Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability
Gaelle Benais-Pont, Anu Punn, Catalina Flores-Maldonado, Judith Eckert, Graca Raposo, Tom P Fleming, Marcelino Cereijido, Maria S Balda, Karl Matter, Gaelle Benais-Pont, Anu Punn, Catalina Flores-Maldonado, Judith Eckert, Graca Raposo, Tom P Fleming, Marcelino Cereijido, Maria S Balda, Karl Matter
Abstract
Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.
Figures
References
- Adamson, P., S. Etienne, P.O. Couraud, V. Calder, and J. Greenwood. 1999. Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway. J. Immunol. 162:2964–2973.
- Anderson, J.M. 2001. Molecular structure of tight junctions and their role in epithelial transport. News Physiol. Sci. 16:126–130.
- Anderson, J.M., B.R. Stevenson, L.A. Jesaitis, D.A. Goodenough, and M.S. Mooseker. 1988. Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J. Cell Biol. 106:1141–1149.
- Balda, M.S., and K. Matter. 2000. a. The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression. EMBO J. 19:2024–2033.
- Balda, M.S., and K. Matter. 2000. b. Transmembrane proteins of tight junctions. Semin. Cell Dev. Biol. 11:281–289.
- Balda, M.S., J.A. Whitney, C. Flores, S. González, M. Cereijido, and K. Matter. 1996. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol. 134:1031–1049.
- Balda, M.S., C. Flores-Maldonado, M. Cereijido, and K. Matter. 2000. Multiple domains of occludin are involved in the regulation of paracellular permeability. J. Cell. Biochem. 78:85–96.
- Benais-Pont, G., K. Matter, and M.S. Balda. 2001. Intracellular signaling in classical and new tight junction functions. Tight Junctions. J.M. Anderson and M. Cereijido, editors. CRC Press, Boca Roton, FL. 367–394.
- Benard, V., B.P. Bohl, and G.M. Bokoch. 1999. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274:13198–13204.
- Blum, M.S., E. Toninelli, J.M. Anderson, M.S. Balda, J. Zhou, L. O'Donnell, R. Pardi, and J.R. Bender. 1997. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am. J. Physiol. 273:H286–H294.
- Cereijido, M., L. González-Mariscal, and G. Contreras. 1989. Tight junction: barrier between higher organisms and environment. News Physiol. Sci. 4:72–74.
- Cereijido, M., L. Shoshani, and R.G. Contreras. 2000. Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight junctions and epithelial polarity. Am. J. Physiol. Gastrointest. Liver Physiol. 279:G477–G482.
- Claude, P. 1978. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J. Membr. Biol. 39:219–232.
- Cordenonsi, M., F. D'Atri, E. Hammar, D.A. Parry, J. Kendrick-Jones, D. Shore, and S. Citi. 1999. Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin. J. Cell Biol. 147:1569–1582.
- D'Atri, F., and S. Citi. 2001. Cingulin interacts with F-actin in vitro. FEBS Lett. 507:21–24.
- D'Atri, F., and S. Citi. 2002. Molecular complexity of vertebrate tight junctions. Mol. Membr. Biol. 19:103–112.
- Fanning, A.S. 2001. Organization and the regulation of the tight junction by the actin-myosin cytoskeleton. Tight Junctions. J.M. Anderson and M. Cereijido, editors. CRC Press, Boca Roton, FL. 265–284.
- Fanning, A.S., and J.M. Anderson. 1998. The tight junction protein ZO-1 establishes a link between the membrane protein occludin and the actin cytoskeleton. J. Biol. Chem. 273:29745–29753.
- Fleming, T.P., B. Sheth, and I. Fesenko. 2001. Cell adhesion in the preimplantation mammalian embryo and its role in trophectoderm differentiation and blastocyst morphogenesis. Front. Biosci. 6:D1000–D1007.
- Fujita, H., H. Katoh, H. Hasegawa, H. Yasui, J. Aoki, Y. Yamaguchi, and M. Negishi. 2000. Molecular decipherment of Rho effector pathways regulating tight-junction permeability. Biochem. J. 346:617–622.
- Fukata, M., and K. Kaibuchi. 2001. Rho-family GTPases in cadherin-mediated cell-cell adhesion. Nat. Rev. Mol. Cell Biol. 2:887–897.
