Figure 1.

Tumor cells degrade substrate-bound hyaluronan (HA) at focal adhesions (FAs).A, contact-dependent in situ degradation of substrate-immobilized HA by DU145, BT474, U2OS, and TRAMP-C2 cells. Cells were cultured for 16 h on the FA-HA substrate as described in Experimental procedures section. In situ HA degradation activity is detected as black areas in the fluorescent substratum. The scale bar represents 5 μm. B, colocalization of vinculin and sites of HA degradation. Cells cultured on the FA-HA substrate were immunostained with anti-vinculin antibody. The scale bar represents 2 μm. C, transmembrane protein 2 (TMEM2) is the predominant hyaluronidase that mediates contact-dependent in situ HA degradation. Hyaluronidases expressed in U2OS cells (TMEM2, KIAA1199, HYAL1, and HYAL2; see Table 1 for expression levels) were individually knocked down by siRNA treatment, and in situ HA degradation assays were performed with siRNA-treated cells. Alexa Fluor 594–conjugated wheat germ agglutinin (WGA) was used to visualize cell morphology. Note that depletion of TMEM2 markedly attenuates in situ HA degradation, whereas depletion of other hyaluronidases does not. The scale bar represents 10 μm. D, mCherry-fused mouse TMEM2 also induces FA-associated in situ HA degradation. U2OS cells stably expressing mCherry-mTMEM2 (endogenous human TMEM is depleted by siRNA treatment) were examined via in situ HA degradation assays and immunostained for vinculin. Note that mCherry-mTMEM2 creates a pattern of in situ HA degradation indistinguishable from that created by endogenous TMEM2 (left panel), and that mCherry signals are present at the sites of HA removal and vinculin immunoreactivities (right panels). The scale bar represents 10 μm (left panel) and 2 μm (right panels).