Journal: The Journal of Cell Biology

Article Title: Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

doi: 10.1083/jcb.200810179

Figure Lengend Snippet: FGF2 and S4 clustering activate Rac1 via RhoG. (a) RFPECs were cultured in 0.5% FBS/DME for 24 h before RhoG activity assay. Cells were treated with 50 ng/ml FGF2 for the indicated times before lysis and subsequent RhoG activity assay. Peak RhoG activity is observed at 10 min. (right) Quantification from three experiments is shown. *, P = 0.018. (b) RFPECs stably expressing the S4-FcR chimera were likewise cultured for 24 h in 0.5% FBS/DME before RhoG activity assay. The S4-FcR chimera was clustered for the indicated times. (right) Quantification from three experiments is shown. *, P = 0.042. (c) RFPECs were transfected with the indicated RhoG constructs for 48 h before assay. 24 h after transfection, cells were cultured in 0.5% FBS/DME. Each condition represents no stimulation (−) or 10 min of FGF2 stimulation at a concentration of 50 ng/ml (+). Rac1 activity assays were performed three times and quantified using a modified ELISA technique (see Materials and methods). * (left to right), P = 0.0008, 0.014, and 0.002. (d) RFPECs were plated on glass coverslips and transfected with Raichu-Rac1 along with the indicated shRNA constructs in a 1:4 molar ratio. Cells were serum starved with 0.5% FBS in 1:1 F12/DME for 12 h before imaging. FGF2 was added at the indicated time points, and the cells were imaged once per minute. The images show the pseudocolored FRET ratios calculated as YFP fluorescence/CFP fluorescence after background subtraction at each pixel. Higher ratio values (red) correlate with higher Rac1 activity. Bars, 10 µm. (e) Quantification of whole cell FRET ratios across six cells were performed in each condition shown in d. FGF2 stimulation (+) is for 10 min. *, P = 0.037. (f) RFPECs were transfected with the indicated constructs and plated on fibronectin-coated plastic dishes. The cells were grown to a confluent monolayer, at which time they were serum starved, and a scratch was introduced to disrupt the monolayer. The images and quantification represent the area migrated, averaged over three experiments with 24 frames measured per condition, 24 h after monolayer disruption. All results were normalized to the migration of nonstimulated control cells (leftmost condition). Constitutively active RhoG (V12) was used as a positive control for migration. * (bottom to top), P = 0.048 and 0.008. Bars, 75 µm. P-values in all experiments were calculated using a two-sample equal variance t test. Error bars represent SEM.

Article Snippet: Mouse monoclonal anti-RhoG antibodies and GST-tagged RhoGDI1 were purchased from Cytoskeleton, Inc. Biotinylated S4 tail peptide (biotin-MKKKDEGSYDLGKKPIYKKAPTNEFYA) corresponding to the C-terminal protein sequence was synthesized by Syngene and verified by mass spectrometry.

Techniques: Cell Culture, Activity Assay, Lysis, Stable Transfection, Expressing, Transfection, Construct, Concentration Assay, Modification, Enzyme-linked Immunosorbent Assay, shRNA, Imaging, Fluorescence, Migration, Positive Control

