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

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases

(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.<p><b>Copyright information:</b></p><p>Taken from "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA"</p><p></p><p>The Journal of Cell Biology 2008;181(6):985-998.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426946.</p><p></p

(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.<p><b>Copyright information:</b></p><p>Taken from "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA"</p><p></p><p>The Journal of Cell Biology 2008;181(6):985-998.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426946.</p><p></p

(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 ). (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.<p><b>Copyright information:</b></p><p>Taken from "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA"</p><p></p><p>The Journal of Cell Biology 2008;181(6):985-998.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426946.</p><p></p

(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).<p><b>Copyright information:</b></p><p>Taken from "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA"</p><p></p><p>The Journal of Cell Biology 2008;181(6):985-998.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426946.</p><p></p

(a) The expression levels of MT1-MMP (MT1), Sec6, Sec8, and Sec10 were analyzed by immunoblotting with specific antibodies in lysates of MDA-MT1ch cells (i

E., MDA-MB-231 cells stably transfected with a construct encoding mCherry-tagged MT1-MMP) treated with the indicated siRNAs for 72 h. After immunoblotting, membranes were stained with Coomassie Brilliant blue (CBB) to control for equal loading. The open arrowhead indicates the position of mCherry-tagged MT1-MMP (MT1Ch). The closed arrowhead points to a processed, catalytically active (60 kD) form of endogenous MT1-MMP (endo MT1). Of note, depletion of the Sec8 exocyst complex subunit led to a reduction of the Sec6 level (arrow). Residual levels of knocked down proteins, as calculated based on densitometric analysis of Western blots, are indicated underneath the blots with respect to the level of mock-treated cells set to 100. The asterisk indicates a nonspecific band detected with anti-Sec6 antibodies that was not depleted upon treatment with Sec6 siRNAs. Molecular masses are indicated in kilodaltons. (b) Effect of MT1-MMP and exocyst subunit-specific siRNAs on the capacity of MDA-MT1ch cells to degrade fluorescent gelatin. MDA-MT1ch cells treated with the indicated siRNAs for 72 h were further incubated on FITC-gelatin for 4 h. Then, cells were fixed and stained with fluorescent phalloidin to label F-actin in all cells. Data are represented as normalized degradation (degradation index), which was calculated as the area of degraded matrix per cell relative to mock-treated cells (mean percentage ± SEM [error bars]) from at least three independent experiments with two coverslips each. The number of cells analyzed for each siRNA is indicated above the bars. (c) Representative images of MDA-MT1ch cells treated with the indicated siRNA and cultured on FITC-gelatin for 4 h. Merged images of F-actin (red) and gelatin (green) are shown. Bars, 10 μm. (d) Effect of MT1-MMP and exocyst subunit-specific siRNAs on the capacity of parental MDA-MB-231 cells to cross a layer of matrigel in a transwell chamber. Data are represented as normalized invasion (invasion index) relative to mock-treated cells calculated as described in Materials and methods (mean percentage ± SEM; each siRNA-treated cell population was analyzed in triplicate in at least three independent experiments).<p><b>Copyright information:</b></p><p>Taken from "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA"</p><p></p><p>The Journal of Cell Biology 2008;181(6):985-998.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426946.</p><p></p

Migrants dans une ville portuaire : Le Havre (xvie-xxie siècle)

Dans quelle mesure l'existence de liaisons maritimes entraîne-t-elle l'établissement de personnes étrangères dans la ville portuaire ? Cette ville est-elle, en raison de sa vocation, un milieu humain particulièrement propice à les intégrer ? Telles sont les questions auxquelles ont souhaité répondre, dans le cadre des débats et des rencontres organisés à l'occasion des Journées nationales « Mémoire des migrations et villes portuaires », une équipe de chercheurs ayant investi de longue date le chantier de l'immigration au Havre. Pour y répondre, ce sont les itinéraires de nombreuses personnes, migrants de l'intérieur et migrants étrangers, riches et miséreux, qui ont été minutieusement reconstitués, entre le xvie et le xxie siècle. Il ressort de ce périple une réalité située loin du cliché de la ville portuaire cosmopolite et ouverte, réalité qui, in fine, tend à mettre en exergue, dans la récurrence des difficultés rencontrées par les migrants, la propension à faire d'eux, quelles que soient les époques, des « étrangers à la cité »

Towards a green molecular chemistry

National audienceThe organic synthesis challenge is now to manage the demands for efficiency and selectivity with the development of a "green and sustainable" organic chemistry. The goals are to obtain complex molecular architectures by reducing the environmental impact, by developing atom- and step-economic new methodologies, and by developing smart alternatives to usual organic solvents