Figure 5.

Cells Expressing Exo70-Binding-Deficient Mutant GIV Adhere, but Fail to Spread, Degrade Matrix, and Invade

(A) Schematic summarizing how polarized exocytosis of membrane and cargo (in this case MT1-MMP) impacts cell adhesion, spreading, and invasion. Check, dependent; cross, independent.

(B) MDA-MB 231 cell lines were analyzed for cell adhesion by growing on poly-D-lysine-coated surface, resuspending, and then plating on 12-well collagen-coated plates for 30 min before fixing with 4% PFA and staining with crystal violet. Cells were visualized and imaged by light microscopy. (Left) Representative images are shown. Scale bar, 250 μm. (Right) Bar graphs display the relative numbers of adherent cells per field, as determined using ImageJ. Error bars represent standard error of mean; ± SEM (n = 4); ∗∗p value < 0.01; ns, not significant.

(C) Schematic summarizing the phases of cell spreading during adhesion.

(D) Adherent MDA-MB 231 cells in (B) were further analyzed for attachment-induced cell spreading at higher magnification. Representative images are shown. Scale bar, 250 μm.

(E) Bar graphs display the quantification of % spreading in (D). Error bars represent standard error of mean; ± SEM (n = 4); ∗∗∗∗p value < 0.0001.

(F) Schematic diagram showing the serum gradient-induced haptotaxis assay conditions used in (G and H).

(G) MDA-MB 231 cell lines in (A) were analyzed for the ability to invade through Matrigel-coated transwells. The number of cells that successfully invaded within 24 h was imaged. Representative images are shown. Scale bar, 250 μm.

(H) Bar graphs display the number of invading cells in (G), as determined using ImageJ. Error bars represent standard error of mean; ± SEM (n = 4); ∗∗∗p value < 0.001, ∗∗∗∗p value < 0.0001.