Journal: The Journal of Cell Biology

Article Title: Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

doi: 10.1083/jcb.200810179

Figure Lengend Snippet: S4 mediates baseline RhoG activity and associates with synectin, RhoGDI1, and RhoG. (a) Murine pulmonary microvascular endothelial cells from WT and S4 knockout mice were cultured in 0.5% FBS/DME for 24 h before assay of RhoG activity. β-Actin and RhoGDI were used as loading controls. (right) Quantification from three experiments is shown. *, P = 0.009. Error bar indicates SEM. (b) A synthetic biotinylated peptide corresponding to the transmembrane domain and cytoplasmic tail of S4 was used to pull down proteins after conjugation to streptavidin beads. Pull-downs were immunoblotted and probed for synectin (top) and RhoGDI (bottom). − indicates unconjugated streptavidin beads. In both cases, the target proteins were pulled down only when incubated with the S4 tail peptide. (c) Coimmunoprecipitations were performed with antibodies against RhoGDI and synectin in three cell lines: WT RFPECs (left), RFPECs expressing the S4-FcR chimera (middle), and RFPECs expressing the S4-FcR (PDZ−) chimera (right). β-Actin was used as a loading control. IP, immunoprecipitation. (d) Glutathione beads conjugated with recombinant RhoGDI1 (right) or without the recombinant protein (left) were incubated with lysates of RFPECs for 12 h at 4°C. After washing, proteins were denatured by boiling and subjected to SDS-PAGE. The proteins were transferred to a membrane and probed for RhoG and synectin. (e) RFPECs were transfected with the S4-FcR chimera. Cells were incubated with FITC-conjugated human IgG to stain the chimera (left), washed twice with PBS, fixed, permeabilized, and stained with antibodies against RhoGDI1 and RhoG (middle and right, respectively). Arrows indicate regions of colocalization. Bars, 10 µm.

Article Snippet: Mouse monoclonal anti-RhoG antibodies and GST-tagged RhoGDI1 were purchased from Cytoskeleton, Inc. Biotinylated S4 tail peptide (biotin-MKKKDEGSYDLGKKPIYKKAPTNEFYA) corresponding to the C-terminal protein sequence was synthesized by Syngene and verified by mass spectrometry.

Techniques: Activity Assay, Knock-Out, Cell Culture, Conjugation Assay, Incubation, Expressing, Immunoprecipitation, Recombinant, SDS Page, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

doi: 10.1083/jcb.200810179

Figure Lengend Snippet: Synectin and RhoGDI1 are required for RhoG suppression at baseline. (a) WT murine pulmonary microvascular endothelial cells were transfected with either GFP- or 4His-tagged synectin 48 h before lysis and RhoG activity assay. Cells were serum starved in 0.5% FBS/DME for 24 h before lysis. (bottom) Quantification from three experiments is shown. *, P = 0.052. (b) Mock-transfected RFPECs (left and middle) and those transfected with 4His-tagged synectin (right) were lysed 48 h after transfection. Pull-downs were performed using glutathione beads conjugated with GST-RhoGDI1 (middle and right) or glutathione beads alone (right). (c) Lysates from WT and synectin knockout murine pulmonary microvascular endothelial cells were normalized to equal protein concentrations and incubated with monoclonal anti-RhoGDI antibodies and protein A/G beads (+) or beads alone (−). After washing and protein elution, the samples were analyzed by SDS-PAGE and probed with an anti-RhoG antibody. IP, immunoprecipitation. (d) RFPECs were transfected with a control shRNA sequence or shRNA specific for RhoGDI1. 24 h after transfection, cells were placed in 0.5% FBS/DME for an additional 24 h, at which time they were lysed and assayed for RhoG activity. The quantifications show the mean of three experiments and reveal a more than twofold increase in baseline RhoG activity in cells treated with RhoGDI1 shRNA. * (bottom to top), P = 0.034 and 0.008. Error bars indicate SEM.

Article Snippet: Mouse monoclonal anti-RhoG antibodies and GST-tagged RhoGDI1 were purchased from Cytoskeleton, Inc. Biotinylated S4 tail peptide (biotin-MKKKDEGSYDLGKKPIYKKAPTNEFYA) corresponding to the C-terminal protein sequence was synthesized by Syngene and verified by mass spectrometry.

Techniques: Transfection, Lysis, Activity Assay, Knock-Out, Incubation, SDS Page, Immunoprecipitation, shRNA, Sequencing

Journal: The Journal of Cell Biology

Article Title: Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

doi: 10.1083/jcb.200810179

Figure Lengend Snippet: PKCα phosphorylation of RhoGDI1 at Ser 96 induces RhoG activation. (a) RFPECs were transduced with GFP or GFP-myristoylated PKCα adenoviral constructs for 48 h before RhoG activity assay. The cells were serum starved in 0.5% FBS/DME 24 h after transfection, at which time lysates were collected. (bottom) Quantification for three experiments is shown. *, P = 0.048 (two-sample equal variance t test). (b) RFPECs were transfected with the indicated constructs for 24 h and serum starved in 0.5% FBS/DME for an additional 24 h before RhoG activity assay. Expression of phosphomimetic RhoGDI1 (S96D) led to RhoG activation. Quantification represents the mean of three experiments. *, P = 0.038. (c) RFPECs with stable expression of the S4-FcR chimera were used to cluster the transmembrane and cytosolic domains of S4. These cells were treated with 2 µg/ml nonimmune human IgG and washed twice with PBS. Clustering was initiated with anti–human IgG F(ab′) 2 fragments at 3 µg/ml for the indicated times. Cells were lysed and analyzed by immunoblotting. A rabbit polyclonal antibody specific for the phosphorylation of Ser 96 was used for the top panel. (d) HeLa cells overexpressing the indicated GFP-tagged RhoGDI1 mutants were lysed 72 h after transfection and subjected to immunoprecipitation (IP) with anti-GFP antibodies conjugated to protein A/G beads. The immunoblot was probed for RhoG. RhoGDI1 (S96D) leads to decreased RhoG–RhoGDI1 interaction. (e) RFPECs were transfected with the indicated constructs and serum starved. FGF2 treatment was performed for 1 min. Lysates were subsequently analyzed by Western blotting using anti–phospho-RhoGDI (Ser 96 ) antibodies. Both FGF2 and (myristoylated) constitutively active (CA) PKCα resulted in RhoGDI1 phosphorylation Ser 96 , whereas kinase-dead (KD) PKCα resulted in a lower baseline level of RhoGDI phosphorylation at this residue. Error bars represent SEM.

Article Snippet: Mouse monoclonal anti-RhoG antibodies and GST-tagged RhoGDI1 were purchased from Cytoskeleton, Inc. Biotinylated S4 tail peptide (biotin-MKKKDEGSYDLGKKPIYKKAPTNEFYA) corresponding to the C-terminal protein sequence was synthesized by Syngene and verified by mass spectrometry.

Techniques: Activation Assay, Transduction, Construct, Activity Assay, Transfection, Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

doi: 10.1083/jcb.200810179

Figure Lengend Snippet: RhoG activation is regulated by a S4–synectin–RhoGDI1 complex. RhoG activity is suppressed at baseline by a complex consisting of S4, synectin, and RhoGDI1. Within this complex, S4 enhances synectin–RhoGDI1 binding, and synectin increases the affinity of RhoGDI1 for RhoG. Upon FGF2 stimulation and subsequent S4 oligomerization, PKCα becomes activated, which phosphorylates RhoGDI at its Ser 96 residue. This induces the dissociation of RhoG from RhoGDI1, after which RhoG becomes activated. Active RhoG associates with ELMO1 and Dock180 to form a functional GEF complex, which is required for Rac1 activation in this pathway.

Article Snippet: Mouse monoclonal anti-RhoG antibodies and GST-tagged RhoGDI1 were purchased from Cytoskeleton, Inc. Biotinylated S4 tail peptide (biotin-MKKKDEGSYDLGKKPIYKKAPTNEFYA) corresponding to the C-terminal protein sequence was synthesized by Syngene and verified by mass spectrometry.

