Supplemental Material

This article contains the following supporting material:

• Figure S1 - Immunofluorescent Staining of COS1 Cells Degrading Collagen. Untransfected COS1 cells and COS1 cells transfected with either the catalytically active FLAG-MT1-MMP (wild-type) or the catalytically inactive mutant FLAG-MT1-MMP (E240A mutant) were cultured on biotinylated reconstituted rat-tail tendon collagen for 72h. Collagen degradation was observed as a loss of green fluorescence after staining with streptavidin-Alexa Fluor 488 in the FLAG-MT1-MMP expressing cell cultures, but no significant degradation could be seen with the untransfected cells or with the cells transfected with the E240A mutant. The cells were located by F-actin staining with phalloidin (blue) and expression of the MT1-MMPs with anti-FLAG antibodies (red). Note the increased green fluorescence indicating that collagen is being pulled by the cells expressing WT and mutant MT1-MMP.

• Figure S2 - Internalization of Collagen Fibres. To demonstrate that the biotinylated collagen substrate stained with streptavidin-Alexa Fluor 488 (green) was being internalized by the HGFs, serial optical sections were prepared from ConA-treated cells cultured on rat-tail tendon collagen for 72 h and stained for F-actin with phalloidin (blue). Two examples are shown. A. Within a bundle of collagen fibres that are distributed with the F-actin, a number of fibres (white arrows) can be seen to appear and disappear indicating that they have been segregated within the cell. B. In this series the appearance and disappearance of a group of fibres (yellow arrows) occurs through a series of sections. In these images the stain for F-actin is merged with the biotinylated collagen showing the internalized collagen in relation to the cell and the collagen substratum. Optical sections, 0.6 μm.