plasmid constructs gst iqgap1  (Addgene inc)

Journal: The Journal of Cell Biology

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

doi: 10.1083/jcb.200709076

Figure Lengend Snippet: IQGAP1 and Sec8 are required for invadopodial proteolysis of the matrix and MT1-MMP accumulation at invadopodia. (a) MDA-MB-231 cells were plated on fluorescent FITC-gelatin for 6 h, subjected to surface labeling using anti–MT1-MMP antibody, fixed with PFA, and then permeabilized and stained for F-actin and endogenous IQGAP1. IQGAP1, red; MT1-MMP, green; F-actin, blue. The bottom left panel corresponds to the boxed area in the top panel. Black and white panels show individual images for surface-labeled MT1-MMP, IQGAP1, and F-actin that are all merged in the bottom left panel as well as FITC-gelatin. Of note, the punctuate accumulation of cell surface MT1-MMP at invadopodia was clearly detected by anti–MT1-MMP antibody over unspecific labeling of the gelatin (which appears as small dots visible even in regions of the gelatin free of cells; not depicted). Arrows indicate surface-labeled MT1-MMP at invadopodia on top of areas with various degrees of matrix degradation. Bars: (top) 2 μm; (bottom) 10 μm. (b) Expression levels of IQGAP1, Sec8, and MT1-MMP in MDA-MB-231 cells treated with specific siRNAs as indicated. Membranes were stained with Coomassie Brilliant blue (CBB) to control for equal loading. Molecular masses are indicated in kilodaltons. (c) Effect of IQGAP1 or Sec8 knockdown on matrix degradation of MDA-MB-231 cells. Degradation indexes calculated as in represent the mean ± SEM (error bars) of three independent experiments. The number of cells analyzed in each dataset is indicated on top of the graph. All siRNA-treated cell populations are significantly different as compared with mock-treated cells (P ≤ 0.01). (d) For each siRNA, matrix-degrading cells were scored for the presence of invadopodia, which were defined as surface-labeled MT-MMP accumulations lying on spots of degraded gelatin. Values are given as number ± SEM of MT1-MMP–positive invadopodia per cell from three independent experiments. All siRNA-treated cell populations are significantly different as compared with mock-treated cells (P ≤ 0.01).

Article Snippet: GST-IQGAP1 fusion proteins expressed in Escherichia coli (BL21 DE3) were purified using glutathione–Sepharose 4B (GE Healthcare).

Techniques: Labeling, Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

doi: 10.1083/jcb.200709076

Figure Lengend Snippet: Sec3 and Sec8 exocyst complex subunits interact with a C-terminal region of IQGAP1. (a) Schematic representation of the C-terminal fragments of human IQGAP1 used. GRD, Ras GTPase-activating protein–related domain (binding site for GTP-Rac1/Cdc42); CC, coiled-coil domain predicted by COILS (version 2.2); RasGAP_C, RasGAP C terminus; Sec3 BD, minimal overlapping region of nine independent IQGAP1 clones isolated in a yeast two-hybrid screen using human Sec3 as bait (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200709076/DC1 ). (b) Lysates of HeLa cells transfected with HA-tagged human Sec3, Sec8, Exo70, or Exo84 were incubated with the indicated C-terminal fragment of IQGAP1 fused with GST immobilized on beads, and bound proteins were analyzed by immunoblotting with anti-HA antibody. 1% of lysates was loaded as a control (input). The bottom panel shows the different GST-IQGAP1 fragments separated by SDS-PAGE and stained with Coomassie Brilliant blue (CBB). Arrowheads indicate intact GST fusion proteins. (c) Effect of Sec3/Sec8 depletion on binding of the exocyst to IQGAP1-CTer2. HeLa cells were treated for 48 h with Sec3- or Sec8-specific siRNA alone or in combination as indicated and were further transfected with a construct encoding HA-tagged human Exo70 for 18 h. Lysates were prepared and incubated with GST-IQGAP1-CTer2, and bound proteins were analyzed by immunoblotting with anti-HA (top) or anti-Sec8 (middle) antibodies. The bottom panel shows GST and GST-IQGAP1-CTer2 proteins separated by SDS-PAGE and stained with Coomassie Brilliant blue. Residual levels of knocked down proteins and of proteins bound to GST-IQGAP1/CTer2 are indicated underneath the blots with respect to the level of mock-treated cells based on densitometric analysis. The efficiency of Sec3 depletion with siSec3 is documented in Fig. S2 b. Molecular masses are indicated in kilodaltons.

