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© The Rockefeller University Press, 0021-9525/1998//101 $5.00
The Journal of Cell Biology, Volume 142, Number 1, , 1998 101-115

Articles

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



Tzuu-Shuh Jou*, Eveline E. Schneeberger{ddagger}, and W. James Nelson*

* Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345; {ddagger} Department of Pathology, Massachusetts General Hospital, Boston, MA 02114

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.

Key Words: GTPase • RhoA • epithelial cells • tight junction • polarity

Abbreviations used in this paper: DC, doxycycline; TER, transepithelial electrical resistance; TJ, tight junctions.

We are very grateful to the following colleagues who made various reagents available to us: Dr. Rong-Guo Qiu (Onyx Pharmaceuticals, Richmond, CA) for the RhoA and Rac1 mutant plasmids; Dr. Gordon Cann (Stanford University, Stanford, CA) for the mouse anti-myc hybridoma, 9E10; Dr. Inke Nathke (Stanford University, Stanford, CA) for the rabbit anti-β-catenin polyclonal antibody; and Dr. Charles Yeaman (Stanford University, Stanford, CA) for the mouse anti-p75NTR antibody and help with the adenoviral expression system. We thank members of the Nelson laboratory and Marc Symons (Onyx Pharmaceuticals, Richmond, CA) for their criticisms and help during the course of this work.

Address all correspondence to W. James Nelson, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Beckman Center, B121, Stanford, CA 94305-5345. Tel.: 650-725-7596; Fax: 650-498-5286; E-mail: wjnelson{at}leland.stanford.edu


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