Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases - PubMed (original) (raw)

Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases

T S Jou et al. J Cell Biol. 1998.

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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Figures

Figure 1

Development of TER in MDCK cells expressing constitutively active or dominant negative RhoA or Rac1 mutants. Control MDCK cells (T23), mock-transfected MDCK cells (pUHD10-3), and cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 under control of the tetracycline repressible transactivator were grown in different concentrations of DC for 16 h at low cell density. Cells were trypsinized, plated on Transwell™ filters as an instant confluent monolayer in LCM containing different amounts of DC for 4 h, and then cell–cell contacts were synchronously induced by switching to HCM containing different amounts of DC. TER (ohms per cm2) was measured at different times after induction of cell–cell contacts (see Materials and Methods). TER was measured in four filters for each cell line and each DC concentration, and the data are reported as the mean ± SEM. At the end of the experiment, filters were fixed and stained with Hoechst 33342 (Molecular Probes, Inc.) to check the integrity of each monolayer.

Figure 3

Freeze-fracture EM of tight junction strand organization in MDCK cells expressing RhoA mutants. (A and B) Representative P fracture face of a TJ from MDCK cells expressing RhoAV14 +DC (A) or −DC (B). Fracture planes are oriented with the apical membrane at the top (from left to right), and lateral membranes are oriented downwards (bracket). Note the chaotically distributed TJ strands that extend basally from a disorganized TJ (arrowheads) in RhoAV14 -DC cells (B). (C and D) Representative P fracture face of a TJ from MDCK cells expressing RhoAN19 +DC (C) or −DC (D). The strand organization of the TJs in RhoAN19 +DC or −DC are similar. Bars, 200 nm.

Figure 2

RhoA and Rac1 mutants disrupt TJ barrier to paracellular diffusion of [3H]inulin, FITC-conjugated 3K dextran, and Texas Red–conjugated 40K dextran. Control MDCK cells (T23), mock-transfected MDCK cells (pUHD), and cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 under the control of the tetracycline repressible transactivator, were cultured with 20 ng/ml DC or without DC at low cell density. Cells were trypsinized and plated on Transwell™ filters at an instant confluent monolayer density in LCM (± DC) for 4 h, and were then switched to normal DMEM/FBS (± DC). 16 h later, paracellular tracer diffusion was measured in four parallel cultures for each cell line ± DC as described in Materials and Methods; the data are shown as the mean ± SEM.

Figure 4

Freeze-fracture EM of tight junction strand organization in MDCK cells expressing Rac1 mutants. (A, B, and _B_′) Representative P fracture face of a TJ from Rac1V12 +DC cells (A) or E and P fracture face of Rac1V12-DC cells (B and _B_′, respectively). Fracture planes are oriented with the apical membrane at the top (from left to right) and lateral membranes oriented downwards (bracket). Note that strands extend from the tight junction network down the lateral membrane to the base of the cells in Rac1V12 -DC (B and _B_′). (C and D) Representative P fracture face of a TJ from Rac1N17 +DC cells (C) and E fracture face of a TJ from Rac1N17 −DC cells (D). There is a relative paucity of cross-bridges in the TJ network, and a few TJ strands extend basally on the lateral membrane surface in Rac1N17 −DC. Bars, 200 nm.

Figure 5

Quantitative analysis of TJ strand counts from freeze-fracture replicas of cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 (−DC) and their matched controls (+DC); see Materials and Methods for details.

Figure 6

Indirect immunofluorescence microscopy of occludin and ZO-1 in MDCK cells expressing RhoA and Rac1 mutants. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were grown in the presence (control, + 20 ng/ml DC) or absence of DC (−DC) for 36 h at a confluent cell density on collagen-coated glass coverslips. Cells were processed for immunofluorescence with either occludin or ZO-1 antibodies, as described in detail in Materials and Methods. Stained cells were viewed with a Axioplan microscope (Carl Zeiss, Inc.) equipped for epifluorescence. Bar, 10 μm.

Figure 7

Confocal microscopy analysis of occludin and ZO-1 distribution in MDCK cells expressing RhoA and Rac1 mutants. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were grown in the presence (control, + 20ng/ml DC) or absence of DC (−DC) for 36 h at a confluent cell density on collagen I–coated glass coverslips. Cells were fixed, permeabilized, and double-stained with rabbit antioccludin antibody and rat anti-ZO-1 R40.76 hybridoma supernatant, followed by rhodamine-conjugated goat anti–rabbit and FITC-conjugated goat anti–rat secondary antibodies. The images were collected with a laser scanning confocal microscope MultiProbe 2010™ (Molecular Dynamics, Inc.). Bar, 10 μm.

