RhoA-induced changes in fibroblasts cultured on organic monolayers

Substantial previous work indicates that adherent cell morphology in cultur e is modulated by surface chemistry. Activation of the intracellular small molecular weight GTPase, RhoA, has recently been shown to play an essential role in controlling initiation of key integrin-mediated events in surface adhesion and proliferation. RhoA is interconvertible between an active, mem brane-bound form and an inactive, cytosolic RhoGDI-bound form in response t o integrin stimulation. This study reports the use of self-assembled functi onalized organic alkylthiol monolayers (SAMs) as well-defined cell culture substrates to investigate the relationships between surface chemistry, RhoA activation and subsequent cell morphological and molecular level signal tr ansduction responses in cells attaching to derivatized SAMs. Well-controlled alkylthiol surface chemistries were used to monitor and mod ulate the activation state of RhoA in attaching cells. Activation states we re determined indirectly by fractionating cell lysates into membrane and cy tosolic fractions by ultracentrifugation. Western blots were then performed , showing RhoA localization to be surface chemistry-dependent. RhoGDI level s and its intracellular localization were also shown to be surface-chemistr y dependent. Cells cultured on -CH3, terminated SAMs, which normally exhibi t a low-growth phenotype, were transfected with a constitutively active mut ant form of RhoA. Subsequent cell morphological changes were observed on SA M surfaces by fluorescence microscopy. Results support surface chemistry in fluences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of t his activation state. (C) 1999 Elsevier Science Ltd. All rights reserved.