Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy

Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.     

Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion

The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice.     

Macrophage adhesion on fibronectin evokes an increase in the elastic property of the cell membrane and cytoskeleton: an atomic force microscopy study

Interactions between cells and microenvironments are essential to cellular functions such as survival, exocytosis and differentiation. Cell adhesion to the extracellular matrix (ECM) evokes a variety of biophysical changes in cellular organization, including modification of the cytoskeleton and plasma membrane. In fact, the cytoskeleton and plasma membrane are structures that mediate adherent contacts with the ECM; therefore, they are closely correlated. Considering that the mechanical properties of the cell could be affected by cell adhesion-induced changes in the cytoskeleton, the purpose of this study was to investigate the influence of the ECM on the elastic properties of fixed macrophage cells using atomic force microscopy. The results showed that there was an increase (~50 %) in the Young’s modulus of macrophages adhered to an ECM-coated substrate as compared with an uncoated glass substrate. In addition, cytochalasin D-treated cells had a 1.8-fold reduction of the Young’s modulus of the cells, indicating the contribution of the actin cytoskeleton to the elastic properties of the cell. Our findings show that cell adhesion influences the mechanical properties of the plasma membrane, providing new information toward understanding the influence of the ECM on elastic alterations of macrophage cell membranes.     

Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments

In probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPalpha, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments.     

Cellular contractility changes are sufficient to drive epithelial scattering

Epithelial scattering occurs when cells disassemble cell–cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial–mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell–cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell–cell interactions occurs by remodeling of cell–cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell–cell adhesion between cells in suspension, suggesting that changes in cell–cell adhesion strength might not accompany epithelial scattering. Instead, cell–substrate adhesion seems to play a bigger role, as cell–substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFβ is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells? ability to scatter. Further consistent with changes in substrate adhesion being required for cell–cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell–cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell–cell detachment during epithelial scattering.     

Investigating differential cell‐matrix adhesion by directly comparative single‐cell force spectroscopy

Tissue‐embedded cells are often exposed to a complex mixture of extracellular matrix (ECM) molecules, to which they bind with different cell adhesion receptors and affinities. Differential cell adhesion to ECM components is believed to regulate many aspects of tissue function, such as the sorting of specific cell types into different tissue compartments or ECM niches. In turn, aberrant switches in cell adhesion preferences may contribute to cell misplacement, tissue invasion, and metastasis. Methods to determine differential adhesion profiles of single cells are therefore desirable, but established bulk assays usually only test cell population adhesion to a single type of ECM molecule. We have recently demonstrated that atomic force microscopy‐based single‐cell force spectroscopy (SCFS), performed on bifunctional, microstructured adhesion substrates, provides a useful tool for accurately quantitating differential matrix adhesion of single Chinese hamster ovary cells to laminin and collagen I. Here, we have extended this approach to include additional ECM substrates, such as bifunctional collagen I/collagen IV surfaces, as well as adhesion‐passivated control surfaces. We investigate differential single cell adhesion to these substrates and analyze in detail suitable experimental conditions for comparative SCFS, including optimal cell‐substrate contact times and the impact of force cycle repetitions on single cell adhesion force statistics. Insight gained through these experiments may help in adapting this technique to other ECM molecules and cell systems, making directly comparative SCFS a versatile tool for comparing receptor‐mediated cell adhesion to different matrix molecules in a wide range of biological contexts. Copyright © 2013 John Wiley & Sons, Ltd.     

Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.     

Atomic force microscopy of animal cells: Advances and prospects

We review the advances of the method of atomic force microscopy (AFM) for investigating the animal cells and analyze its development, paying much attention to studies of living cells. We consider the specific features and tasks of AFM, and a number of special AFM-based techniques. We discuss the choice of probe geometry for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and analysis of the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches, as well as combining AFM with optical and laser scanning confocal microscopy, and the possible applications of AFM in biotechnology and medicine.     

Toward single cell traction microscopy within 3D collagen matrices

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.     

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.     

Study of the time effect on the strength of cell-cell adhesion force by a novel nano-picker

Cell’s adhesion is important to cell’s interaction and activates. In this paper, a novel method for cell–cell adhesion force measurement was proposed by using a nano-picker. The effect of the contact time on the cell–cell adhesion force was studied. The nano-picker was fabricated from an atomic force microscopy (AFM) cantilever by nano fabrication technique. The cell–cell adhesion force was measured based on the deflection of the nano-picker beam. The result suggests that the adhesion force between cells increased with the increasing of contact time at the first few minutes. After that, the force became constant. This measurement methodology was based on the nanorobotic manipulation system inside an environmental scanning electron microscope. It can realize both the observation and manipulation of a single cell at nanoscale. The quantitative and precise cell–cell adhesion force result can be obtained by this method. It would help us to understand the single cell interaction with time and would benefit the research in medical and biological fields potentially.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

