Figure 3. The impact of palmitoylated cld7 on cell motility.

(A) Wt, kd and rescue ASML cells (2 × 10⁴ in RPMI/1% BSA) were seeded in the upper part of a Boyden chamber; the lower part, separated by a 0.8 μm pore size membrane contained RPMI/20% FCS. Recovery of cells on the lower membrane site was evaluated after 16 h by crystal violet staining. The percent ± SD of migrating cells compared to the total input are shown. (B) Wt, kd and rescue ASML cells were seeded in 24-well plates. When cultures reached a subconfluent stage, the monolayer was scratched with a pipette tip. Wound healing was followed for 72 h. Examples (scale bar: 250 μm) and the mean percent ± SD of the wound area compared to the 0 time point are shown. (C) Cells as above were seeded in 6-well plates coated with LN111. Pictures were taken every 20 min for 24 h. Migration of 20 individual cells was recorded. An example of migration of a single cell as well as the mean migration ± SD of 20 cells/well is presented. (A–C) Significant differences as compared to ASML^wt cells: *. (D) Wt, kd and rescue ASML cells were stained with anti-ezrin (green) or anti-RhoA (green) and anti-EpC (red) or anti-cld7 (red). Staining was evaluated by confocal microscopy; digital overlays of staining are shown (scale bar: 10 μm). The indicated area (white square) was amplified 10-fold for better discrimination. The Pearson correlation coefficient is shown for the encircled membrane area. (E) Lysates of cells as above were precipitated with anti-α3, -α6β4 (B5.5), -ezrin and -tubulin and were blotted with anti-cld7 or were precipitated with anti-cld7 and blotted with -RhoA and -Tspan8. The relative signal strength of cld7 precipitates is indicated. The strength of the cld7 signal in ASML wt was arbitrarily set as 1.0. WB of α3, β4, ezrin, tubulin and Rho are included as controls. Migration of ASML-cld7^kd and -cld7^mPalm, but not of -cld7^resc cells is severely reduced. Impaired migration is accompanied by reduced association of cld7^mPalm with ezrin, and, less pronounced, α3, α6β4, tubulin, RhoA and Tspan8. In the absence of (palmitoylated) cld7, EpC does poorly colocalize/associate with RhoA.