RhoA-induced changes in fibroblasts cultured on organic monolayers.
Substantial previous work indicates that adherent cell morphology in culture 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, membrane-bound form and an inactive, cytosolic RhoGDI-bound form in response to integrin stimulation. This study reports the use of self-assembled functionalized 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 transduction responses in cells attaching to derivatized SAMs. Well-controlled alkylthiol surface chemistries were used to monitor and modulate the activation state of RhoA in attaching cells. Activation states were determined indirectly by fractionating cell lysates into membrane and cytosolic fractions by ultracentrifugation. Western blots were then performed, showing RhoA localization to be surface chemistry-dependent. RhoGDI levels and its intracellular localization were also shown to be surface-chemistry dependent. Cells cultured on -CH3 terminated SAMs, which normally exhibit a low-growth phenotype, were transfected with a constitutively active mutant form of RhoA. Subsequent cell morphological changes were observed on SAM surfaces by fluorescence microscopy. Results support surface chemistry influences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of this activation state.
McClary
K B
KB
Department of Biochemistry, Colorado State University, Ft. Collins 80523-1872, USA.
Grainger
D W
DW
D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins; D002455:Cell Division; D048429:Cell Size; D003600:Cytosol / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D051379:Mice; D015398:Signal Transduction; D013499:Surface Properties; D013997:Time Factors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D005353:Fibronectins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein