1Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322; 2Department of General Surgery, University of Muenster, 48149 Muenster, Germany; and 3Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637
Submitted 11 February 2004 ; accepted in final form 22 March 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells
Proteins constituting the TJ complex include the transmembrane proteins occludin, junctional adhesion molecule (JAM), claudin family members, and linker proteins such as ZO-1 that affiliate with the underlying actin cytoskeleton (6, 9, 10, 25). It was shown previously that claudin-1 and -2 are important for TJ strand formation (8). The AJ is immediately subjacent to the TJ and is essential for cell-cell recognition and cell sorting (13).
TJ assembly and function can be modulated by a number of signaling molecules, including cAMP, Ca2+, PKC, G proteins, phospholipase C, and diacylglycerol (1, 27, 36). Recently, the Rho family of small GTPases, comprising Rho, Rac, and Cdc42, has been shown to be important in the regulation of epithelial structure, function, and assembly (3, 18, 29, 45) and in the regulation of F-actin dynamics (15, 28, 33). Rho GTPases cycle between an active GTP-bound state and an inactive GDP-bound state (2, 16). Transitions between GTP- and GDP-bound forms of small GTPases are controlled by specialized regulators (2), and by cycling between GTP- and GDP-bound states RhoA family GTPases are able to transduce signals between cell surface receptors and intracellular target molecules (16).
Diverse pharmacological and molecular tools that interfere with Rho protein function have offered valuable insights into how Rho GTPases regulate epithelial permeability. Our initial investigations (29) utilized a modified cell-permeant chimeric toxin consisting of the Clostridium botulinum toxin C3 transferase (to inhibit RhoA activity through ADP-ribosylation of Asp41) and the receptor binding domain of diphtheria toxin to facilitate internalization. In this system, barrier function of T84 intestinal epithelial cells was compromised with reductions in transepithelial resistance (TER), enhancements in paracellular permeability, and redistribution of ZO-1 and occludin away from the TJ membrane (29). Moreover, ZO-1 and occludin, which are known to be affiliated with membrane microdomains or detergent-insoluble glycolipid rafts (DIGs), were redistributed from membrane microdomains on incubation with C. botulinum toxin (32). Related work has focused on the impact of dominant-active and dominant-negative small GTPase mutants on barrier function in Madin-Darby canine kidney (MDCK) epithelial cells and has demonstrated that either inactivation or activation of RhoA, Rac1, or Cdc42 perturbs TJ gate and fence function (22, 34). These changes were associated with redistribution of occludin and ZO-1 away from the lateral membrane and abnormal TJ strand morphology after activation of the above GTPases, whereas TJ structure remained intact after inactivation of these GTPases. Similarly, cytotoxic necrotizing factor (CNF)-1, a bacterial toxin that constitutively activates Rho GTPases via inhibition of GTP hydrolysis (7, 24, 39), was shown to disrupt barrier function by displacement of occludin and ZO-1 and reorganization of JAM-1 away from the TJ membrane (19). In contrast, another study described decreased localization of occludin and ZO-1 at cell junctions during RhoA inactivation, whereas constitutive RhoA signaling caused an accumulation of these proteins at cell junctions (11). However, continuous expression of the same construct was reported to have no effect on ZO-1 localization at MDCK cell contact sites (41). Ambiguities such as those mentioned directly above highlight the importance of solid model systems in any investigation of the role of Rho proteins in barrier function. Cellular signaling from Rho proteins is likely to be tightly regulated at all times, especially in light of the fact that any imbalances in Rho activity (whether activation or inactivation) evoke similar disturbances in function. Thus our study utilized a tetracycline-repressible system to exert tight control over the expression of Rho, Rac, and Cdc42 GTPases in MDCK epithelial cells. We used this model system to dissect out in detail the effects of Rho GTPases on TJ structure/function and the contribution of the actin cytoskeleton to this process, to resolve ambiguities regarding the contribution of Rho proteins to barrier function in vitro. We present a number of novel aspects in our study including an important role for claudin-1 and -2 in the regulation of barrier function by Rho proteins. Although increased paracellular permeability in dominant-negative RhoA was associated with changes in the apical actin only, we observed a substantial internalization of TJ transmembrane proteins in constitutively active GTPases, dominant-negative Rac1, and dominant-negative Cdc42. These structural changes were associated with an increased detergent solubility of claudin-1 and -2 in all constitutively active GTPases, whereas constitutively active RhoA alone reduced claudin-2 and ZO-1 partitioning into detergent-insoluble membrane rafts and JAM-1 from membrane raft-containing fractions.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophysiology and permeability assays. TER was checked in all monolayers before each experiment with an epithelial voltohmmeter (EVOM/EndOhm; World Precision Instruments, Sarasota, FL). Paracellular permeability to fluoresceinated dextran (mol wt 3,000; FD-3) was assessed as previously described (38). Briefly, cells grown on 0.33-cm2 permeable supports for 1848 h in medium either containing 20 ng/ml DC or lacking DC were washed in Hanks' balanced salt solution-10 mM HEPES (HBSS+) and equilibrated at 37°C for 10 min on an orbital shaker. Monolayers were loaded apically with 1 mg/ml FD-3 (Molecular Probes, Eugene, OR). Basolateral samples were collected at t = 0 and 120 min, and fluorescence intensity was analyzed on a fluorescent plate reader (Fluostar; BMG Labtechnologies, Durham, NC). FD-3 concentrations transported were extrapolated from a standard curve and expressed as micromolar FD-3 transported per square centimeter per hour. Monolayers permeabilized with Triton X-100 (TX-100; 1%) for 10 min were used as positive controls.
Immunofluorescent localization of c-myc, junctional proteins, and F-actin.
Monolayers of MDCK cells grown for 18 or 48 h on 0.33-cm2 permeable supports were washed in HBSS+, fixed/permeabilized with either ethanol at 20°C for 20 min or 3.7% paraformaldehyde [10 min, room temperature (RT)] and 0.5% TX-100. Nonspecific background was blocked with 5% BSA (1 h, RT). Monolayers were incubated with primary antibodies to occludin, ZO-1, claudin-1, claudin-2 (Zymed, San Francisco, CA), JAM-1 (J10.4; Ref. 25), E-cadherin, -catenin (Transduction Laboratories, Lexington, KY), or c-myc (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Monolayers were washed and probed with Alexa (Molecular Probes)- or FITC (Jackson Labs, West Grove, PA)-conjugated secondary antibodies. All monolayers were visualized on an LSM510 confocal microscope (Carl Zeiss Microimaging, Thornwood, NY).
Immunoblotting for TJ/AJ proteins in epithelial cells. MDCK cells were grown on 5-cm2 permeable supports for 18 or 48 h as described above, washed in HBSS+, and scraped into extraction buffer (in mM: 100 KCl, 3 NaCl, 3.5 MgCl2, and 10 HEPES pH 7.4) containing 1% TX-100, protease inhibitors (250 µM PMSF, 5 µg/ml leupeptin, 10 µg/ml chymostatin, 0.25 µg/ml pepstatin, and 2 µg/ml aprotinin), and phosphatase inhibitors (in mM: 25 sodium fluoride, 10 sodium orthovanadate). Cell lysates were centrifuged (4,000 g, 5 min, 4°C), and equivalent protein concentrations (10 µg/lane) from induced and noninduced monolayers were subjected to SDS-PAGE. Western blots were analyzed for TJ/AJ proteins, Rac1, Cdc42, and RhoA.
Differential detergent extraction of AJC proteins. MDCK cells were grown on 5-cm2 permeable supports for 18 or 48 h as described above, washed, and incubated for 30 min at 4°C with 1% TX-100 extraction buffer as above. The TX-100-soluble fraction was subjected to low-speed centrifugation to remove cell debris and added to an equal amount of 2x sample buffer (3% SDS, 0.75 M Tris pH 8.8, 20% glycerol, and 20 mM DTT). The TX-100-insoluble fraction was scraped into an equal amount of SDS sample buffer. Equal volumes of each fraction were analyzed by SDS-PAGE and immunoblotting for TJ/AJ proteins as previously described (30). Densitometry was performed with the UN-SCAN-IT automated digitizing system (Silk Scientific, Orem, UT).
Isolation of DIGs by sucrose gradient fractionation. Transfected MDCK cell lines were grown on 45-cm2 permeable supports for 1870 h and harvested into HBSS+-containing 1% TX-100 and protease inhibitors as above. The sucrose concentration of the cell lysate was adjusted to 40%, placed at the bottom of an ultracentrifuge tube, and overlaid with a 530% (wt/wt) linear sucrose gradient as previously described (31). Gradients were ultracentrifuged (19 h, 39,000 rpm, 4°C), fractionated, and analyzed for sucrose concentration, light scattering at 600 nm, protein concentration, and alkaline phosphatase activity as previously described (31, 32). TJ/AJ protein profiles in raft fractions were determined by SDS-PAGE and Western blotting.
Statistics. Results are expressed as means ± SE. Student's t-tests or Welch tests were used to compare results, with statistical significance assumed at P < 0.05. Individual experiments were performed in triplicate or greater, and each experiment was performed independently three or more times.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the second set of conditions, mutant GTPase expression was induced at early time points after cell-cell contact had occurred (18 h). Effects of each mutant on paracellular permeability are shown in Fig. 1. TER was significantly lower (P < 0.01) in all GTPase mutants relative to cells with an empty vector (pUHD) or GTPases without mutant induction (data not shown) 18 h after initiation of cell-cell contacts (Fig. 1A). After peaking at 18 h after seeding, the TER of control cells consistently dropped to low but stable values by 48 h (90
·cm2; data not shown). At this time point, TER was not affected by induction of any mutant GTPase, including Rac1N17.
|
Expression levels of mutant GTPase induction remained constant during the experimental time course for each cell line and are shown by immunoblot in Fig. 2A. Uniformity of expression in epithelial monolayers was confirmed by immunofluorescence staining of c-myc-tagged mutant GTPases (Fig. 2B).
|
|
Effects of mutant GTPases on distribution of TJ and AJ proteins. Because we observed that all GTPase mutants showed an increase in paracellular permeability and this function is primarily regulated by epithelial TJs, we examined the distribution of TJ and AJ proteins with immunofluorescence confocal microscopy. In control polarized MDCK cells expressing only the empty vector (pUHD) or noninduced GTPases (data not shown) all of the TJ proteins (Fig. 4, A1, B1, C1, D1, and E1) were appropriately localized to their respective intercellular junctions. Thus, in the en face or horizontal plane, they presented the characteristic "chicken wire" staining pattern typical for TJ-associated proteins. Expression of dominant-negative RhoA (Fig. 4, A2, B2, C2, D2, and E2) did not influence distribution of TJ proteins despite an increase in paracellular permeability 18 h after plating on inserts. In RacN17 (Fig. 4, A3, B3, C3, D3, and E3) and Cdc42N17 (Fig. 4, A4, B4, C4, D4, and E4) cells, occludin and ZO-1 localization was similar to control cells. Moreover, Cdc42N17 cells showed intact ring structures at the TJ membrane for both claudin-1 and JAM-1 (Fig. 4, C4 and E4) with only a minor decrease in staining intensity for JAM-1 relative to control cells. In contrast, the characteristic ring structure of claudin-2 was disturbed at some tricellular corners in cells expressing Cdc42N17 and internalization of claudin-2 was observed (Fig. 4D4). Rac1N17-expressing cells displayed claudin-1 and -2 and JAM-1 reorganization in the TJ plane, manifested as decreased localization of the respective TJ protein at the TJ membrane as well as internalization of claudin-1 and -2 (Fig. 4, C3, D3, and E3).
|
Immediately subjacent to the TJ, the AJ proteins -catenin (Fig. 5A1) and E-cadherin (Fig. 5B1) were also visualized in a ring pattern in control cells by en face confocal imaging. Subtle diffusions of both
-catenin and E-cadherin away from the membrane were observed in cells expressing RhoAV14 (Fig. 5, A5 and B5) and Cdc42V12 (Fig. 5, A7 and B7). In contrast, all other induced GTPases evoked staining patterns similar to those in control cells. Analogous to TJ proteins, total cellular levels of AJ proteins were not changed by GTPases activation (data not shown).
|
|
Influence of GTPase inactivation/activation on affiliation of AJC proteins with membrane rafts.
TX-100 insolubility has also been described for proteins partitioning to membrane microdomains or DIGs (31, 46). Because we previously showed affiliation of TJ proteins with membrane rafts (31) and inactivation of Rho GTPases by Clostridium difficile toxins induced redistribution of occludin and ZO-1 from detergent-insoluble membrane microdomains (32), we analyzed the influence of inactivation/activation of GTPases on TJ/AJ protein affiliation with membrane rafts. Light scattering at 600 nm, alkaline phosphatase activity, and protein profiles were not significantly different in GTPase-inactivated and -activated cells, suggesting that the overall biophysical properties of membrane rafts were not altered (data not shown). In cells with an empty vector (pUHD) or in noninduced cells (data not shown), hyperphosphorylated occludin, ZO-1, and claudin-2 were predominantly localized in membrane raft-containing light fractions (Fig. 7). In contrast, a significant pool of claudin-1, -catenin, and E-cadherin localized in non-DIG fractions at the bottom of the gradient. Induction of RhoAV14 promoted an increase in the proportion of ZO-1 and claudin-2 in high-density fractions at the bottom of the gradients. In contrast, associations of other TJ or AJ proteins with DIG fractions were minimally affected by activation or inactivation of RhoA, Rac1, or Cdc42 (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using MDCK cell lines that express constitutively active or dominant-negative RhoA, Rac1, or Cdc42 under the control of the tetracycline-repressible transactivator (12, 21), we show that activation as well as inactivation of each GTPase enhances paracellular permeability. Because TJs are key structural components that regulate epithelial paracellular permeability, we analyzed mechanisms by which activation or inactivation may regulate such properties in epithelial cells. It was previously documented that the apical perijunctional F-actin ring plays a central role in regulating TJ function (26). F-actin filaments in this ring are themselves intimately associated with the actin filaments of the terminal web into which the F-actin-rich microvillous core rootlets are linked in a myosin-dependent manner (20, 42). In our study, enhanced paracellular permeability induced by both activation and inactivation of RhoA, Rac1, or Cdc42 was paralleled by profound reorganization of the F-actin cytoskeleton and/or TJ proteins. RhoAN19 expression evoked significant reductions in F-actin staining intensity at the level of both the perijunctional ring and the basal stress fibers without affecting TJ protein localization and distribution in membrane rafts. Our previous results (29) showed that inactivation of RhoA with C. botulinum transferase was associated with reorganization of F-actin in the perijunctional F-actin ring as well as redistribution of ZO-1 from the TJ membrane. Moreover, using C. difficile toxins A or B to inactivate RhoA, Rac1, and Cdc42, we showed that enhanced paracellular permeability was associated with reduction of hyperphosphorylated occludin and displacement of ZO-1 from membrane rafts (32). In contrast, disturbances in barrier function induced on inhibition of Rho kinase, a downstream effector of Rho GTPases, were characterized by reorganization of apical F-actin without alterations in TJ protein localization (44). Discrepancies in these results may be due to several factors. First, effects of different toxins, pharmacological agents, or GTPase mutants can be different. In this regard, it should be noted that the expression level of dominant-negative RhoA in our study was relatively low, inducing a leaky TJ gate function without any apparent effects on TJ structure/function. In contrast, inactivation of RhoA by the above-mentioned toxins induced a dramatic increase in paracellular permeability after short-term incubation that was more profound than the respective changes observed after inhibition of Rho kinase signaling (29, 44). Second, activated Rho can engage several different efforts in addition to Rho kinase, thereby accounting for this difference in effect on TJ protein organization. Third, the combined inactivation of RhoA, Rac1, and Cdc42 by C. difficile toxins is likely to affect TJ structure/function more dramatically than inactivation of RhoA alone. Therefore, it seems likely that only dramatic or toxic effects on barrier function induce displacement of TJ proteins from membrane rafts.
However, RhoA inactivation could be clinically relevant as a therapeutic approach for inflammatory disorders such as inflammatory bowel disease (IBD), in which TJ proteins are disrupted and proinflammatory cytokines such as tumor necrosis factor- and interferon-
are elevated (4, 23). It was shown recently that RhoA activation levels are increased in the inflamed mucosa of IBD patients (40). Our in vitro results as well as a recent report by Hopkins et al. (19) showing dramatic effects of CNF-1 on the localization of the TJ proteins occludin, ZO-1, and JAM-1 support the above hypothesis.
Furthermore, internalization of claudin-1 and -2 and changes in their biochemical properties induced by constitutively active GTPases could explain alterations in the formation of TJ strands, although TX-100 solubility for other TJ proteins such as occludin and ZO-1 remained unaltered in our model and in a previous report (22). Disruption of the occludin gene in embryonic stem cells has revealed functional TJ strand formation in the absence of occludin (37), whereas claudin-1 and -2 have been shown to be essential for TJ strand formation (8). In our model, JAM-1 localization was also disturbed but, unlike the other TJ proteins, did not virtually "disappear" from the plane of the TJ membrane in response to RhoA activation. Instead, some diffusion of JAM-1 away from the membrane was evident in en face confocal images. It is intriguing to speculate why the disruption of JAM-1 is not as drastic as that of the other TJ proteins. This might relate to a necessity for JAM-1 localization at the TJ membrane for correct assembly of the TJ protein complex (25). Loss of this protein could prove detrimental for the reestablishment of barrier function after transient insult by RhoA activation. Another feature in RhoAV14-expressing cells was loss of polarity, in which cells lost their parallel orientations relative to each other and occasionally formed multilayers. This may suggest that dominant-active RhoA expression interferes not only with cell-cell but also with cell-matrix adhesion.