Article Snippet: GST-IQGAP1 fusion proteins expressed in Escherichia coli (BL21 DE3) were purified using glutathione–Sepharose 4B (GE Healthcare).

Techniques: Binding Assay, Clone Assay, Isolation, Two Hybrid Screening, Transfection, Incubation, Western Blot, SDS Page, Staining, Construct

Journal: The Journal of Cell Biology

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

doi: 10.1083/jcb.200709076

Figure Lengend Snippet: In vivo interaction of IQGAP1 with Sec8 and Sec3 is regulated by Cdc42 and RhoA. (a) Endogenous association of IQGAP1 with Sec8 in MD-MB-231 cells. 2 mg lysates of MDA-MB-231 cells was immunoprecipitated with control, anti-Sec8, or anti-IQGAP1 IgGs, and bound proteins were analyzed by immunoblotting with the indicated antibodies. 1% of total lysate was loaded as a control (input). Densitometric analysis showed that ∼2% of immunoprecipitated Sec8 was in complex with IQGAP1, and ∼7% of immunoprecipitated IQGAP1 was associated with Sec8 in typical experiments. (b) Activated Cdc42 and RhoA promote IQGAP1 association with Sec8 in transfected HEK293 cells. HA-tagged Sec8 was transiently coexpressed with GFP-tagged wild-type (WT) or mutant IQGAP1-T1050AX2 (IQGAP1-T) in HEK293 cells together with myc-tagged Cdc42, Rac1, or RhoA GTPases either in their GTP-bound form (L [Cdc42-Q61L], Rac1-Q61L, or RhoA-Q63L) or GDP-bound form (N [Cdc42-T17N], Rac1-T17N, or RhoA-T19N). IQGAP1-T harbors mutations in the GRD domain that abolish binding to GTP-Cdc42/Rac1 . Approximately 1 mg of cellular extract was immunoprecipitated with anti-GFP antibodies and analyzed by anti-HA (top), anti-GFP (middle), or anti-myc (bottom) immunoblotting as indicated (lanes 1–12). Control IPs with irrelevant IgGs are shown in Fig. S3 b. Protein expression levels in 10 μg of total cell extracts are shown in the right panel (input, lanes 13–24). Of note, mycRhoA-T19N was consistently expressed to a lower extent as compared with mycRhoA-Q63L. The open arrowhead indicates the position of IgG light chain, and closed arrowheads point to myc-tagged Rho GTPases. (c) Cdc42 and RhoA activities are required for IQGAP1–Sec8 complex formation in MDA-MB-231 cells. The same amount of cell lysates prepared from MDA-MB-231 cells either mock treated (lanes 1 and 2), depleted for 72 h with combined siRNAs for Cdc42 and RhoA (lanes 3 and 4), or serum starved for 48 h (lanes 5 and 6) were immunoprecipitated with control IgGs or anti-Sec8 antibodies, and bound proteins were analyzed by immunoblotting with the indicated antibodies. A fraction (1%) of the lysates was analyzed as a control (input, lanes 7–9). (d) Sec3 and Sec8 form a complex with and dependent on GTP-bound Cdc42. HEK293 cells were transiently transfected with HA-tagged Sec3, V5-tagged Sec8, and myc-tagged Cdc42-Q61L (L) or -T17N (N) as indicated, and ∼1 mg of cellular extracts was immunoprecipitated with anti-myc antibodies (lanes 1–6). Bound proteins were analyzed by anti-HA, anti-V5, and anti-myc immunoblotting as indicated. Protein expression levels in 10 μg of total cell extracts are shown in the right panel (input, lanes 7–12). The open arrowheads indicate IgG light chain, and closed arrowheads indicate myc-tagged Cdc42. Control IPs with irrelevant IgGs are shown in Fig. S3 c (available at http://www.jcb.org/cgi/content/full/jcb.200709076/DC1 ). Molecular masses are indicated in kilodaltons.

Article Snippet: GST-IQGAP1 fusion proteins expressed in Escherichia coli (BL21 DE3) were purified using glutathione–Sepharose 4B (GE Healthcare).

