Figure 5. TOM1L1 regulates MT1-MMP activity.

(a) BT-474 cells infected with shCtrl or shTOM1L1 encoding viruses were transfected with mCherry–MT1-MMP and GFP, TOM1L1–GFP or TOM1L1ΔGAT–GFP then plated on a gelatin/Oregon Green 488 gelatin mix and imaged by (xz) or (xy) confocal microscopy. Note the more basal localization of MT1-MMP when co-expressed with TOM1L1, but not with TOM1L1ΔGAT (arrowheads). Scale bar, 10 μm (See also Supplementary Movie 1). (b) Quantification of a. The fraction of mCherry–MT1-MMP in contact with the gelatin layer was evaluated as indicated in the upper panel. Lower panel shows fluorescence quantifications in indicated conditions (n=8). Mean±s.e.m., *P≤0.05; **P≤0.01 (Student's t-test). (c) 48 h after transfection with mCherry–MT1-MMP, BT-474 cells infected with the indicated viruses were seeded on plasma-cleaned glass coverslips coated with gelatin and imaged by epifluorescence or TIRF. Shown is a representative image out of 10. Scale bar, 10μm. (d) Confocal orthogonal imaging (x/z) of BT-474 shTOM1L1-infected cells were transfected with control (Ctrl) or TOLLIP siRNA, then transfected with mCherry–MT1-MMP and GFP–TOM1L1 and plated on gelatin-coated coverslips. Cells were treated with DMSO or 1 μM Lapatinib for 3 h and imaged by confocal orthogonal imaging (x/z). Note the strong co-localization (arrowheads) of GFP–TOM1L1 and mCherry–MT1-MMP at the basal plane (dotted lines) of cells and the loss of this localization after TOLLIP depletion or Lapatinib treatment. Scale bar, 10 μm. (e) Quantification of d. The fraction of mCherry–MT1-MMP in contact with the gelatin layer was evaluated as in b. Graph shows mean±s.e.m. of fluorescence quantifications in indicated conditions (n=4–10). **P≤0.01 (Student's t-test). (f) Western blot showing the efficiency of TOLLIP depletion 48 h after transfection of TOLLIP or control siRNAs in BT-474 cells.