Another interesting observation from our study was the delay in formation of a confluent monolayer in cells expressing dominant-negative Rac1. It was shown previously that Rac1 inhibition in fibroblasts completely prevents cell movement (17). However, because cells in our epithelial model did eventually achieve confluence, it seems that Rac1 is not the only participant in the complex sequence of events leading to coordinated spreading/migration of epithelial sheets. The importance of Rac1 in epithelial sheet movement is demonstrated by the fact that sustained Rac1 activation has been implicated in keratinocyte migration induced by epidermal growth factor (35). However, we cannot exclude the possibility that Rac1 inhibition also interferes with cell-cell (as well as cell-matrix) attachment in our model. Our results do conflict with a previous report by Jou et al. (22) in the same inducible GTPase system, where Rac1N17-expressing cells formed a monolayer of uniformly shaped cells 18 h after induction of cell-cell contacts. This discrepancy may arise from differences in cell seeding density, because the same group showed that Rac1N17-expressing cells grown at low density rounded up and spread less on the substratum, a feature that we also observed in our immunofluorescence staining for F-actin. Moreover, Rac1N17 expression resulted in substantial reductions in the microvillous F-actin pool and some submembranous diffusion away from the perijunctional ring. These changes were associated with alterations in claudin-1, claudin-2, and JAM-1 localization, whereas occludin and ZO-1 remained unaltered in cells expressing Rac1N17 as described previously (22).
In conclusion, we have shown that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active vs. resting GTPase levels rather than simply loss or gain of GTPase activity.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bourne HR. GTPases. A turn-on and a surprise. Nature 366: 628629, 1993.[CrossRef][ISI][Medline]
3. Braga VM, Machesky LM, Hall A, and Hotchin NA. The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J Cell Biol 137: 14211431, 1997.
4. Bruewer M, Luegering A, Kucharzik T, Parkos CA, Madara JL, Hopkins AM, and Nusrat A. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunol 171: 61646172, 2003.
5. Dejana E and Del Maschio A. Molecular organization and functional regulation of cell to cell junctions in the endothelium. Thromb Haemost 74: 309312, 1995.[ISI][Medline]
6. Fanning AS, Jameson BJ, Jesaitis LA, and Anderson JM. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273: 2974529753, 1998.
7. Flatau G, Lemichez E, Gauthier M, Chardin P, Paris S, Fiorentini C, and Boquet P. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387: 729733, 1997.[CrossRef][ISI][Medline]
8. Furuse M, Fujita K, Hiiragi T, Fujimoto K, and Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 15391550, 1998.
9. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, and Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123: 17771788, 1993.[Abstract]
10. Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, and Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127: 16171626, 1994.[Abstract]
11. Gopalakrishnan S, Raman N, Atkinson SJ, and Marrs JA. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol Cell Physiol 275: C798C809, 1998.[Abstract]
12. Gossen M and Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 55475551, 1992.[Abstract]
13. Gumbiner B. Generation and maintenance of epithelial cell polarity. Curr Opin Cell Biol 2: 881887, 1990.[Medline]
14. Gumbiner BM. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84: 345357, 1996.[ISI][Medline]
15. Hall A. Rho GTPases and the actin cytoskeleton. Science 279: 509514, 1998.
16. Hall A. G proteins and small GTPases: distant relatives keep in touch. Science 280: 20742075, 1998.
17. Hall A and Nobes CD. Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos Trans R Soc Lond B Biol Sci 355: 965970, 2000.[CrossRef][ISI][Medline]
18. Hopkins AM, Li D, Mrsny RJ, Walsh SV, and Nusrat A. Modulation of tight junction function by G protein-coupled events. Adv Drug Delivery Res 41: 329340, 2000.[CrossRef][ISI][Medline]
19. Hopkins AM, Walsh SV, Verkade P, Boquet P, and Nusrat A. Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function. J Cell Sci 116: 725742, 2003.
20. Hull BE and Staehelin LA. The terminal web. A reevaluation of its structure and function. J Cell Biol 81: 6782, 1979.[Abstract]
21. Jou TS and Nelson WJ. Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity. J Cell Biol 142: 85100, 1998.
22. Jou TS, Schneeberger EE, and Nelson WJ. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol 142: 101115, 1998.
23. Kucharzik T, Walsh SV, Chen J, Parkos CA, and Nusrat A. Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins. Am J Pathol 159: 20012009, 2001.