- Glaven, J.A., I.P. Whitehead, T. Nomanbhoy, R. Kay, and R.A. Cerione. 1996. Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein. J. Biol. Chem. 271:27374–27381.
- Glaven, J.A., I. Whitehead, S. Bagrodia, R. Kay, and R.A. Cerione. 1999. The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signaling pathways in cells. J. Biol. Chem. 274:2279–2285.
- Glotzer, M. 2001. Animal cell cytokinesis. Annu. Rev. Cell Dev. Biol. 17:351–381.
- Gopalakrishnan, S., N. Raman, S.J. Atkinson, and J.A. Marrs. 1998. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am. J. Physiol. 275:C798–C809.
- Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science. 279:509–514.
- Hasegawa, H., H. Fujita, H. Katoh, J. Aoki, K. Nakamura, A. Ichikawa, and M. Negishi. 1999. Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells. J. Biol. Chem. 274:20982–20988.
- Hauri, H.-P., E.E. Sterchi, D. Bienz, J.A.M. Fransen, and A. Marxer. 1985. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J. Cell Biol. 101:838–851.
- Hecht, G., L. Pestic, G. Nikcevic, A. Koutsouris, J. Tripuraneni, D.D. Lorimer, G. Nowak, V. Guerriero, Jr., E.L. Elson, and P.D. Lanerolle. 1996. Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability. Am. J. Physiol. 271:C1678–C1684.
- Herrenknecht, K., M. Ozawa, C. Eckerskorn, F. Lottspeich, M. Lenter, and R. Kemler. 1991. The uvomorulin-anchorage protein alpha catenin is a vinculin homologue. Proc. Natl. Acad. Sci. USA. 88:9156–9160.
- Herz, J., N. Flint, K. Stanley, R. Frank, and B. Dobberstein. 1990. The 68 kDa protein of signal recognition particle contains a glycine-rich region also found in certain RNA-binding proteins. FEBS Lett. 276:103–107.
- Hirase, T., S. Kwashima, E.Y. Wong, T. Ueyama, Y. Rikitake, S. Tsukita, M. Yokoyama, and J.M. Staddon. 2001. Regulation of tight junction permeability and occludin phosphorylation by RhoA-p160ROCK-dependent and -independent mechanisms. J. Biol. Chem. 276:10423–10431.
- Hirokawa, N., and L.G. Tilney. 1982. Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear. J. Cell Biol. 95:249–261.
- Itoh, M., A. Nagafuchi, S. Moroi, and S. Tsukita. 1997. Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments. J. Cell Biol. 138:181–192.
- Itoh, M., K. Morita, and S. Tsukita. 1999. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J. Biol. Chem. 274:5981–5986.
- Jou, T.S., E.E. Schneeberger, and W.J. Nelson. 1998. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J. Cell Biol. 142:101–115.
- Kreis, T.E. 1987. Microtubules containing detyrosinated tubulin are less dynamic. EMBO J. 6:2597–2606.
- Krendel, M., F.T. Zenke, and G.M. Bokoch. 2002. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat. Cell Biol. 4:294–301.
- Lawrence, D.W., K.M. Comerford, and S.P. Colgan. 2002. Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch. Am. J. Physiol. Cell Physiol. 282:C1235–C1245.
- Liou, W., H.J. Geuze, and J.W. Slot. 1996. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106:41–58.
- Madara, J.L. 1987. Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am. J. Physiol. 253:C171–C175.
- Mattagajasingh, S.N., S.-H. Huang, J.S. Hartenstein, and E.J. Benz. 2000. Characterization of the interaction between protein 4.1R and ZO-2: a possible link between the tight junction and the actin cytoskeleton. J. Biol. Chem. 275:30573–30585.
- Matter, K., E.M. Yamamoto, and I. Mellman. 1994. Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J. Cell Biol. 126:991–1004.
- Meza, I., M. Sabanero, E. Stefani, and M. Cereijido. 1982. Occluding junctions in MDCK cells: modulation of transepithelial permeability by the cytoskeleton. J. Cell. Biochem. 18:407–421.
- Nakagawa, M., M. Fukata, M. Yamaga, N. Itoh, and K. Kaibuchi. 2001. Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites. J. Cell Sci. 114:1829–1838.
- Nusrat, A., M. Giry, J.R. Turner, S.P. Colgan, C.A. Parkos, D. Carnes, E. Lemichez, P. Boquet, and J.L. Madara. 1995. Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc. Natl. Acad. Sci. USA. 92:10629–10633.
