In contrast, depletion of CGNL1 alone resulted in decreased junctional labeling for MgcRacGAP only in Eph4 (Supplemental Figure S2C) but not in SKCO-15 cells (single arrowhead in Supplemental Figure S2B, CGNL1-si) or human breast cancer (MCF-7) cells (unpublished data)
In contrast, depletion of CGNL1 alone resulted in decreased junctional labeling for MgcRacGAP only in Eph4 (Supplemental Figure S2C) but not in SKCO-15 cells (single arrowhead in Supplemental Figure S2B, CGNL1-si) or human breast cancer (MCF-7) cells (unpublished data). Taken together, these observations demonstrate that CGN, in all the epithelial cell types we tested, and CGNL1, in all the cell types we tested with the exception of SKCO-15 and MCF-7 cells, are required for the recruitment of MgcRacGAP to the AJC. Cingulin and paracingulin interact with MgcRacGAP To explore whether the requirement of CGN and CGNL1 for the junctional recruitment of MgcRacGAP depends on an conversation of MgcRacGAP with these proteins, we carried out immunoprecipitation and GST pull-down experiments. 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 Calcineurin Autoinhibitory Peptide 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 Calcineurin Autoinhibitory Peptide and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP. INTRODUCTION The precise spatiotemporal control of the activity of Rho-family GTPases is essential in many cellular processes, including the establishment and maintenance of cellCcell junctions and the formation of epithelial barriers (Nusrat of the low-speed Calcineurin Autoinhibitory Peptide supernatant. (C) Immunoblotting of total (RIPA) lysates from three impartial double-KD rescue clones (aCc) 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 Physique S1E. (E) TER profile in the calcium switch for the stable clones described in D. Clones b and c are shown in Supplemental Physique S1F. Next we tested the hypothesis that this decreased expression of MgcRacGAP plays a mechanistic role in the increased Rac1 activation and normal development of the epithelial paracellular permeability barrier of double-KD cells. We established stable lines expressing exogenous FLAG-tagged MgcRacGAP in the background of double-KD cells (Physique 2C) and asked whether the exogenous MgcRacGAP expression could revert the phenotype of double-KD cells to that of CGNL1(?), single-KD cells. GST pull-down analysis of activated Rac1 showed that when exogenous MgcRacGAP was expressed, the increased Rac1 activation detected at different time points during the calcium switch in double-KD cells was suppressed, thus reverting the phenotype to that of single-KD, CGNL1(?) cells (Physique 2D and Supplemental Physique S1E). Furthermore, Rac1 inactivation induced by exogenous MgcRacGAP expression correlated with a strong reduction in the peak of TER detected at 8 h after the calcium switch in double-KD cells, resulting in a phenotype that was comparable to that of single-KD, CGNL1(?) cells (Physique 2E and Supplemental Physique S1F). We also attempted to generate stable lines depleted of MgcRacGAP through shRNA expression in order to test directly the role of MgcRacGAP in junction assembly in WT cells. However, such lines could not be isolated, probably due to the essential role of MgcRacGAP in cytokinesis (Glotzer, 2009 ). Cingulin and paracingulin are required for efficient junctional recruitment of MgcRacGAP in different types of epithelial cells Having established that modulating MgcRacGAP expression levels affects Rac1 activation and the dynamics of establishment of the TJ barrier to ions, we asked whether MgcRacGAP is usually localized at TJs through conversation with CGN, CGNL1, or both. To do this, we examined the localization of MgcRacGAP in epithelial cells in which CGN, CGNL1, or Calcineurin Autoinhibitory Peptide both were depleted through either shRNA or small interfering RNA (siRNA). In addition, we studied Calcineurin Autoinhibitory Peptide the localization of MgcRacGAP in mixed cultures of primary keratinocytes isolated from WT and CGN-KO mice (Physique 3). Open in a separate window Physique 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 (mpkCCDCl4) cells, after siRNA control, si-CGN, si-CGNL1, and si-double (CGN and CGNL1) treatment. Cells were labeled also with rat antiCZO-1 to identify junctions. Arrows, junctions labeled by both MgcRacGAP and CGN antibodies. Double arrowheads, junctions with decreased labeling BNIP3 for both CGN and MgcRacGAP and normal labeling for ZO-1. The square area in A and.