Techniques: In Vivo, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Binding Assay, Expressing

Journal: The Journal of Cell Biology

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

doi: 10.1083/jcb.200709076

Figure Lengend Snippet: Localization of IQGAP1 and Sec8 at F-actin–rich invadopodia in MDA-MB-231 cells plated on cross-linked fluorescent gelatin. (a) MDA-MB-231 cells were plated on fluorescent FITC-gelatin, and, after 5 h, cells were fixed and stained for immunofluorescence microscopy with anti-IQGAP1 antibodies and fluorescent phalloidin. F-actin and endogenous IQGAP1 colocalize at invadopodia corresponding to proteolytic holes in the fluorescent gelatin matrix (right). (b) Phalloidin and anti-V5 staining of MDA-MB-231 cells transiently expressing V5-tagged Sec8 after 5 h on FITC-gelatin showing colocalization of F-actin and overexpressed Sec8 at invadopodia. (c) MDA-MB-231 cells transiently transfected with V5-tagged Sec8 together with HA–MT1-MMP and Y527F c-Src were plated for 4 h on AlexaFluor350-gelatin and stained for immunofluorescence microscopy with the indicated antibodies. Higher magnification views of the boxed areas are shown underneath each image. Bars, 10 μm.

Article Snippet: GST-IQGAP1 fusion proteins expressed in Escherichia coli (BL21 DE3) were purified using glutathione–Sepharose 4B (GE Healthcare).

Techniques: Staining, Immunofluorescence, Microscopy, Expressing, Transfection

Journal: The Journal of Cell Biology

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

doi: 10.1083/jcb.200709076

Figure Lengend Snippet: Stimulation of invadopodial proteolysis by a constitutively active IQGAP1 mutant requires the Sec3/Sec8-binding domain. (a) Schematic representation of IQGAP1, IQGAP1-T1050AX2 (IQGAP1-T) mutant, and its C-terminal deletion mutants. CHD, calponin homology domain; WW, polyproline-binding domain; IQ, calmodulin-binding motif; GRD, Ras GTPase-activating protein–related domain; CC, predicted coiled-coil domain; RasGAP_C, RasGAP C terminus. The stars indicate mutations in the GRD domain, which abolish binding to GTP-Cdc42/Rac1 . (b) Evaluation of fluorescent matrix degradation in MDA-MT1ch cells transfected for 24 h with the indicated constructs and analyzed after 4 h of incubation on AlexaFluor350-labeled gelatin. Data are represented as normalized degradation (degradation index), calculated as the area of degraded matrix per cell relative to GFP-expressing cells (mean ± SEM [error bars] from three independent experiments). The number of cells analyzed for each construction is indicated above the graph. (c) Localization of IQGAP1 and IQGAP1-T to invadopodia. After 4 h on AlexaFluor350-labeled gelatin, MDA-MT1ch cells transfected with the indicated construct were fixed and processed for immunofluorescence analysis by staining with AlexaFluor633-phalloidin to visualize polymerized actin. GFP-tagged IQGAP1 proteins localize at F-actin–rich invadopodia lying on top of degraded areas of the fluorescent gelatin matrix (arrows). In contrast, the localization of GFP-IQGAP1-TΔCC appears more diffuse. Higher magnification views of the boxed area are shown. Bars, 10 μm.

Article Snippet: GST-IQGAP1 fusion proteins expressed in Escherichia coli (BL21 DE3) were purified using glutathione–Sepharose 4B (GE Healthcare).

Techniques: Mutagenesis, Binding Assay, Transfection, Construct, Incubation, Labeling, Expressing, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

doi: 10.1083/jcb.200709076

Figure Lengend Snippet: Model for Rho-GTPase signaling control of invadopodial formation and function. In response to the interaction of tumor cells with their 3D matrix environment, Rho-GTPases are activated locally, triggering actin assembly through activation of the Arp2/3 complex and leading to invadopodia protrusion within the ECM. IQGAP1, which is downstream of Cdc42 and RhoA, plays a central role in invadopodia function through the coordination of actin assembly with the exocytic machinery via the vesicle-docking exocyst complex and possibly through microtubule plus end anchoring (see Discussion for details).

Article Snippet: GST-IQGAP1 fusion proteins expressed in Escherichia coli (BL21 DE3) were purified using glutathione–Sepharose 4B (GE Healthcare).

Techniques: Activation Assay