MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly

Abstract

The regulation of Rho-family GTPases is crucial to direct the formation of cell-cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin-Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.

FIGURE 1:

CGN(−)/CGNL1(−) (double-KD) cells show normal Rac1 activation and TJ assembly, despite reduced junctional Tiam1. (A, B) Rac1 activation at steady-state (A) and Rac1 and RhoA activation during the calcium switch (B), as determined by GST pull-down analysis. (C) TER (ohm⋅cm²) of WT, single-KD (CGNL1(−)), and double-KD (CGN(−)/CGNL1(−)) cells during the calcium switch. (D) Occludin immunofluorescence, showing a similar pattern of occludin accumulation at junctions in WT, control, and double-KD cells during the calcium switch (for single-KD CGNL1(−), see Guillemot et al., 2008). (E) Immunofluorescence analysis of exogenous hemagglutinin (HA)-tagged Tiam1 in WT and double-KD cells, showing reduced junctional recruitment of Tiam1 in double-KD cells (exposure time in double-KD cells increased fourfold; see key in each panel). Bar, 10 μm. (F) Immunoblotting with anti-HA (exogenous Tiam1) and anti-actin antibodies of fractionated lysates of WT and double-KD cells (Guillemot et al., 2008). See Supplemental Figure S1 for additional data on single-KD cells.

FIGURE 2:

MgcRacGAP levels are reduced in double-KD MDCK cells, and rescue of MgcRacGAP expression inhibits Rac1 activation and the peak in TER during development of the TJ barrier. (A) Histogram showing the relative mRNA levels for Asef, Vav2, Rich-1, and MgcRacGAP in double-KD cells vs. WT cells, taking WT levels as 100, as determined by quantitative RT-PCR. (B) Immunoblotting analysis of either total lysates (RIPA) or fractionated lysates of WT and double-KD cells. Low, pellet after centrifugation at low speed (13,000 × g). High, pellet after high-speed (100,000 × g) centrifugation of the supernatant obtained after low-speed centrifugation. Soluble, Triton-soluble supernatant after centrifugation at 100,000 × g of the low-speed supernatant. (C) Immunoblotting of total (RIPA) lysates from three independent double-KD rescue clones (a–c) stably expressing or not (−) an exogenous human (h) FLAG-tagged MgcRacGAP. (D) Rac1 activation in either single-KD (CGNL1(−)) or double-KD cells expressing (clone a) or not the exogenous MgcRacGAP protein during the calcium switch. Clones b and c are shown in Supplemental Figure S1E. (E) TER profile in the calcium switch for the stable clones described in D. Clones b and c are shown in Supplemental Figure S1F.

FIGURE 3:

CGN and CGNL1 are required for the efficient recruitment of MgcRacGAP to epithelial junctions. (A, B) Double immunofluorescence of CGN and MgcRacGAP (Mgc) in WT MDCK cells in cocultures of WT and CGN-KD MDCK cells, WT and double-KD MDCK cells (A), or mouse kidney (mpkCCD_Cl4) cells, after siRNA control, si-CGN, si-CGNL1, and si-double (CGN and CGNL1) treatment. Cells were labeled also with rat anti–ZO-1 to identify junctions. Arrows, junctions labeled by both MgcRacGAP and CGN antibodies. Double arrowheads, junctions with decreased labeling for both CGN and MgcRacGAP and normal labeling for ZO-1. The square area in A and magnified inset shows labeling for MgcRacGAP (arrowheads) in the mitotic spindle. Asterisks, positions of nuclei of KD cells. Single arrowheads, junctions with reduced CGNL1 staining and normal MgcRacGAP staining. n, nuclear labeling for MgcRacGAP. (C) Semiquantitative analysis of junctional labeling intensity for MgcRacGAP (expressed as a ratio of MgcRacGAP to ZO-1 pixel intensity in the same junctional areas) in WT, CGN-KD, dKD junctions of MDCK clonal lines or WT, CGNL1-KD, dKD si-treated mouse kidney cells. (D) Double immunofluorescence of CGN and MgcRacGAP in cocultures of primary keratinocytes derived from either WT or CGN KO mice. Magnified insets in D′ and D′′ show intensity-adjusted images of junctions, to show the lower levels (but not absence) of MgcRacGAP in junctional areas between KO cells (D′) vs. junctions between WT cells (D′′). Bar, 5 μm.

FIGURE 4:

CGN and CGNL1 form a complex and interact directly with McRacGAP. (A) Immunoblotting of CGN (top, from MDCK cell lysates) or CGNL1 (bottom, from Eph4 cell lysates) immunoprecipitates, using antibodies against CGN/MgcRacGAP or CGNL1/MgcRacGAP, respectively. Input: 1/40 of volume used for immunoprecipitation. MgcRacGAP shows different mobility and forms in MDCK vs. Eph4 cells. (B) Domain organization of MgcRacGAP and immunoblotting analysis of CGN in GST pull downs of full-length CGN interacting with either full-length (FL) or truncated constructs (1–110, 111–632) of MgcRacGAP fused to GST. (C, D) Domain organization of CGN (C) and CGNL1 (D) and immunoblotting analysis of myc-tagged MgcRacGAP in GST pull downs of the indicated fragments of either CGN or CGNL1 fused to GST. Images of Ponceau red–labeled membranes below immunoblots show protein loadings for GST fusion proteins.

FIGURE 5:

Depletion of CGN and CGNL1 does not affect the junctional recruitment of the Rho GEF ECT2. (A) Double immunofluorescence analysis (CGN, red; ECT2, green; 4′,6-diamidino-2-phenylindole [DAPI], blue; in merge images) of WT keratinocytes (top) or mixed cultures of WT and CGN-KO keratinocytes (bottom). Additional images are shown in Supplemental Figure S4A. Note the presence of ECT2 (arrow) in cells lacking CGN (arrowhead). (B, C) Double immunofluorescence analysis (CGN, red; ECT2, green; DAPI, blue; in merge images) of Eph4 cells treated with siRNA against CGNL1 or SKCO-15 cells treated with siRNA against both CGN and CGNL1 (C), showing both WT cells, containing junctional CGNL1/CGN (arrow) and CGNL1(CGN)-depleted cells (arrowhead). Note the presence of ECT2 (arrow) in cells lacking CGNL1/CGN. (D) Double immunofluorescence analysis (CGN, red; MKLP1, green) of confluent SKCO-15 cells, showing that MKLP1 is not detected at junctions (arrow), but only at the cleavage furrow (CF) of dividing cells. Asterisks, positions of nuclei of siRNA-CGN–depleted/KO cells. Single arrowheads, sites with reduced CGN/CGNL1 labeling (KD or KO) but detectable ECT2 labeling. Arrows, junctions between WT cells showing both CGN/CGNL1 and ECT2 labeling. Bar, 5 μm (B–D), 2 μm (A).

FIGURE 6:

The control of Rac1 signaling at the apical junctional complex. Simplified scheme showing the AJC and the putative molecular interactions of GEFs and GAPs with CGN, CGNL1, and other junctional proteins. Green lines indicate activation; red lines indicate inhibition. A key for the graphical objects is shown at the bottom.