Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTPbinding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rhodependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing changes in Rho activity from the structural contributions of ECM to the formation of focal adhesions. To address these questions, we established an assay for GTP-bound cellular Rho. Plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase was greatly enhanced by serum. In serum-starved adherent cells, LPA induced transient Rho activation, whereas in suspended cells Rho activation was sustained. Furthermore, suspended cells showed higher Rho activity than adherent cells in the presence of serum. These data indicate the existence of an adhesion-dependent negative-feedback loop. We also observed that both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. Our results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity.

M.A.del Pozo and L.S.Price contributed equally to this workThe small GTPase Rac regulates cytoskeletal organization, cell cycle progression, gene expression and oncogenic transformation, processes that depend upon both soluble growth factors and adhesion to the extracellular matrix (ECM). We now show that growth factors and adhesion to the ECM both contribute independently and approximately equally to Rac activation. However, activated Rac in non-adherent cells failed to stimulate the Rac effector PAK. V12 Rac or Rac activated by serum translocated to the membrane fraction of adherent cells but remained mainly cytoplasmic in suspended cells. An activated Rac mutant lacking a membrane-targeting sequence did not activate PAK in adherent cells, while mutations that forced membrane targeting restored PAK activation in suspended cells. In vitro, V12 Rac showed greater binding to membranes from adherent relative to suspended cells, indicating that cell adhesion regulated membrane binding sites for Rac. These results show that ECM regulates the ability of Rac to couple with PAK via an effect on membrane binding sites that facilitate their interaction.

To successfully induce tissue repair or regeneration in vivo, bioengineered constructs must possess both optimal bioactivity and mechanical strength. This is because cell interaction with the extracellular matrix (ECM) produces two different but concurrent signaling mechanisms: ligation-induced signaling, which depends on ECM biological stimuli, and traction-induced signaling, which depends on ECM mechanical stimuli. In this report, we provide a fundamental understanding of how alterations in mechanical stimuli alone, produced by varying the viscoelastic properties of our bioengineered construct, modulate phenotypic behavior at the whole-cell level. Using a physiologically relevant ECM mimic composed of hyaluronan and fibronectin, we found that adult human dermal fibroblasts modify their mechanical response in order to match substrate stiffness. More specifically, the cells on stiffer substrates had higher modulus and a more stretched and organized actin cytoskeleton (and vice versa), which translated into larger traction forces exerted on the substrate. This modulation of cellular mechanics had contrasting effects on migration and proliferation, where cells migrated faster on softer substrates while proliferating preferentially on the stiffer ones. These findings implicate substrate rigidity as a critical design parameter in the development of bioengineered constructs aimed at eliciting maximal cell and tissue function.

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The MAP kinase pathway is a major regulator of both normal and oncogenic growth. We report that activation of the MAP kinase ERK2 by serum or purified growth factors is strongly dependent on cell adhesion to extracellular matrix proteins. This effect is specific to soluble growth factors, since suspended cells still activate ERK2 in response to plating on fibronectin, and is reversible. Analysis of endogenous Ras and Raf show that these proteins are still activated by serum in suspended cells, whereas MEK activity is inhibited. Conversely, activation of ERK2 by activated mutants of Ras and Raf is still adhesion‐dependent but activation by MEK is not. Consistent with these results, activated MEK enhances growth of ras‐transformed cells in suspension but not when adherent. These results identify a novel synergism between cell adhesion‐ and growth factor‐regulated pathways, and explain how oncogenic activation of MAP kinases induces both serum‐ and anchorage‐independent growth.

Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

A key step in the elimination of pathogens from the body is the covalent binding of complement proteins C3 and C4 to their surfaces. Proteolytic activation of these proteins results in a conformational change, and an internal thioester is exposed which reacts with amino or hydroxyl groups on the target surface to form amide or ester bonds, or is hydrolysed. We report here that the binding of the human C4A isotype involves a direct reaction between amino-nucleophiles and the thioester. A two-step mechanism is used by the C4B isotype. The histidine at position 1,106(aspartic acid in C4A) first attacks the thioester to form an acyl-imidazole intermediate. The released thiol then acts as a base to catalyse the transfer of the acyl group to amino- and hydroxyl-nucleophiles, including water.

Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP-or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown. INTRODUCTIONThe small GTPase Rho plays a key role in the regulation of actin filament polymerization and cell growth (Hall, 1994;Olson et al., 1995). Like other GTPases, Rho proteins cycle between a GDP-bound inactive state and a GTP-bound active state. In the GTP-bound state, Rho activates downstream effector(s), leading to the assembly of actin stress fibers and focal adhesions. These effects can be blocked by ADP-ribosylation of endogenous Rho protein within the putative effectorbinding region (amino acids 32-42) with C3 exoenzyme from Clostridium botulinum (Paterson et al., 1990;Ridley and Hall, 1992). Rho proteins (193 amino acids)