- Patrie, K.M., A.J. Drescher, A. Welihinda, P. Mundel, and B. Margolis. 2002. Interaction of two actin-binding proteins, synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1. J. Biol. Chem. 277:30183–30190.
- Raposo, G., M.J. Kleijmeer, G. Posthuma, J.W. Slot, and H.J. Geuze. 1997. Immunogold labeling of ultrathin cryosections: application in immunology. Handbook Exp. Immunol. 4:1–11.
- Ren, X.D., W.B. Kiosses, and M.A. Schwartz. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18:578–585.
- Ren, Y., R. Li, Y. Zheng, and H. Busch. 1998. Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases. J. Biol. Chem. 273:34954–34960.
- Rosenblatt, J., M.C. Raff, and L.P. Cramer. 2001. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr. Biol. 11:1847–1857.
- Sahai, E., and C.J. Marshall. 2002. ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat. Cell Biol. 4:408–415.
- Schneeberger, E.E., and R.D. Lynch. 2001. Ultrastructure and immuno-labeling of the tight junction. Tight Junctions. J.M. Anderson and M. Cereijido, editors. CRC Press, Boca Roton, FL. 19–37.
- Sheth, B., I. Fesenko, J.E. Collins, B. Moran, A.E. Wild, J.M. Anderson, and T.P. Fleming. 1997. Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform. Development. 124:2027–2037.
- Sheth, B., B. Moran, J.M. Anderson, and T.P. Fleming. 2000. Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation. Development. 127:831–840.
- Takaishi, K., T. Sasaki, H. Kotani, H. Nishioka, and Y. Takai. 1997. Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells. J. Cell Biol. 139:1047–1059.
- Takakuwa, R., Y. Kokai, T. Kojima, T. Akatsuka, H. Tobioka, N. Sawada, and M. Mori. 2000. Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B. Exp. Cell Res. 257:238–244.
- Takumi, T., and H.F. Lodish. 1994. Rapid cDNA cloning by PCR screening. Biotechniques. 17:443–444.
- Tsukamoto, T., and S.K. Nigam. 1997. Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly. J. Biol. Chem. 272:16133–16139.
- Tsukita, S., M. Furuse, and M. Itoh. 1999. Structural and signalling molecules come together at tight junctions. Curr. Opin. Cell Biol. 11:628–633.
- Tsukita, S., M. Furuse, and M. Itoh. 2001. Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell Biol. 2:285–293.
- Turner, J.R., B.K. Rill, S.L. Carlson, D. Carnes, R. Kerner, R.J. Mrsny, and J.L. Madara. 1997. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am. J. Physiol. 273:C1378–C1385.
- Walsh, S.V., A.M. Hopkins, J. Chen, S. Narumiya, C.A. Parkos, and A. Nusrat. 2001. Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia. Gastroenterology. 121:566–579.
- Whitehead, I., H. Kirk, C. Tognon, G. Trigo-Gonzalez, and R. Kay. 1995. Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C. J. Biol. Chem. 270:18388–18395.
- Whitehead, I.P., S. Campbell, K.L. Rossman, and C.J. Der. 1997. Dbl family proteins. Biochim. Biophys. Acta. 1332:F1–F23.
- Wittchen, E.S., J. Haskins, and B.R. Stevenson. 1999. Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J. Biol. Chem. 274:35179–35185.
- Wojciak-Stothard, B., S. Potempa, T. Eichholtz, and A.J. Ridley. 2001. Rho and Rac but not Cdc42 regulate endothelial cell permeability. J. Cell Sci. 114:1343–1355.
- Yu, J.Y., S.L. DeRuiter, and D.L. Turner. 2002. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA. 99:6047–6052.
- Zahraoui, A., D. Louvard, and T. Galli. 2000. Tight junction, a platform for trafficking and signaling protein complexes. J. Cell Biol. 151:F31–F36.
- Zhong, C., M.S. Kinch, and K. Burridge. 1997. Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells. Mol. Biol. Cell. 8:2329–2344.
- Zondag, G.C., E.E. Evers, J.P. ten Klooster, L. Janssen, R.A. van der Kammen, and J.G. Collard. 2000. Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition. J. Cell Biol. 149:775–782.
Source: PubMed