Figure 8

DC switch experiment to examine the reversibility of defects in TJ function caused by RhoA and Rac1 mutants. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were divided into two populations. One population of each cell line (+DC → −DC group) was grown initially in DMEM/FBS + 20 ng/ml DC, and then switched to medium without DC at the indicated time (arrowhead). The other population (−DC → +DC group) was grown at first without DC and then switched to medium containing 20 ng/ml DC (arrowhead). 2.5 mM sodium butyrate was added between 56 and 96 h to enhance RhoA and Rac1 mutant expression. TER was measured in quadruplicate cultures, and is reported as mean ± SEM.

Figure 9

Indirect immunofluorescence of myc-tagged RhoA or Rac1 mutants, occludin, and ZO-1 distributions at the end of the DC switch experiment shown in Fig. 8. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were divided into two populations. One population of each cell line was grown initially in DMEM/FBS + 20 ng/ml DC, and then switched to medium without DC 32 h later (+DC → −DC group). The other population (−DC → +DC group) was grown at first without DC and then switched to medium containing 20 ng/ml DC. 2.5 mM sodium butyrate was added between 56 and 96 h to enhance RhoA and Rac1 mutant expression. Immunofluorescence was performed at the end of the DC switch experiment by staining the filters with mouse anti-myc antibody (to identify the myc-tagged RhoA or Rac1 mutant), rabbit anti-occludin antibody, and rat anti-ZO-1 (R40.76 hybridoma) antibody. Cells were examined in a Axioplan™ microscope (Carl Zeiss, Inc.) equipped with epifluorescence. Bar, 10 μm.

Figure 10

Diffusion of fluorescent lipid from the apical to lateral membranes in cells expressing RhoA and Rac1 mutants. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were cultured on Transwell™ filters in the presence (+DC) or absence (−DC) of DC. The apical surface was incubated in BODIPY-sphingomyelin/BSA complex solution on ice for 10 min (see Materials and Methods). The cells were washed three times in P buffer, and were then either immediately processed for microscopy (0 min) or incubated for an additional 60 min (60 min) on ice before processing. The distribution of fluorescent lipid was determined with a Molecular Dynamics confocal microscope, and representative xz projections are shown. Bar, 10 μm.

Figure 11

Distribution of the apical membrane protein, p75NTR, in MDCK cells expressing RhoA and Rac1 mutants. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were grown in the presence (+DC) or absence (−DC) of 20 ng/ml DC at low cell density for 20 h, trypsinized, and plated at confluent cell density on Transwell™ filters. Cells were incubated for a further 48 h in the presence or absence of 20 ng/ml DC. p75NTR-expressing adenovirus was applied to the monolayer. 24 h after adenovirus infection, filters were fixed, permeabilized, and double-stained with mouse anti-p75 and rabbit anti-myc antibody followed by FITC-conjugated goat anti–mouse and rhodamine-conjugated goat anti–rabbit secondary antibodies. Cells were examined with a Molecular Dynamics confocal microscope, and representative stacked xy and xz projections of p75NTR staining are shown. Bar, 10 μm.

Figure 12

Western blot analysis of occludin, ZO-1, and ZO-2 levels in MDCK cells expressing RhoA and Rac1 mutants. 30 μg of total protein lysates from MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 grown in the presence (+DC) or absence (−DC) of 20 ng/ml DC was separated by SDS-PAGE, proteins were transferred electrophoretically to nitrocellulose filters and probed with antibodies specific for ZO-1, ZO-2, and occludin. For details, see Materials and Methods.

Figure 13

Triton X-100 solubility of occludin, ZO-1, and ZO-2 in MDCK cells expressing RhoA or Rac1 mutants. MDCK cells expressing RhoAV14, RhoAN19, Rac1V12, or Rac1N17 were grown in the presence (+DC) or absence (-DC) of 20 ng/ml DC on Transwell™ filters. Cells were extracted in buffer containing 1% Triton X-100 on ice for 20 min, and equal portions of the pellet (P) and supernatant (S) were separated by high-speed centrifugation. Proteins in equal amounts of pellet and supernatant samples were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose filters, and probed with antibodies specific for ZO-1, ZO-2, and occludin. For details, see Materials and Methods.

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Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases - PubMed (original) (raw)