The elastic modulus of Matrigel as determined by atomic force microscopy

Recent studies indicate that the biophysical properties of the cellular microenvironment strongly influence a variety of fundamental cell behaviors. The extracellular matrix’s (ECM) response to mechanical force, described mathematically as the elastic modulus, is believed to play a particularly critical role in regulatory and pathological cell behaviors. The basement membrane (BM) is a specialization of the ECM that serves as the immediate interface for many cell types (e.g. all epithelial cells) and through which cells are connected to the underlying stroma. Matrigel is a commercially available BM-like complex and serves as an easily accessible experimental simulant of native BMs. However, the local elastic modulus of Matrigel has not been defined under physiological conditions. Here we present the procedures and results of indentation tests performed on Matrigel with atomic force microscopy (AFM) in an aqueous, temperature controlled environment. The average modulus value was found to be approximately 450 Pa. However, this result is considerably higher than macroscopic shear storage moduli reported in the scientific literature. The reason for this discrepancy is believed to result from differences in test methods and the tendency of Matrigel to soften at temperatures below 37° C.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Cell viability and probe-cell membrane interactions of XR1 glial cells imaged by atomic force microscopy.

As atomic force microscopy (AFM) imaging of live specimens becomes more commonplace, at least two important questions arise: 1) do live specimens remain viable during and after AFM, and 2) is there transfer of membrane components from the cell to the AFM probe during probe-membrane interactions? We imaged live XR1 glial cells in culture by single- or dual-pass contact or tapping-mode AFM, examined cell viability at various postimaging times, and report that AFM-imaged live XR1 cells remained viable up to 48 h postimaging and that cell death rates did not increase. To determine if nonlethal, transient interactions between the AFM probe and cell membrane led to transfer of XR1 cell membrane phospholipid components on the probe, we treated the scanned probes with the lipid-binding fluorophore FM 1-43. Confocal microscopy revealed that phospholipid membrane components did accumulate on the probe, and to a generally greater extent during contact-mode imaging than during tapping-mode imaging. Moreover, membrane accumulations on the probe were greater when live XR1 cells were damaged or perturbed, yet membrane did not accumulate in fluorescently detectable quantities during repeated "force curves" during control experiments. Taken together, our data indicate that although AFM imaging of live cells in culture does not affect long-term cell viability, there are substantial probe-membrane interactions that lead to transfer of membrane components to the probe.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

Involvement of a protein kinase C and protein phosphatases in adhesion of CD4+ T cells to and detachment from extracellular matrix proteins.

For immune surveillance and function to be effective, T lymphocytes constantly recirculate via lymph and blood between lymphoid organs and body tissues. To enable efficient cell movement and migration, cell adhesion to components of the basement membrane and the extracellular matrix (ECM) must be a rapid and transitory process. Whether phosphorylation and dephosphorylation of cellular proteins are involved in this phenomena was explored by monitoring the adhesion of T cells to immobilized ECM proteins. A short exposure of 51Cr-labeled human CD4+ T cells to phorbol esters in vitro induced a rapid beta 1-integrin-mediated adhesion to both fibronectin and laminin, as determined by inhibition with anti-integrin antibodies. Adhesion was reversible; detachment from the immobilized ECM ligands occurred between 20 and 120 min without further intervention. This T cell adhesion was regulated by the activation of protein kinase C because (a) staurosporine and H-7 inhibitors of protein kinase C suppressed T cell adhesion, and (b) PMA-induced down-regulation of intracellular levels of protein kinase C was associated with the abrogation of the T cell adhesiveness to fibronectin and laminin. Furthermore, inhibition of protein phosphatases activity by okadaic acid delayed the detachment of the T cells from fibronectin or laminin. Thus, we suggest that T cell-ECM interactions such as adhesion and detachment are regulated, respectively, by protein kinase C and protein phosphatases.     

Revealing non-genetic adhesive variations in clonal populations by comparative single-cell force spectroscopy

Cell populations often display heterogeneous behavior, including cell-to-cell variations in morphology, adhesion and spreading. However, better understanding the significance of such cell variations for the function of the population as a whole requires quantitative single-cell assays. To investigate adhesion variability in a CHO cell population in detail, we measured integrin-mediated adhesion to laminin and collagen, two ubiquitous ECM components, by AFM-based single-cell force spectroscopy (SCFS). CHO cells generally adhered more strongly to laminin than collagen but population adhesion force distributions to both ECM components were broad and partially overlapped. To determine the levels of laminin and collagen binding in individual cells directly, we alternatingly measured single cells on adjacent microstripes of collagen and laminin arrayed on the same adhesion substrate. In repeated measurements (≥60) individual cells showed a stable and ECM type-specific adhesion response. All tested cells bound laminin more strongly, but the scale of laminin over collagen binding varied between cells. Together, this demonstrates that adhesion levels to different ECM components are tightly yet differently set in each cell of the population. Adhesion variability to laminin was non-genetic and cell cycle-independent but scaled with the range of α6 integrin expression on the cell surface. Adhesive cell-to-cell variations due to varying receptor expression levels thus appear to be an inherent feature of cell populations and should to be considered when fully characterizing population adhesion. In this approach, SCFS performed on multifunctional adhesion substrates can provide quantitative single-cell information not obtainable from population-averaging measurements on homogeneous adhesion substrates.