Modulation of cell-matrix adhesions to affect mechanotransduction

Abstract

In cultured fibroblasts three types of cell-matrix adhesion with specific cellular localization and protein composition can be distinguished: focal complexes, focal adhesions and fibrillar adhesions. The formation of fibrillar adhesions is known to be required for fibronectin fibrillogenesis. However, little is known about specific signaling from these adhesion sites. Previous experiments showed that ILK (integrin-linked kinase) knockout fibroblasts are defective in RhoA-dependent responses to cyclic strain. In addition these cells did not form fibrillar adhesions and did not assemble fibronectin. We therefore speculated that pericellular fibronectin and the fibrillar adhesions are important to activate the RhoA/ROCK pathway by mechanical stress. To test this hypothesis, we generated fibronectin knockdown fibroblasts and analyzed their ability to activate specific RhoA dependent responses to cyclic strain in the absence and presence of exogenous fibronectin. Normal fibroblasts seeded on vitronectin in fibronectin-depleted medium deposited their own fibronectin matrix and in response to cyclic strain, activated RhoA, formed stress fibers, translocated MAL (megakaryocytic leukemia protein) to the nucleus, and induced tenascin-C. By contrast, these responses were suppressed in fibronectin knockdown or knockout cells grown under identical conditions. Interestingly, on vitronectin substrate, fibronectin-deficient cells lacked integrin a5â1-positive fibrillar adhesions. However, when fibronectin-deficient fibroblasts were plated on exogenous fibronectin, their defects in adhesions and mechanotransduction were restored. Studies with fibronectin fragments indicated that both, the RGD-synergy site and the adjacent heparin-binding region were required for full activity in mechanotransduction, but not its ability to self-assemble. In contrast to RhoA-mediated responses, activation of Erk1/2 and PKB/Akt by cyclic strain was not affected in fibronectin-deficient cells. Our results indicate that activation of the RhoA/ROCK pathway by mechanical stress originates from fibrillar adhesions connected to fibronectin in the extracellular matrix. In a second project we tried to get more insight in signaling by focal complexes. These cellmatrix adhesions are the first to be formed by adhering fibroblasts. They are clearly smaller than more mature focal adhesions and are uniquely found at the cell border of lamellipodia. Using patterns with square-shaped, RGD-coupled gold dots of a limited size, we attempted to inhibit maturation of focal complexes into focal adhesions. Indeed, on RGD-coated goldsquares smaller than 1im, cells uniquely formed focal complexes indicated by low recruitment of a5 integrin. Cells formed excessive amounts of lamellipodia and assembled actin only into a fine meshwork. However, on squares equal or larger than 1 im cells exhibited focal adhesions, spread normally and assembled actin into thick fibers. Our results show that at the level of focal complexes cell adhesion maturation can be inhibited by restricting size. In addition, according to the lamellipodia which are seen with cells on patterns smaller than 1 im, let suggest increased Rac signaling deriving from these focal complexes.