24. Lerm M, Schmidt G, Goehring UM, Schirmer J, and Aktories K. Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1). J Biol Chem 274: 2899929004, 1999.
25. Liu Y, Nusrat A, Schnell FJ, Reaves TA, Walsh S, Pochet M, and Parkos CA. Human junction adhesion molecule regulates tight junction resealing in epithelia. J Cell Sci 113: 23632374, 2000.
26. Madara JL. Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am J Physiol Cell Physiol 253: C171C175, 1987.
27. Mullin JM, Kampherstein JA, Laughlin KV, Clarkin CE, Miller RD, Szallasi Z, Kachar B, Soler AP, and Rosson D. Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia. Am J Physiol Cell Physiol 275: C544C554, 1998.
28. Nobes CD and Hall A. Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility. Biochem Soc Trans 23: 456459, 1995.[ISI][Medline]
29. Nusrat A, Giry M, Turner JR, Colgan SP, Parkos CA, Carnes D, Lemichez E, Boquet P, and Madara JL. Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc Natl Acad Sci USA 92: 1062910633, 1995.[Abstract]
30. Nusrat A, Parkos CA, Bacarra AE, Godowski PJ, Delp-Archer C, Rosen EM, and Madara JL. Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest 93: 20562065, 1994.[ISI][Medline]
31. Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, and Madara JL. Tight junctions are membrane microdomains. J Cell Sci 113: 17711781, 2000.
32. Nusrat A, von Eichel-Streiber C, Turner JR, Verkade P, Madara JL, and Parkos CA Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun 69: 13291336, 2001.
33. Ridley AJ and Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389399, 1992.[ISI][Medline]
34. Rojas R, Ruiz WG, Leung SM, Jou TS, and Apodaca G. Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell 12: 22572274, 2001.
35. Russell AJ, Fincher EF, Millman L, Smith R, Vela V, Waterman EA, Dey CN, Guide S, Weaver VM, and Marinkovich MP. 6
4 Integrin regulates keratinocyte chemotaxis through differential GTPase activation and antagonism of
3
1 integrin. J Cell Sci 116: 35433556, 2003.
36. Saha C, Nigam SK, and Denker BM. Involvement of Gi2 in the maintenance and biogenesis of epithelial cell tight junctions. J Biol Chem 273: 2162921633, 1998.
37. Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T, and Tsukita S. Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol 141: 397408, 1998.
38. Sanders SE, Madara JL, McGuirk DK, Gelman DS, and Colgan SP. Assessment of inflammatory events in epithelial permeability: a rapid screening method using fluorescein dextrans. Epithelial Cell Biol 4: 2534, 1995.[ISI][Medline]
39. Schmidt G, Sehr P, Wilm M, Selzer J, Mann M, and Aktories K. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387: 725729, 1997.[CrossRef][ISI][Medline]
40. Segain JP, Raingeard de la Bletiere D, Sauzeau V, Bourreille A, Hilaret G, Cario-Toumaniantz C, Pacaud P, Galmiche JP, and Loirand G. Rho kinase blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in Crohn's disease and experimental colitis. Gastroenterology 124: 11801187, 2003.[CrossRef][ISI][Medline]
41. Takaishi K, Sasaki T, Kotani H, Nishioka H, and Takai Y. Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells. J Cell Biol 139: 10471059, 1997.
42. Temm-Grove C, Helbing D, Wiegand C, Honer B, and Jockusch BM. The upright position of brush border-type microvilli depends on myosin filaments. J Cell Sci 101: 599610, 1992.[Abstract]
43. Troyanovsky SM. Mechanism of cell-cell adhesion complex assembly. Curr Opin Cell Biol 11: 561566, 1999.[CrossRef][ISI][Medline]
44. Walsh SV, Hopkins AM, Chen J, Narumiya S, Parkos CA, and Nusrat A. Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia. Gastroenterology 121: 566579, 2001.[ISI][Medline]
45. Weber E, Berta G, Tousson A, St John P, Green MW, Gopalokrishnan U, Jilling T, Sorscher EJ, Elton TS, Abrahamson DR, and Kirk KL. Expression and polarized targeting of a rab3 isoform in epithelial cells. J Cell Biol 125: 583594, 1994.[Abstract]
46. Wong V. Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am J Physiol Cell Physiol 273: C1859C1867, 1997.