1. Binding of GEF-H1 to the Tight Junction-Associated Adaptor Cingulin Results in Inhibition of Rho Signaling and G1/S Phase Transition 
Saima Aijaz, Fabio D’Atri, Sandra Citi, Maria S. Balda, Karl Matter 
Developmental Cell 
Volume 8, Issue 5, Pages (May 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 GEF-H1 Directly Interacts with Cingulin
(A) Association of endogenous GEF-H1 and cingulin. MDCK cell extracts were immunoprecipitated with the anti-GEF-H1 mAb B4/7 (IP B4/7), a control antibody (mAb), or empty beads. The precipitates and total cell extract were analyzed by immunoblotting using the anti-GEF-H1 antibody B4/7 or an anti-cingulin polyclonal antibody. The positions of GEF-H1, cingulin, and the antibody heavy chain (γ) are indicated.
(B) Domain map of GEF-H1. The C1, the Dbl homology (DH), the pleckstrin homology (PH), and the C-terminal (CTD) domain are indicated.
(C) Mapping of the interacting domain in GEF-H1. MDCK cell extracts were loaded on beads conjugated with either GST or GST fusion proteins containing different domains of GEF-H1. The pull-downs were analyzed by immunoblotting for cingulin using an antibody generated against the C-terminal half of cingulin. The asterisk marks the position of a cingulin degradation product.
(D) Domain map of cingulin. Indicated are the head, the central rod, and the C-terminal tail domain along with the number of the amino acid residues forming the domain borders.
(E and F) Identification of the GEF-H1 binding site in cingulin. Pull-down assays with cingulin/GST fusion proteins of recombinant His6-PH domain of GEF-H1 were analyzed by immunoblotting with an anti-His6 or anti-GST antibodies. (F) Increasing concentrations of His6-PH domain were added to test saturation of binding.
(G) Colocalization of GEF-H1 and cingulin at the junctional complex. Semiconfluent MDCK cells were stained with a mAb against GEF-H1 (FITC) and a rabbit polyclonal antibody specific for cingulin (Cy3). The sample was analyzed by optical sectioning with a confocal microscope. Shown is an xy-section taken at the level of tight junctions and an xz-section to show the distribution along the z-axis. The colored panels represent the corresponding overlays.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 Redistribution of GEF-H1 by Transfection of Cingulin
(A and B) Wild-type MDCK cells or a cell line overexpressing VSV-tagged GEF-H1 (C) were transfected with myc-cingulin or truncated mutants containing the indicated domains. The cells were processed for double immunofluorescence with a rabbit anti-myc antibody to detect transfected cingulin and either monoclonal antibodies against GEF-H1 (A), ZO-1 (B), or the VSV-epitope to stain transfected GEF-H1 (C). In (C), myc-cingulinR+T transfection, a GEF-H1-VSV-expressing (arrow) and a nonexpressing (asterisk) cell are shown. (D) VSV-tagged PH domain of GEF-H1 was transiently expressed in MDCK cells, and the cells were stained with anti-VSV and anti-cingulin. Shown are confocal XY, upper two panels, and XZ sections, lower two panels. (E) cDNAs coding for VSV-tagged PH domain with a W563 to A substitution or a GEF-H1 mutant with an internal deletion of the PH domain were transiently transfected and localized by immunofluorescence.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3 The C1 Domain of GEF-H1 Modulates Junctional Targeting
(A) MDCK cell lines expressing GEF-H1-VSV or truncated proteins lacking either the C- (GEF-ΔCTD-VSV) or the N-terminal domain (GEF-ΔNTD-VSV) under the control of a tetracycline-regulated promoter were induced for 24 hr. The cells were processed for double immunofluorescence using anti-VSV and anti-cingulin antibodies.
(B) MDCK cells were transiently transfected with cDNAs coding for either GEF-H1-VSV or a construct containing the N-terminal domain of GEF-H1, which contains the C1 domain as well as the sequences between the C1 and DH domains (C1/ID-VSV). After 24 hr, the cells were stained with antibodies against the VSV-epitope and α-tubulin.
(C) In vitro binding of the C1 to the PH domain. GST with or without the C1 domain were bound to glutathione beads and used to precipitate recombinant His6-PH domain of GEF-H1. The pull-downs were analyzed by immunoblotting with an anti-His6 monoclonal antibody.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4 Inhibition of GEF-H1 and Rho Activation by Cingulin
(A) MDCK cells grown to 20%, 50%, or 100% confluence were lyzed and equal amounts of protein were analyzed by immunoblotting for the expression of GEF-H1, cingulin, and, as a loading control, α-tubulin. Note the pronounced upregulation of cingulin with cell confluence.
(B) MDCK cells were transiently transfected with a truncated cingulin construct lacking the head domain (myc-cingulinR+T). Cells were stained with antibodies against myc, GEF-H1, and fluorescent phalloidin. Note the reduced appearance of stress fibers in transfected cells.
