Figure 8.

Accumulation and internalization of biotinylated collagen by HGFs. Confocal images of optical sections of single HGFs stimulated with ConA for 72 h in the presence of 10 μM E-64d and grown on biotinylated collagen (Biotin-COL). (A) After 24 h, increased collagen fluorescence is occasionally observed beneath the cells as shown for a single cell in this image. The collagen around areas of collagen degradation (arrows) is often seen to be immunoreactive to an antibody recognizing denatured collagen (red). (B) After 48 h, the increased collagen fluorescence is observed more frequently and collagen fibers extending perpendicular to the cell can be seen in this image. Stronger staining for unfolded collagen is evident around a more extensive area of collagen degradation that lies within the area of increased collagen fluorescence. (C) Immunostaining for MT1-MMP (red) is concentrated on the cell surface, whereas some biotinylated collagen (green) seems to be in the initial stages of phagocytosis (arrow). (D) Biotinylated collagen fibers (green) are shown in the same focal plane as phalloidin-stained actin. The position of the collagen fibers is indicated by an arrow. (E) Biotinylated collagen fibers occurring within cells (arrows) are shown in relation to collagen stained for an epitope exposed by cathepsin B (red). In the bottom panels, z-stacked images of the same cell are shown. (F) Relationship between biotinylated collagen and lysosomes is shown using LysoTracker Red to identify the lysosomes. A punctate pattern typical of lysosomes is seen (red) surrounding a large fiber (arrow) that seems to be within the cell, as indicated also in z-stacked images shown in the bottom panels.