Metastatic State of Cancer Cells May Be Indicated by Adhesion Strength.

Cancer cells within a tumor are heterogeneous and only a small fraction are able to form secondary tumors. Universal biological markers that clearly identify potentially metastatic cells are limited, which complicates isolation and further study. However, using physical rather than biological characteristics, we have identified Mg2+- and Ca2+-mediated differences in adhesion strength between metastatic and nonmetastatic mammary epithelial cell lines, which occur over concentration ranges similar to those found in tumor stroma. Metastatic cells exhibit remarkable heterogeneity in their adhesion strength under stromal-like conditions, unlike their nonmetastatic counterparts, which exhibit Mg2+- and Ca2+-insensitive adhesion. This heterogeneity is the result of increased sensitivity to Mg2+- and Ca2+-mediated focal adhesion disassembly in metastatic cells, rather than changes in integrin expression or focal adhesion phosphorylation. Strongly adherent metastatic cells exhibit less migratory behavior, similar to nonmetastatic cell lines but contrary to the unselected metastatic cell population. Adhesion strength heterogeneity was observed across multiple cancer cell lines as well as isogenically, suggesting that adhesion strength may serve as a general marker of metastatic cells.

Haptotaxis is cell type specific and limited by substrate adhesiveness.

Motile cells navigate through tissue by relying on tactile cues from gradients provided by extracellular matrix (ECM) such as ligand density or stiffness. Mesenchymal stem cells (MSCs) and fibroblasts encounter adhesive or 'haptotactic' gradients at the interface between healthy and fibrotic tissue as they migrate towards an injury site. Mimicking this phenomenon, we developed tunable RGD and collagen gradients in polyacrylamide hydrogels of physiologically relevant stiffness using density gradient multilayer polymerization (DGMP) to better understand how such ligand gradients regulate migratory behaviors. Independent of ligand composition and fiber deformation, haptotaxis was observed in mouse 3T3 fibroblasts. Human MSCs however, haptotaxed only when cell-substrate adhesion was indirectly reduced via addition of free soluble matrix ligand mimetic peptides. Under basal conditions, MSCs were more contractile than fibroblasts. However, the presence of soluble adhesive peptides reduced MSC-induced substrate deformations; increased contractility may contribute to limited migration, but modulating cytoskeletal assembly was ineffective at promoting MSC haptotaxis. When introduced to gradients of increased absolute ligand concentrations, 3T3s displayed increased contractility and no longer haptotaxed. These data suggest that haptotactic behaviors are limited by adhesion and that although both cell types may home to tissue to aid in repair, fibroblasts may be more responsive to ligand gradients than MSCs.

The cytoskeleton regulates cell attachment strength.

Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature-only during shear application-also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.

Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart.

The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. We present the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts.

Acute shear stress direction dictates adherent cell remodeling and verifies shear profile of spinning disk assays.

Several methods have been developed to quantify population level changes in cell attachment strength given its large heterogeneity. One such method is the rotating disk chamber or 'spinning disk' in which a range of shear forces are applied to attached cells to quantify detachment force, i.e. attachment strength, which can be heterogeneous within cell populations. However, computing the exact force vectors that act upon cells is complicated by complex flow fields and variable cell morphologies. Recent observations suggest that cells may remodel their morphology and align during acute shear exposure, but contrary to intuition, shear is not orthogonal to the radial direction. Here we theoretically derive the magnitude and direction of applied shear and demonstrate that cells, under certain physiological conditions, align in this direction within minutes. Shear force magnitude is also experimentally verified which validates that for spread cells shear forces and not torque or drag dominate in this assay, and demonstrates that the applied force per cell area is largely independent of initial morphology. These findings suggest that direct quantified comparison of the effects of shear on a wide array of cell types and conditions can be made with confidence using this assay without the need for computational or numerical modeling.

Interplay of matrix stiffness and protein tethering in stem cell differentiation.

Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

Cation type specific cell remodeling regulates attachment strength.

Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α5-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions.

In situ mechanotransduction via vinculin regulates stem cell differentiation.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate.

A physical sciences network characterization of non-tumorigenic and metastatic cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

The assembly of nonadhesive fibrinogen matrices depends on the αC regions of the fibrinogen molecule.

Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated adhesion of platelets and leukocytes with implications for the surface-mediated control of thrombus growth and blood compatibility of biomaterials. The underlying mechanism of this process is surface-induced aggregation of fibrinogen, resulting in the assembly of a nanoscale multilayered matrix. The matrix is extensible, which makes it incapable of transducing strong mechanical forces via cellular integrins, resulting in insufficient intracellular signaling and weak cell adhesion. To determine the mechanism of the multilayer formation, the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal (rFg), and fibrinogen with the truncated αC regions (FgAα251) were compared. Using atomic force microscopy and force spectroscopy, we show that whereas hFg and rFg generated the matrices with a thickness of ∼8 nm consisting of 7-8 molecular layers, the deposition of FgAα251 was terminated at two layers, indicating that the αC regions are essential for the multilayer formation. The extensibility of the matrix prepared from FgAα251 was 2-fold lower than that formed from hFg and rFg. In agreement with previous findings that cell adhesion inversely correlates with the extensibility of the fibrinogen matrix, the less extensible FgAα251 matrix and matrices generated from human fibrinogen variants lacking the αC regions supported sustained adhesion of leukocytes and platelets. The persistent adhesiveness of matrices formed from fibrinogen derivatives without the αC regions may have implications for conditions in which elevated levels of these molecules are found, including vascular pathologies, diabetes, thrombolytic therapy, and dysfibrinogenemia.

Long lifetime of hydrogen-bonded DNA basepairs by force spectroscopy.

Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ~2 s for two-hydrogen bonds (A-T) and ~4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements.

Measuring passive myocardial stiffness in Drosophila melanogaster to investigate diastolic dysfunction.

Aging is marked by a decline in LV diastolic function, which encompasses abnormalities in diastolic relaxation, chamber filling and/or passive myocardial stiffness. Genetic tractability and short life span make Drosophila melanogaster an ideal organism to study the effects of aging on heart function, including senescent-associated changes in gene expression and in passive myocardial stiffness. However, use of the Drosophila heart tube to probe deterioration of diastolic performance is subject to at least two challenges: the extent of genetic homology to mammals and the ability to resolve mechanical properties of the bilayered fly heart, which consists of a ventral muscle layer that covers the contractile cardiomyocytes. Here, we argue for widespread use of Drosophila as a novel myocardial aging model by (1) describing diastolic dysfunction in flies, (2) discussing how critical pathways involved in dysfunction are conserved across species and (3) demonstrating the advantage of an atomic force microscopy-based analysis method to measure stiffness of the multilayered Drosophila heart tube versus isolated myocytes from other model systems. By using powerful Drosophila genetic tools, we aim to efficiently alter changes observed in factors that contribute to diastolic dysfunction to understand how one might improve diastolic performance at advanced ages in humans.

In situ mechanical analysis of myofibrillar perturbation and aging on soft, bilayered Drosophila myocardium.

Drosophila melanogaster is a genetically malleable organism with a short life span, making it a tractable system in which to study mechanical effects of genetic perturbation and aging on tissues, e.g., impaired heart function. However, Drosophila heart-tube studies can be hampered by its bilayered structure: a ventral muscle layer covers the contractile cardiomyocytes. Here we propose an atomic force microscopy-based analysis that uses a linearized-Hertz method to measure individual mechanical components of soft composite materials. The technique was verified using bilayered polydimethylsiloxane. We further demonstrated its biological utility via its ability to resolve stiffness changes due to RNA interference to reduce myofibrillar content or due to aging in Drosophila myocardial layers. This protocol provides a platform to assess the mechanics of soft biological composite systems and, to our knowledge, for the first time, permits direct measurement of how genetic perturbations, aging, and disease can impact cardiac function in situ.

Antibody-unfolding and metastable-state binding in force spectroscopy and recognition imaging.

Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance-possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.

Identifying single bases in a DNA oligomer with electron tunnelling.

It has been proposed that single molecules of DNA could be sequenced by measuring the physical properties of the bases as they pass through a nanopore. Theoretical calculations suggest that electron tunnelling can identify bases in single-stranded DNA without enzymatic processing, and it was recently experimentally shown that tunnelling can sense individual nucleotides and nucleosides. Here, we report that tunnelling electrodes functionalized with recognition reagents can identify a single base flanked by other bases in short DNA oligomers. The residence time of a single base in a recognition junction is on the order of a second, but pulling the DNA through the junction with a force of tens of piconewtons would yield reading speeds of tens of bases per second.

