Zymography analyses of MMP-2 and MMP-9 secreted from MDA-MB-231 (Y527F c-Src) cells overexpressing Exo70ΔC (amino acids 1–408) or full-length Exo70. (A) After transfection with GFP-tagged Exo70ΔC or Exo70 for 18 h, cell culture media were collected and analyzed by gelatin zymography. Overexpression of Exo70ΔC caused a dramatic decrease in the amounts of MMP-2 and MMP-9 secreted in the media. Overexpression of Exo70 stimulated the secretion of MMPs (bottom panel). Cell lysates were collected for the detection of extragenically expressed GFP-tagged Exo70 proteins (top panel). Actin staining was shown as a loading control (middle panel). (B) Quantification of MMP secretion. Error bars, SD. *p < 0.01.

Localization of Exo70 at the focal degradation sites. (A) MDA-MB-231 cells were plated on fluorescent Alexa 568–labeled gelatin (red) for 20 h. After fixation, cells were stained for Exo70 (green). Individual and merged images are shown. Endogenous Exo70 showed colocalization with the “degradation holes” in the fluorescent gelatin matrix, along with some enrichment at the plasma membrane. (B) The localization of GFP-tagged Exo70 expressed at low levels using the pJ3-GFP vector was examined in MDA-MB-231 cells. GFP-tagged Exo70 partially overlapped with the degrading spots. Higher magnification views of the boxed areas are shown underneath each image. Arrows, colocalization of Exo70 and invadopodia-forming sites. Scale bars, 5 μm.

Overexpression of Exo70 stimulates invadopodial activity. (A) MDA-MB-231 cells were transfected with GFP-tagged Exo70 (GFP-Exo70), plated on fluorescent Alexa 568–labeled gelatin film for 4 h, and then processed for microscopy. Individual and merged images of GFP-Exo70 fluorescence (green), F-actin (blue), and gelatin (red) are shown. Overexpression of GFP-Exo70 stimulated focal degradation in MDA-MB-231 cells (top panel). Cells transfected with GFP alone were used as negative control (bottom panel). (B) Quantification of degradation areas in cells overexpressing Exo70. Three independent measurements (∼70 cells each) for each treatment were carried out. Error bars, SD. *p < 0.01. Scale bar, 5 μm.

The exocyst is required for the secretion of MMPs in MDA-MB-231 (Y527F c-Src) cells. (A) Secretion of MMP-2 and MMP-9 was examined in MDA-MB-231 (Y527F c-Src) cells with Exo70 or Sec8 RNAi knockdown. Cell culture media were collected and concentrated. MMP-2 and MMP-9 activities were analyzed by gelatin zymography. Knockdown of Exo70 or Sec8 led to dramatic decreases of MMP-2 and MMP-9 secreted in the media (top panel). Expression of rat Exo70 rescued MMP secretion in Exo70 knockdown cells (siEXO70(1) + rExo70). The amounts of endogenous Exo70 and Sec8, and the amount of extragenically expressed GFP-tagged rat Exo70 were detected by Western blot using anti-Exo70 and Sec8 monoclonal antibodies (middle panel). Actin was used as the loading control (bottom panel). A and B are duplicates of each experiment. (B) Quantification of secreted MMP levels was performed based on three independent experiments for each group. Secreted MMPs were represented as normalized secretion index, which was calculated as the amount of secreted MMP relative to that of the control cells. Error bars, SD. *p < 0.01.

The interaction between Exo70 and the Arp2/3 complex is required for invadopodia formation. (A) The interaction between Exo70 and the Arp2/3 complex was examined in cells with different levels of invasiveness. GST-CA pulldown assay was performed in HeLa cells, MDA-MB-231 parental and c-Src (Y527F)-transfected cells as described in Materials and Methods. Although GST–CA–Sepharose (the CA domain of N-WASP) pulled down similar amounts of Arp2/3 in HeLa, MDA-MB-231 parental and c-Src (Y527F)-transfected cells, more Exo70 bound to Arp2/3 in c-Src (Y527F)-transfected cells than in MDA-MB-231 parental cells and less Exo70 bound to Arp2/3 in HeLa cells than in MDA-MB-231 parental cells. The inputs and the bound Exo70 and Arp2/3 were analyzed by Western blot using anti-Exo70 monoclonal and anti-Arp3 polyclonal antibodies, respectively. GST alone was used as a negative control in the binding assay. (B) Matrix degradation assay was performed in MDA-MB-231 (Y527F c-Src) cells transfected with GFP-tagged Exo70^Δ628–630 or GFP alone. After transfection with GFP-tagged Exo70^Δ628–630 or GFP for 18 h, cells were cultured on Alexa 568–labeled gelatin for 4 h and then processed for microscopy. Individual and merged images of GFP-Exo70 fluorescence (green), F-actin (blue), and gelatin (red) were shown. Overexpression of GFP-tagged Exo70^Δ628–630 in Y527F c-Src cells largely inhibited matrix degradation, whereas transfection with GFP did not affect invadopodia formation. (C) Quantification of percentages of cells with different degradation levels in each treatment in B. Error bars, SD. *p < 0.01.

Exo70 is involved in Arp2/3-mediated actin polymerization in the cell. (A) Lysates were prepared from MDA-MB-231 (Y527F c-Src) cells transfected with indicated siRNA oligos. Exo70 and Sec8 knockdown levels were examined by Western blot, along with the amounts of actin and Arp3 in cell lysates. (B) The ability of the lysates to stimulate actin polymerization was analyzed by pyrene actin assay in the presence or absence of the VCA domain of N-WASP as described in Materials and Methods. The initial rate of actin polymerization, an indicator of actin nucleation by Arp2/3, was two fold lower in Exo70 knockdown cells than in the control cells. Sec8 knockdown slightly decreased the initial rate of actin polymerization. Cell lysates in the absence of VCA had little actin polymerization activity. (C) Normalized initial polymerization rates in each treatment in A were calculated by dividing the actin polymerization rate of each treatment by that of the control siRNA-treated cells. Three independent measurements for each treatment were carried out. Error bars, SD. *p < 0.01. (n = 3). (D) Actin polymerization kinetics was analyzed in lysates from MDA-MB-231 (Y527F c-Src) cells transfected with GFP-Exo70 and GFP-Exo70^Δ628–630 mutant. The levels of Exo70, actin, and Arp3 in cell lysates were detected by Western blot. (E) Lysates from cells expressing GFP-Exo70^Δ628–630 had a lower initial actin polymerization rate compared with GFP-transfected cells. Cells overexpressing GFP-Exo70 had an increased actin polymerization rate compared with the control cells. (F) Quantification of normalized polymerization rate of each treatment in D. Error bars, SD. *p < 0.01; n = 3.

Knockdown of the exocyst inhibits invadopodial activity. (A) Expression levels of the exocyst subunits Exo70 and Sec8 in MDA-MB-231 (Y527F c-Src) cells transfected with siRNAs. Lysates were prepared from cells transfected with siRNA oligos targeting Luciferase (Control siRNA), Exo70 (two different oligos: siEXO70(1) and siEXO70(2)), or Sec8 (siSEC8). siRNA treatments led to significant reduction in the amounts of Exo70 and Sec8 in the cells. The levels of Exo70 and Sec8 in cell lysates were analyzed by Western blot (left panel). In the rescue experiment, siEXO70(1) knockdown cells were transfected with GFP-tagged rat Exo70 (GFP-rExo70). The amounts of endogenous Exo70 and GFP-rExo70 were detected by Western blot (right panel). The amounts of actin were also examined as loading controls. (B) MDA-MB-231 (Y527F c-Src) cells with RNAi knockdown were cultured on Alexa 568–labeled gelatin film for 4 h. Cells were then fixed and stained with phalloidin-Alexa 488 to label F-actin. Individual and merged images of F-actin (green) and gelatin (red) were shown. RNAi knockdown of Exo70 or Sec8 decreased the amount of focal degradation (the black dots). (C) Gelatin degradation assay was performed in siEXO70(1) knockdown cells expressing GFP-rExo70. Individual and merged images of GFP-rExo70 fluorescence (green), F-actin (blue), and gelatin (red) are shown. The expression of GFP-rExo70 in siEXO70(1) knockdown cells rescued invadopodia formation in these cells. (D) Comparison of focal degradation in cells with exocyst knockdown. Four independent measurements (50 cells each) for each siRNA treatment (left panel) and rescue experiment (right panel) were carried out. Error bars, SD. *p < 0.01. Scale bar, 5 μm.