(C) Inhibition of SRE-driven transcription by cingulin. ARPE-19 cells were cotransfected with a plasmid containing a SRE driving firefly luciferase expression, one with a control promoter regulating renilla luciferase expression, and the indicated expression vectors. After 30 hr, the luciferases were assayed and the ratios of the values for firefly divided by those from renilla luciferase calculated. Shown are the means ± 1 SD of a typical experiment performed in triplicates. Both panels were normalized to plasmid controls without GEF-H1 cotransfection. Asterisks indicate p values smaller than 0.05 that were calculated with two-tailed t tests comparing the single transfections to plasmid controls and the double transfections to GEF-H1 transfections.
(D) Inhibitin of RhoA activation by cingulin. MDCK cells were transfected with pRaichu-RBD, a Rho-specific FRET probe, and the indicated expression vectors. TAT-C3 labels cells that were incubated with TAT-modified C3 between the transfection and cell lysis. After cell lysis, the emission for YFP (530 nm) and CFP (475 nm) was measured with an excitation wavelength of 430 nm, and the ratios were calculated. Shown are the means ± 1 SD of a typical experiment (n = 4). Asterisks label p values smaller than 0.05 that refer to comparisons of single transfections with plasmid controls and double transfections with GEF-H1-transfected samples. Note, increased YFP emission indicates Rho inactivation.
(E) Downregulation of GEF-H1 in MDCK cells by tetracycline-regulated RNA interference. Confluent MDCK cells expressing control RNA duplexes or GEF-H1-directed RNA duplexes were treated with tetracycline for 3 days. Expression of GEF-H1 and α-tubulin was then analyzed by immunoblotting with monoclonal antibodies B4/7 and 1A2, respectively.
(F) The Rho-specific FRET probe pRaichu-RBD was transfected into tetracycline treated control or GEF-H1 RNAi cells together with empty expression vector or full-length or truncated cingulin. The extracts were then analyzed as in (D) (asterisks, p < 0.05).
(G). FRET probes specific for RhoA, Rac1, or Cdc42 were transfected into MDCK cells together with the indicated cingulin constructs (asterisks, p < 0.05). Note, decreased YFP emission indicates inactivation.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5 GEF-H1 Regulates G1/S Phase Transition
(A) Wild-type, GEF-H1 RNAi, and control RNAi cells were plated on coverslips synchronized by incubation in 0.1% serum in the presence or absence of tetracycline for 2 days. Serum was then added back together with bromodeoxyuridine to trigger cell cycle progression and label cells in S phase. After 6 hr, the cells were fixed and stained with antibodies against bromodeoxyuridine and Hoechst The percentages of cells were then determined by counting all cells and bromodeoxyuridine-positive cells in at least 12 fields per experiment and sample. Shown are means ± 1 SD of three experiments (asterisks, p < 0.05). White bars, bromodeoxyuridine positive; gray bars, bromodeoxyuridine negative.
(B) MDCK cells were transiently transfected with the indicated cingulin constructs and then plated on coverslips, synchronized, and labeled with bromodeoxyuridine as in (A). Shown are means ± 1 SD of three experiments (asterisks, p < 0.05).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
Regulated Depletion of Cingulin Affects Cell Morphology and GEF-H1 Localization
(A) Stable MDCK cells expressing control or cingulin-directed RNA duplexes under the control of a tetracycline-inducible promoter were induced for 40 hr. Expression of cingulin, α-tubulin, and GEF-H1 was then analyzed by immunoblotting.
(B) Depletion of cingulin and cell morphology. Control- and Cingulin-RNAi cells were cultured at 40% confluence with or without tetracycline. After 2 days, the cell morphology was analyzed by phase contrast microscopy.
(C and D) Cingulin-RNAi cells were cultured without or with tetracycline for 40 hr before processing for immunofluorescence with anti-GEF-H1 and anti-cingulin antibodies, or antibodies specific for ZO-1, occludin, or β-catenin. Note, cingulin depletion results in increased cytoplasmic staining of GEF-H1 but not ZO-1, occludin, and β-catenin.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7 Depletion of Cingulin and RhoA Activation
(A) MDCK cells were transfected with the Rho-specific FRET probe and the indicated vectors. After 30 hr, emission for YFP and CFP was measured (excitation, 430 nm) and the ratios were calculated. Shown are the means ± 1 SD of a typical experiment (n = 6).
(B) Wild-type, two different clones of cingulin RNAi cells (CinRiTc1/c2), as well as control RNAi cells (ConRiT) were cultured without or with tetracycline for 40 hr. Cell extracts were then incubated with GST with or without the Rho binding domain of rhotekin. Samples of total cell extracts and pellets were analyzed by immunoblotting using anti-RhoA antibodies.
(C) GEF-H1-VSV expressing cells, control RNAi cells (ConRiT), and two different clones of cingulin RNAi cells (CinRiTc1/c2) were cultured without or with tetracycline for 40 hr, lysed, and analyzed by immunoblotting with antibodies against phosphorylated (p-MYP) or total myosin light chain phosphatase (MYP). The graph shows quantification of the immunoblots by densitometric scanning. Shown are the means ± 1 SD (n = 4).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions