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Development of biochips containing living cells for biodetection, drug screening and tissue engineering applications is limited by a lack of reconfigurable material interfaces and actuators. Here we describe a new class of nanostructured magnetizable materials created with a femtosecond laser surface etching technique that function as multiplexed magnetic field gradient concentrators. When combined with magnetic microbeads coated with cell adhesion ligands, these materials form microarrays of 'virtual' adhesive islands that can support cell attachment, resist cell traction forces and maintain cell viability. A cell death (apoptosis) response can then be actuated on command by removing the applied magnetic field, thereby causing cell retraction, rounding and detachment. This simple technology may be used to create reconfigurable interfaces that allow users to selectively discard contaminated or exhausted cellular sensor elements, and to replace them with new living cellular components for continued operation in future biomedical microdevices and biodetectors.

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell–material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell–material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

The mechanism by which vascular smooth muscle (VSM) cells modulate their contractility in response to structural cues from extracellular matrix remains poorly understood. When pulmonary VSM cells were cultured on increasing densities of immobilized fibronectin (FN), cell spreading, myosin light chain (MLC) phosphorylation, cytoskeletal prestress (isometric tension in the cell before vasoagonist stimulation), and the active contractile response to the vasoconstrictor endothelin-1 all increased in parallel. In contrast, MLC phosphorylation did not increase when suspended cells were allowed to bind FN-coated microbeads (4.5-μm diameter) or cultured on micrometer-sized (30 × 30 μm) FN islands surrounded by nonadhesive regions that support integrin binding but prevent cell spreading. Cell spreading and MLC phosphorylation also both decreased in parallel when the mechanical compliance of flexible FN substrates was raised. MLC phosphorylation was inhibited independently of cell shape when cytoskeletal prestress was dissipated using a myosin ATPase inhibitor in fully spread cells, whereas it increased to maximal levels when microtubules were disrupted using nocodazole in cells adherent to FN but not in suspended cells. These data demonstrate that changes in cell-extracellular matrix (ECM) interactions modulate smooth muscle cell contractility at the level of biochemical signal transduction and suggest that the mechanism underlying this regulation may involve physical interplay between ECM and the cytoskeleton, such that cell spreading and generation of cytoskeletal tension feed back to promote MLC phosphorylation and further increase tension generation.

Caldesmon (CaD), a protein component of the actomyosin filament apparatus, modulates cell shape and cytoskeletal structure when overexpressed. When capillary endothelial cells were infected with an adenoviral vector encoding GFP-CaD under Tet-Off control, progressive inhibition of contractility, loss of actin stress fibers, disassembly of focal adhesions, and cell retraction resulted. This was accompanied by a cell shape (rounding)-dependent increase in apoptosis and concomitant inhibition of cell cycle progression. Cell growth also was inhibited in low expressor cells in which cell tension was suppressed independently of significant changes in cell shape, cytoskeletal structure, or focal adhesions. Thus, changes in both cytoskeletal structure and contractility appear to be central to the mechanism by which extracellular matrix-dependent changes in capillary cell shape influence growth and apoptosis during angiogenesis, and hence the cytoskeleton may represent a potential target for anti-angiogenesis therapy.

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130Cas and its subsequent binding to several SH2 domains, which depended on both the p130Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130Cas further increased cell migration on FN and coexpression of the p130Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase implicated in controlling cellular responses to the engagement of cell-surface integrins, including cell spreading and migration, survival and proliferation. Aberrant FAK signaling may contribute to the process of cell transformation by certain oncoproteins, including v-Src. Progress toward elucidating the events leading to FAK activation following integrin-mediated cell adhesion, as well as events downstream of FAK, has come through the identification of FAK phosphorylation sites and interacting proteins. A signaling partnership is formed between FAK and Src-family kinases, leading to tyrosine phosphorylation of FAK and associated 'docking' proteins Cas and paxillin. Subsequent recruitment of proteins containing Src homology 2 domains, including Grb2 and c-Crk, to the complex is likely to trigger adhesion-induced cellular responses, including changes to the actin cytoskeleton and activation of the Ras-MAP kinase pathway.

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.

Background— Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMCs) during hypoxia and may play a protective role in the response of the lung to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia. Methods and Results— To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2–deficient mice to a model of chronic normobaric hypoxia. COX-2–null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling after hypoxia. Pulmonary vascular remodeling in COX-2–deficient mice was characterized by PASMC hypertrophy but not increased proliferation. Furthermore, COX-2–deficient mice had significant upregulation of the endothelin-1 receptor (ET A ) in the lung after hypoxia. Similarly, selective pharmacological inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ET A receptor expression in pulmonary arterioles. The absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2–deficient PASMCs with iloprost, a prostaglandin I 2 analog, and prostaglandin E 2 abrogated the potent contractile response to hypoxia and restored the wild-type phenotype. Conclusions— Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling are exacerbated in the absence of COX-2 with enhanced ET A receptor expression and increased PASMC hypertrophy. COX-2–deficient PASMCs have a maladaptive response to hypoxia manifested by exaggerated contractility, which may be rescued by either COX-2–derived prostaglandin I 2 or prostaglandin E 2 .

Analysis of how cells sense and respond to mechanical stress has been limited by the availability of techniques that can apply controlled mechanical forces to living cells while simultaneously measuring changes in cell and molecular distortion, as well as alterations of intracellular biochemistry. We have confronted this challenge by developing new engineering methods to measure and manipulate the mechanical properties of cells and their internal cytoskeletal and nuclear frameworks, and by combining them with molecular cell biological techniques that rely on microscopic analysis and real-time optical readouts of biochemical signaling. In this chapter, we describe techniques like microcontact printing, magnetic twisting cytometry, and magnetic pulling cytometry that can be systematically used to study the molecular basis of cellular mechanotransduction.

Caldesmon (CaD), a protein component of the actomyosin filament apparatus, modulates cell shape and cytoskeletal structure when overexpressed. When capillary endothelial cells were infected with an adenoviral vector encoding GFP-CaD under Tet-Off control, progressive inhibition of contractility, loss of actin stress fibers, disassembly of focal adhesions, and cell retraction resulted. This was accompanied by a cell shape (rounding)-dependent increase in apoptosis and concomitant inhibition of cell cycle progression. Cell growth also was inhibited in low expressor cells in which cell tension was suppressed independently of significant changes in cell shape, cytoskeletal structure, or focal adhesions. Thus, changes in both cytoskeletal structure and contractility appear to be central to the mechanism by which extracellular matrix-dependent changes in capillary cell shape influence growth and apoptosis during angiogenesis, and hence the cytoskeleton may represent a potential target for anti-angiogenesis therapy.

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

Focal adhesion kinase (FAK) participates in signaling events induced by diverse stimuli including integrin engagement, oncogenic transformation and mitogenic neuropeptides. FAK's signaling function is regulated by tyrosine phosphorylation. The major autophosphorylation site is tyrosine-397, which interacts with the Src homology 2 (SH2) domain of Src-family kinases including Src and Fyn. Full activation of FAK appears to require additional phosphorylation by the associated Src-family kinases. Previously identified Src sites include catalytic domain tyrosines-576 and -577, important for maximal FAK kinase activity, and tyrosine-925, which permits an SH2-mediated association with Grb2. A full understanding of FAK-mediated signaling events will require the identification of all sites of tyrosine phosphorylation. Here we report that tyrosine-861 is the major Src site in the carboxyl-terminal domain of FAK. Phosphotyrosine-861 may function in additional interactions between FAK and SH2-containing proteins.

A novel protein-tyrosine kinase, designated 'Focal Adhesion Kinase' (FAK), has recently been implicated in signal transduction pathways activated by extracellular adhesion molecules and by neuropeptide growth factors. Previously deduced primary structures for chicken and mouse FAK polypeptides differ at their amino-termini, with mouse FAK reported to have a 25 amino acid residue extension not present in chicken FAK. Additional sequence information from the 5'-end region of the chicken FAK transcript now indicates that the amino-terminal extension previously thought to be unique to mouse FAK is, in fact, also predicted for chicken FAK. Thus mouse and chicken FAK polypeptides appear to be structurally similar throughout their lengths. This is further supported by comparison of their electrophoretic mobilities.