The regulated assembly and disassembly of cell–extracellular matrix junctions (focal adhesions) contributes to cell motility and tumour invasion. Rho–ROCK signalling promotes focal adhesion disassembly at the rear of the cell by inducing contractile forces that are dependent on the phosphorylation of myosin light chain-2 (MLC2). Isacke and colleagues now show that the Rho–ROCK–MLC2 signalling pathway is activated to generate contractile forces at distinct subcellular locations through endosomal trafficking.
Using human cancer cell lines the authors compared the small interfering RNA (siRNA)-mediated depletion of the promigratory transmembrane receptor ENDO180 (also known as CD280) and two other endocytic receptors. ENDO180 is constitutively recruited into clathrin-coated pits on the cell surface, which are then internalized into intracellular endosomes. Only the depletion of ENDO180 induced tail-retraction defects, indicative of reduced Rho–ROCK–MLC2-mediated contractile forces. This phenotype was comparable to that observed when cells were treated with Rho or ROCK inhibitors, establishing ENDO180–Rho–ROCK–MLC2 as a functional pathway. Moreover, ENDO180 siRNA or a ROCK inhibitor reduced the phosphorylation of three other ROCK substrates, LIM kinase-1 and -2, and myosin phosphatase-1.
Isacke and colleagues also show that ENDO180 is required for the spatial activation of Rho–ROCK–MLC2. Using cell lines that stably express an ENDO180 mutant that cannot be internalized, the authors demonstrate that ROCK cannot be recruited to focal adhesions. This shows that intracellular ENDO180-containing endosomes are required to activate Rho–ROCK.
ENDO180 is highly expressed in motile cells, particularly fibroblasts, and has an established role in collagen internalization. Bugge and colleagues have shown that mice undergoing polyomavirus-induced mammary carcinogenesis that also have a targeted deletion in ENDO180 have impaired tumour expansion, and therefore a reduced tumour burden. Isacke and colleagues suggest that this reduction in tumour burden could arise both from the inability of ENDO180-null fibroblasts to remodel the extracellular matrix and from impaired motility in the tumour. Indeed, targeting ENDO180 might benefit anticancer therapy by causing the retention of a tumour-inhibitory matrix.
