Cited by 354Barrier Structure and Function StudiesProtein Kinase Regulation and GTPase SignalingCell Adhesion Molecules Research

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Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

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Jou, Tzuu-Shuh, Schneeberger, Eveline E., James Nelson, W.·1998·The Journal of Cell Biology

DOI 10.1083/jcb.142.1.101PMC PMC2133025

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Abstract

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.

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Rho GTPases and the Actin Cytoskeleton
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Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
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“To study roles of RhoA and Rac1 in the structural and functional organization of TJ, we generated MDCK cells that express RhoA and Rac1 mutants under the control of the tetracycline repressible transactivator (Gossen and Bujard, 1992).”
Background
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Authors: Jou, T.-S., Schneeberger, E. E., & James Nelson, W.
Affiliations: Massachusetts General Hospital (US)
*Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345; ‡Department of Pathology, Massachusetts General Hospital, Boston, MA 02114
ORCID: Jou, Tzuu-Shuh
Venue: The Journal of Cell Biology
Publisher: Rockefeller University Press
Published: 1998-07-13
Volume: 142
Issue: 1
Pages: 101-115
ISSN: 0021-9525 (ISSN-L)
1540-8140
DOI: 10.1083/jcb.142.1.101
PMC: PMC2133025
OpenAlex: W2046587126
Cited by: 354
References: 58
Citations by year: 2026: 1 · 2025: 7 · 2024: 5 · 2023: 2 · 2022: 5 · 2021: 6 · 2020: 3 · 2019: 5 · 2018: 3 · 2017: 5 · 2016: 5 · 2015: 4 · 2014: 16 · 2013: 10 · 2012: 14
FWCI: 16.95
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: cc-by-nc-sa
Locations: The Journal of Cell Biology (publishedVersion)
Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
PubMed Central (publishedVersion, cc-by-nc-sa)
PubMed (publishedVersion)
Topics: Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
Keywords: tight junctions, RhoA, Rac1, small GTPases, paracellular permeability, occludin, ZO-1, MDCK cells, epithelial polarity, transepithelial electrical resistance, membrane protein targeting, membrane lateral organization
MeSH terms: Animals, Cell Line, Cell Membrane, Detergents, Dogs, Epithelial Cells, Membrane Proteins, Phosphoproteins, Solubility, Mutagenesis, Octoxynol, Tight Junctions, GTP-Binding Proteins, GTP Phosphohydrolases, rhoA GTP-Binding Protein, rac GTP-Binding Proteins, Occludin, Zonula Occludens-1 Protein, Zonula Occludens-2 Protein

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Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

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