Origin of the nonadhesive properties of fibrinogen matrices probed by force spectroscopy.

The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.

Single-molecule force spectroscopy: a method for quantitative analysis of ligand-receptor interactions.

The quantitative analysis of molecular interactions is of high interest in medical research. Most methods for the investigation of ligand-receptor complexes deal with huge ensembles of biomolecules, but often neglect interactions with low affinity or small subpopulations with different binding properties. Single-molecule force spectroscopy offers fascinating possibilities for the quantitative analysis of ligand-receptor interactions in a wide affinity range and the sensitivity to detect point mutations. Furthermore, this technique allows one to address questions about the related binding energy landscape. In this article, we introduce single-molecule force spectroscopy with a focus on novel developments in both data analysis and theoretical models for the technique. We also demonstrate two examples of the capabilities of this method.

Control of integrin alphaIIb beta3 outside-in signaling and platelet adhesion by sensing the physical properties of fibrin(ogen) substrates.

The physical properties of substrates are known to control cell adhesion via integrin-mediated signaling. Fibrin and fibrinogen, the principal components of hemostatic and pathological thrombi, may represent biologically relevant substrates whose variable physical properties control adhesion of leukocytes and platelets. In our previous work, we have shown that binding of fibrinogen to the surface of fibrin clot prevents cell adhesion by creating an antiadhesive fibrinogen layer. Furthermore, fibrinogen immobilized on various surfaces at high density supports weak cell adhesion whereas at low density it is highly adhesive. To explore the mechanism underlying differential cell adhesion, we examined the structural and physical properties of surfaces prepared by deposition of various concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposition at high density resulted in an aggregated multilayered material characterized by low adhesion forces. In contrast, immobilization of fibrinogen at low density produced a single layer in which molecules were directly attached to the solid surface, resulting in higher adhesion forces. Consistent with their distinct physical properties, low- but not high-density fibrinogen induced strong alpha(IIb)beta(3)-mediated outside-in signaling in platelets, resulting in their spreading. Moreover, while intact fibrin gels induced strong signaling in platelets, deposition of fibrinogen on the surface of fibrin resulted in diminished cell signaling. The data suggest that deposition of a multilayered fibrinogen matrix prevents stable cell adhesion by modifying the physical properties of surfaces, which results in reduced force generation and insufficient signaling. The mechanism whereby circulating fibrinogen alters adhesive properties of fibrin clots may have important implications for control of thrombus formation and thrombogenicity of biomaterials.

Quantitative analysis of single-molecule RNA-protein interaction.

RNA-binding proteins impact gene expression at the posttranscriptional level by interacting with cognate cis elements within the transcripts. Here, we apply dynamic single-molecule force spectroscopy to study the interaction of the Arabidopsis glycine-rich RNA-binding protein AtGRP8 with its RNA target. A dwell-time-dependent analysis of the single-molecule data in combination with competition assays and site-directed mutagenesis of both the RNA target and the RNA-binding domain of the protein allowed us to distinguish and quantify two different binding modes. For dwell times <0.21 s an unspecific complex with a lifetime of 0.56 s is observed, whereas dwell times >0.33 s result in a specific interaction with a lifetime of 208 s. The corresponding reaction lengths are 0.28 nm for the unspecific and 0.55 nm for the specific AtGRP8-RNA interactions, indicating formation of a tighter complex with increasing dwell time. These two binding modes cannot be dissected in ensemble experiments. Quantitative titration in RNA bandshift experiments yields an ensemble-averaged equilibrium constant of dissociation of KD = 2 x 10(-7) M. Assuming comparable on-rates for the specific and nonspecific binding modes allows us to estimate their free energies as DeltaG0 = -42 kJ/mol and DeltaG0 = -28 kJ/mol for the specific and nonspecific binding modes, respectively. Thus, we show that single-molecule force spectroscopy with a refined statistical analysis is a potent tool for the analysis of protein-RNA interactions without the drawback of ensemble averaging. This makes it possible to discriminate between different binding modes or sites and to analyze them quantitatively. We propose that this method could be applied to complex interactions of biomolecules in general, and be of particular interest for the investigation of multivalent binding reactions.