Techniques: Activation Assay, Activity Assay, Binding Assay, Functional Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Tumor necrosis factor-induced ArhGEF10 selectively activates RhoB contributing to human microvascular endothelial cell tight junction disruption

doi: 10.1096/fj.202002783RR

Figure Lengend Snippet: ArhGEF10 knockdown blocks and reverses endothelial permeability and improve barrier function induced by TNF. A, Human dermal microvascular endothelial cells (HDMECs) transfected with siRNA (non-targeting, si-NT or ARHGEF10, si-ARHGEF10) demonstrate efficient knockdown of ARHGEF10 under saline (PBS) and tumor necrosis factor (TNF, 10 ng/mL, 6 hours) stimulation. mRNA levels are expressed as the means relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a standard deviation (n = 3). Concordantly, HDMECs transfected with ARHGEF10 siRNA demonstrate depleted ArhGEF10 (representative western blot B, quantification C, results are expressed as the means relative to control with standard deviation, n = 3). D, E, TEER changes in HDMECs transfected with non-targeting siRNA (si-NT), siRNA for ARHGEF10 (si-ARHGEF10) or siRNA for RHOB was recorded between 48 and 72 hours of transfection stimulated in the presence and absence of TNF (10 ng/mL, 6 hours) without replacing the growth medium at 24-hour post-feeding. The y-axis shows normalized TEER calculated as a ratio of TEER measurement taken post-TNF to the basal TEER level before adding TNF, which is set at 1.0. HDMECs depleted of ArhGEF10, Rho or both demonstrate dramatically less TNF-induced leak. Error bars represent standard deviation (n = 3). Non-parametric Mann-Whitney test was used to compare groups (n = 3)

Article Snippet: Antibodies and reagents Validated, commercially available Taqman probes were used for detecting mRNA species of RHOA (Thermo Fisher, catalog# PN4448892), RHOB (Thermo Fisher, catalog # Hs00269660-s1 PN-4453320), RHOC (Thermo Fisher, catalog # Hs00237129-m1), ARHGEF10 (Thermo Fisher, catalog # Hs0101023-g1), GAPDH (Thermo Fisher, catalog # Hs02786624-g1), and SELE (Thermo Fisher, catalog #Hs00174057-m1).

Techniques: Permeability, Transfection, Standard Deviation, Western Blot, MANN-WHITNEY

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Tumor necrosis factor-induced ArhGEF10 selectively activates RhoB contributing to human microvascular endothelial cell tight junction disruption

doi: 10.1096/fj.202002783RR

Figure Lengend Snippet: ArhGEF10 knockdown Significantly reduces the total amount of GTP-bound active RhoB but not, RhoA or RhoC in whole-cell lysates. A-C, Human dermal microvascular endothelial cells (HDMECs) transfected with non-targeting or ARHGEF10 siRNA demonstrate similar expression of RHOB and RHOC with slightly increased RHOA. Stimulation with TNF (10 ng/mL, 6 hours) reveals no changes in RHOC expression, a similar pattern but significantly less RHOB upregulation and a blunted increase in RHOA expression. mRNA levels are expressed as the means relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a standard deviation (n = 3). D-F, HDMECs transfected with non-targeting siRNA (si-NT) and stimulated with TNF (10 ng/mL, 6 hours) demonstrate increased levels of total (relative to the loading control β-actin) active RhoA and RhoB but not RhoC. In HDMEC depleted of ArhGEF10, there are significant increases in total and active RhoA, but decreases in total and active RhoB (quantification G, J, H, K, I, L). The density of the proteins in each control (NT-PBS) group was used as a standard (1 arbitrary unit) to compare the relative density of the other groups. Non-parametric Mann-Whitney test was used to compare groups (n = 3)

Article Snippet: Antibodies and reagents Validated, commercially available Taqman probes were used for detecting mRNA species of RHOA (Thermo Fisher, catalog# PN4448892), RHOB (Thermo Fisher, catalog # Hs00269660-s1 PN-4453320), RHOC (Thermo Fisher, catalog # Hs00237129-m1), ARHGEF10 (Thermo Fisher, catalog # Hs0101023-g1), GAPDH (Thermo Fisher, catalog # Hs02786624-g1), and SELE (Thermo Fisher, catalog #Hs00174057-m1).

