Journal: Molecular psychiatry

Article Title: Functional Analysis of Rare Variants Found in Schizophrenia Implicates a Critical Role for GIT1-PAK3 Signaling in Neuroplasticity

doi: 10.1038/mp.2016.98

Figure Lengend Snippet: (A) GIT1 structural and functional domains (see Introduction for description). Locations of unique GIT1 coding variants are shown above (SCZ cases; in red) or below (controls; in blue) the domain structure diagram. The SHD domain (Spa2 Homology Domain) is a key protein-protein interaction region known to mediate interactions with ARHGEF6/7-PAK, MAP2K1 (MEK1), FAK, PYK2, PLCO, and PPFIA2. (B) Summary of study design. Coding variants unique to SCZ cases or controls identified in large-scale human exome sequencing studies were tested in a range of biological assays in cell lines and primary cultured neurons to characterize and compare the effect of different variant effects and reveal potentially relevant functional changes. See also Figures S4, Table S1, and Table S2.

Article Snippet: anti-ARHGEF6 (Cell Signaling, #4573), anti-DLG2 (Neuro Mab, #73–284), anti-DLG4 (Neuro Mab, #73–028), anti-GABRG2 (Synaptic Systems, #224 003), anti-GAD1 (Chemicon, #MAB5406), anti-GAPDH (Cell Signaling, #2118), anti-GAPDH (Cell Signaling, #8884), anti-GIT1 (Cell Signaling, #2919), anti-GRIA1 (Millipore, #ABN241), anti-GRIA2 (Millipore, #AB1768), anti-GRIK2/3 (GLUR6/7) (Millipore, #04–921), anti-GRM5 (Millipore, #AB5675), anti-MEK1 (MAP2K1) (Cell Signaling, #9146), anti-NLGN1 (Neuro Mab, #73–158), anti-PAK1 (Cell Signaling, #2602), anti-PAK3 (Cell Signaling, #2609), anti-β-Tubulin (Sigma, #T8660), anti-SLC17A7 (VGLUT1) (Synaptic Systems, #135303).

Techniques: Functional Assay, Sequencing, Cell Culture, Variant Assay

Journal: Molecular psychiatry

Article Title: Functional Analysis of Rare Variants Found in Schizophrenia Implicates a Critical Role for GIT1-PAK3 Signaling in Neuroplasticity

doi: 10.1038/mp.2016.98

Figure Lengend Snippet: (A) Schematic diagram depicting the biochemical basis of the GIT1-PAK3 co-transfection assay. Rac/Cdc42 Guanine Nucleotide Exchange Factor (ARHGEF6/7), also known as the PIX (p21-activated kinase interacting exchange factor) family, bind to the PIX binding domain (PBD) on PAK family members in the presence of GIT1, leading to conformational changes and activation of PAKs. Deactivated PAK dimers are arranged in a head-to-tail manner in an auto-inhibited state due to interaction of one N-terminal Regulatory Domain (RD) composed of an auto-inhibitory kinase domain with the opposing C-terminal Kinase Domain (KD). As reviewed in (63), upon GTP-bound Rac/Cdc42 family GTPase binding to the GTPase Binding Domain (GBD), PAKs undergo a conformational change enabling autophosphorylation in trans that switches PAKs to an active state. Whereas auto-phosphorylation of S144 reduces the interaction of the kinase auto-inhibitory domain and kinase domain, auto-phosphorylation of S199/S204 reduces PAK--ARHGEF6/7 (PIX) interaction. Activation-loop phosphorylation at T423, which is required for full catalytic activity, occurs through autophosphorylation in trans or from the activity of other kinases. (B-D) Effect of wild-type GIT1 and GIT1-R283W on PAK3 and MAPK activation in HEK293 cells. Cells were co-transfected with the indicated expression vectors and 24hrs later cells were harvested in 2X SDS sample buffer. (B) Protein samples were subjected to immunoblot analysis using the indicated antibodies. For pMAPK, the top band corresponds to phospho-MAPK3 (ERK1; p44) T202/Y204 and the bottom band corresponds to phospho-MAPK1 (ERK2; p42) T185/T187. (C) Elevation of phospho-MAPK3, phospho-MAPK1 and Myc-PAK3 levels with wild-type GIT1 but not GIT1-R283W. For immunoblots other than those for the phospho-PAKs, band intensities were normalized to GAPDH band intensities, and then to values observed in wild-type GIT1-transfected cells in order to obtain fold changes (data represent mean + SEM; N = 3; **** and ** = p < 0.0001 and p < 0.01, respectively; two-way ANOVA with post hoc across row comparisons using a Tukey correction). (D) Elevation of phospho-PAK3 levels with wild-type GIT1 but not GIT1-R283W. For phospho-PAK immunoblots, band intensities were normalized to total Myc-PAK3 signals, and then to values for wild-type GIT1-transfected cells, in order to obtain fold changes (data represent mean + SEM; N = 3; **** = p < 0.0001; two-way ANOVA with post hoc across row comparisons using a Tukey correction). (E) GIT1-R283W shows reduced complex formation with PAK3, compared to wild-type GIT1. HEK293 cells were transfected with the indicated expression vectors. One day after transfection, cells were lysed and immunoprecipitated with FLAG-M2 antibody-conjugated agarose. The resulting FLAG-M2-immunoprecipitates were analyzed by immunoblot analysis with anti-FLAG-M2 or anti-Myc antibodies. (F) Quantitation of the decreased association between Myc-PAK3 and wild-type GIT1 or GIT1-R283W. Co-immunoprecipitated PAK3 immunoblot band intensities were normalized to immunoprecipitated GIT1-FLAG values. Data represent mean + SEM; N = 4; **** = p < 0.0001; two-tailed t-test; group variances were not significantly different. See also Figure S2.

