstun ◽  
Taylor B. Poston ◽  
Toni Darville ◽  
...  
Keyword(s):  
Immune Response ◽  
Chlamydia Trachomatis ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Barrier Function ◽  
Epithelial Barrier ◽  
Developmental Cycle ◽  
Epithelial Barrier Function ◽  
Membrane Bound

Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host–pathogen interactions in porcine oviduct epithelial cells (pOECs)—the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

1998 ◽  
Vol 275 (3) ◽  
pp. C798-C809 ◽  
Author(s):  
Shobha Gopalakrishnan ◽  
Narayan Raman ◽  
Simon J. Atkinson ◽  
James A. Marrs
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Rho Gtpase ◽  
Mdck Cells ◽  
Atp Depletion ◽  
Cell Junctions ◽  
Mutant Proteins ◽  
Vitro Model ◽  
The Relationship

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion.

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