Techniques: Transfection, Expressing, Standard Deviation, MANN-WHITNEY

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Tumor necrosis factor-induced ArhGEF10 selectively activates RhoB contributing to human microvascular endothelial cell tight junction disruption

doi: 10.1096/fj.202002783RR

Figure Lengend Snippet: ArhGEF10 is a specific GTP exchange factor for RhoB in cell-free assay. We are able to immunoprecipitate ArhGEF10 protein, which was performed separately immediately prior to each cell-free assay (A, D, J, quantification relative to IgG pulldown F, H, N, n = 3). Immunoprecipitated ArhGEF10 was incubated with recombinant Rho proteins, which contained very little pre-formed active Rho, as assessed by pulldown assays (B, E, K). Pulldown of active Rho after incubation revealed no changes in GTP loading for RhoA (C) or RhoC (L), but a dramatic increase in GTP loaded RhoB (I, quantification relative to GTP loading in the IgG group G, M, O). Results are expressed as the means of three different experiments. The density of the proteins in each control (NT-PBS) group was used as a standard (1 arbitrary unit) to compare the relative density of the other groups. Non-parametric Mann-Whitney test was used to compare groups (n = 3)

Article Snippet: Antibodies and reagents Validated, commercially available Taqman probes were used for detecting mRNA species of RHOA (Thermo Fisher, catalog# PN4448892), RHOB (Thermo Fisher, catalog # Hs00269660-s1 PN-4453320), RHOC (Thermo Fisher, catalog # Hs00237129-m1), ARHGEF10 (Thermo Fisher, catalog # Hs0101023-g1), GAPDH (Thermo Fisher, catalog # Hs02786624-g1), and SELE (Thermo Fisher, catalog #Hs00174057-m1).

Techniques: Cell-Free Assay, Immunoprecipitation, Incubation, Recombinant, MANN-WHITNEY

Journal: Blood

Article Title: RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis

doi: 10.1182/blood.2020008738

Figure Lengend Snippet: RhoG controls F-actin through modulation of Rac1 activity. (A) Graphs comparing F-actin intensity (phalloidin staining) in WT and RhoG-deficient NK-92 (left) and patient-derived T cells (right). (B) Histogram showing activation of Rac1, RhoA, and Cdc42 in WT and RHOG KO NK-92 cells upon stimulation. Activation of GTPases was measured using G-LISA assay (Cytoskeleton, Inc). (C) Histogram comparing activation of Rac1, RhoA, and Cdc42 in WT and RHOG KO NK-92 cells upon treatment with MLN4924. (D) Scheme demonstrating the mechanism of action for the top hit MLN4924 identified in pharmacological screening experiment. (E) Graph showing F-actin intensity in WT and RhoG-deficient NK-92 cells without, and after 16 hours of treatment with MLN4924. (F) Histogram demonstrating the effect of Rac1, RhoA, and Cdc42 inhibitors on degranulation in MLN4924-treated RHOG KO NK-92 cells. ICAM, human recombinant ICAM-1 Fc chimera; PLL, poly-L lysine. Data shown as mean ± SEM. P values were calculated using a multiple Student t test. **P < .01; *****P < .0001.

Article Snippet: Activation of GTPases was measured using G-LISA assay (Cytoskeleton, Inc). (C) Histogram comparing activation of Rac1, RhoA, and Cdc42 in WT and RHOG KO NK-92 cells upon treatment with MLN4924. (D) Scheme demonstrating the mechanism of action for the top hit MLN4924 identified in pharmacological screening experiment. (E) Graph showing F-actin intensity in WT and RhoG-deficient NK-92 cells without, and after 16 hours of treatment with MLN4924. (F) Histogram demonstrating the effect of Rac1, RhoA, and Cdc42 inhibitors on degranulation in MLN4924-treated RHOG KO NK-92 cells.

Techniques: Activity Assay, Staining, Derivative Assay, Activation Assay, Recombinant