Article Snippet: anti-ARHGEF6 (Cell Signaling, #4573), anti-DLG2 (Neuro Mab, #73–284), anti-DLG4 (Neuro Mab, #73–028), anti-GABRG2 (Synaptic Systems, #224 003), anti-GAD1 (Chemicon, #MAB5406), anti-GAPDH (Cell Signaling, #2118), anti-GAPDH (Cell Signaling, #8884), anti-GIT1 (Cell Signaling, #2919), anti-GRIA1 (Millipore, #ABN241), anti-GRIA2 (Millipore, #AB1768), anti-GRIK2/3 (GLUR6/7) (Millipore, #04–921), anti-GRM5 (Millipore, #AB5675), anti-MEK1 (MAP2K1) (Cell Signaling, #9146), anti-NLGN1 (Neuro Mab, #73–158), anti-PAK1 (Cell Signaling, #2602), anti-PAK3 (Cell Signaling, #2609), anti-β-Tubulin (Sigma, #T8660), anti-SLC17A7 (VGLUT1) (Synaptic Systems, #135303).

Techniques: Cotransfection, Binding Assay, Activation Assay, Activity Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Confocal images of primary hippocampal neurons ( DIV14 ) transfected at DIV7 with pGIPZ-scramble, shRNF10 and shRNF10 plus flagRNF10 and immunolabeled for GFP (green); scale bar: 5 μm. ( B - E ) Histograms showing the quantification of dendritic spine density ( B ) (n=6–10; *p<0.05, scramble vs shRNF10; ***p<0.001, shRNF10 vs shRNF10 + flagRNF10; one-way ANOVA, followed by Tukey post-hoc test), dendritic spine length ( C ), dendritic spine head width ( D ) and dendritic spine type ( E ). ( F ) WB analysis from homogenates of primary hippocampal neurons ( DIV14 ) and organotypic hippocampal slices ( DIV14 ) lentivirally infected with pGIPZ-scramble sequence (scramble) as control or with pLKO-shRNF10 (shRNF10). The histogram shows the quantification of the expression levels of GluN2A, GluA1, PSD-95 and GluN2B in shRNF10-infected neurons and slices, normalized on tubulin and expressed as % of scramble (n=6; *p<0.05; **p<0.01; unpaired Student’s t-test). ( G ) mRNA expression levels of genes associated with synaptic transmission or dendritic spine morphology by real-time PCR from DIV14 organotypic hippocampal slices lentivirally infected ( DIV4 ) with pGIPZ-scramble sequence (scramble) as control or with pLKO-shRNF10 (shRNF10) (n=4, ***p<0.001; **p<0.01; *p<0.05; unpaired Student’s t-test). ( H ) WB for ArhGef6, ArhGap4, Ophn1 and tubulin from cell lysates of organotypic hippocampal slices infected with pGIPZ-scramble or with pLKO-shRNF10. The histogram shows the quantification of protein levels from shRNF10 samples with respect to pGIPZ-scramble, following normalization on tubulin (n=3, *p<0.05; **p<0.01; unpaired Student’s t-test). DOI: http://dx.doi.org/10.7554/eLife.12430.006

Article Snippet: The following antibodies were used: monoclonal antibody (mAb) anti-α-calcium/calmodulin-dependent kinase II (αCaMKII), polyclonal antibody (pAb) anti-GluN2A, pAb anti-CREB, mAb anti-Myc, pAb anti-GluA1 and pAb anti-p-CREB (Ser-133) were purchased from Millipore (Billenca, MA,USA); mAb anti-Meox2 and pAb anti-Synaptotagmin were purchased from Abcam (Cambridge, MA, USA); mAb anti-GluR2, mAb anti-GFP, mAb anti-GST and anti-PSD-95 were purchased from NeuroMab (Davis, CA); mAb anti-α-Tubulin and mAb anti-Flag were purchased from Sigma-Aldrich (St. Louis, MO); mAb anti-p21 and mAb anti-Rch1 (importin α1, KPNA2 gene product) were purchased from BD Biosciences (Franklin Lakes, NJ); pAb anti-histone H3 was purchased from Proteintech (Chicago, IL); pAb anti-RNF10 were purchased from ProteinTech and Santacruz; mAb anti-GluN2B and p-GluA1(845) were purchased from Invitrogen (Carlsbad, CA); pAb anti-BDNF was purchased from Genetex (Irvine, CA); mAb anti-Map2 was purchased from Immunological Sciences (Roma, Italy); mAb anti-JL8 was purchased from Clontech (Mountain View, CA); pAb anti-ArhGap4 was purchased from Novus Biologicals (Cambridge, UK); mAb anti-GFAP, pAb anti-Ophn1 and pAb anti-ArhGef6 were purchased from Cell Signaling (Danvers, MA).

Techniques: Transfection, Immunolabeling, Infection, Sequencing, Expressing, Transmission Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation

doi: 10.1172/JCI83535

Figure Lengend Snippet: (A) Colocalization (white, with arrow) of ARHGEF1 (purple) with ROR1 (red) and ROR2 (green) in CLL cells cultured without (–) or with (+) Wnt5a, as indicated on the left margin or each row. Objective, ×100. Scale bars: 5 μm. (B) Colocalization of ARHGEF2 (purple) with ROR1/ROR2 in CLL cells cultured without (–) or with (+) Wnt5a, as in panel A. Objective, ×100. Scale bars: 5 μm. (C) Colocalization of ARHGEF6 (purple) with ROR1/ROR2 in CLL cells cultured without (–) or with (+) Wnt5a, as in A. Objective, ×100. Scale bars: 5 μm.

Article Snippet: Anti-ARHGEF1 antibody (Cell Signaling Technology), anti-ARHGEF2 antibody (Abcam), or anti-ARHGEF6 antibody (Cell Signaling Technology) was added in blocking buffer and incubated overnight at 4°C.

Techniques: Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation

doi: 10.1172/JCI83535

Figure Lengend Snippet: (A) In vitro exchange over time (in minutes) of RhoA (top 2 rows of graphs) or Rac1 (bottom 2 row of graphs) in immune precipitates using mAbs specific for ARHGEF1, ARHGEF2, or ARHGEF6 on lysates of CLL cells cultured with (blue lines) or without (red lines) Wnt5a, as indicated in the lower right of each graph. The green lines depict GTPase activation observed using buffer alone. (B) In vitro exchange assay on RhoA or Rac1 of immune precipitates from lysates of CLL cells cultured with UC-961 (purple lines) or Ctrl-IgG (blue lines), using mAbs specific for ARHGEF1, ARHGEF2, or ARHGEF6, as indicated in the bottom of each graph. The green lines depict GTPase activation using buffer alone. (C) Activation of RhoA or Rac1 following treatment without (–) or with (+) Wnt5a of CLL cells transfected with Ctrl-siRNA or siRNA specific for ARHGEF1, ARHGEF2, or ARHGEF6. Whole-cell lysates were run on parallel gels to determine total RhoA or Rac1. The number beneath each lane is the ratio of band densities for activated versus total GTPase normalized to that of untreated samples.

Article Snippet: Anti-ARHGEF1 antibody (Cell Signaling Technology), anti-ARHGEF2 antibody (Abcam), or anti-ARHGEF6 antibody (Cell Signaling Technology) was added in blocking buffer and incubated overnight at 4°C.

Techniques: In Vitro, Cell Culture, Activation Assay, Transfection

Journal: The Journal of Clinical Investigation

Article Title: Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation

doi: 10.1172/JCI83535

Figure Lengend Snippet: (A) Schematic depicts the structure of ROR1 or truncated forms of ROR1. Interaction of ROR1 with ROR2 was confirmed by immunoblot analysis of anti-ROR1 immune precipitates using lysates from nontransfected MEC1 (Ctrl.), MEC1-ROR1 (ROR1), or MEC1 cells transfected with each of the various truncated forms of ROR1. (B) Colocalization (white, with arrow) of ARHGEF1, ARHGEF2 or ARHGEF6 (purple) with ROR1 (red) and ROR2 (green) in MEC1-ROR1 cells. Objective, ×100. Scale bars: 2 μm. (C) Activated Rac1 or RhoA was measured in MEC1 (Ctrl.), MEC1-ROR1 (ROR1), or MEC1 cells expressing each of the truncated forms of ROR1. (D) Activated Rac1 or RhoA in MEC1 or MEC1-ROR1 cells treated with nonspecific IgG (Crtl-IgG), UC-961, or anti-Wnt5a. Whole-cell lysates were run on parallel gels to determine total RhoA or Rac1. The number beneath each lane is the ratio of band densities for activated versus total GTPase normalized to that of untreated samples.

Article Snippet: Anti-ARHGEF1 antibody (Cell Signaling Technology), anti-ARHGEF2 antibody (Abcam), or anti-ARHGEF6 antibody (Cell Signaling Technology) was added in blocking buffer and incubated overnight at 4°C.

Techniques: Western Blot, Transfection, Expressing