Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Influence of cantilever tip geometry and contact model on AFM elasticity measurement of cells.

We have measured the elastic properties of live cells by Atomic Force Microscope (AFM) using different tip geometries commonly used in AFM studies. Soft 4-sided pyramidal probes (spring constant = 12 and 30 mN/m, radius 20 nm), 3-sided pyramidal probes (spring constant = 100 mN/m, radius 65-75 nm), flat (circular) probes (spring constant = 63 mN/m, radius 290 nm) and spherical probes (spring constant = 43 mN/m, radius 5 µm) have been used. Cells (3T3 fibroblasts) having elastic moduli around 0.5 kPa were investigated. We found that cell measured stiffness shows a systematic dependence on tip geometry: the sharper the tip, the higher the average modulus values. We hypothesize that the blunter the tip, the larger the contact area over which the mechanical response is measured or averaged. If there are small-scale stiffer areas (like actin bundles) they will be easier to pick up by a sharp probe. This effect can be seen in the wider distribution of the histograms of the measured elastic moduli on cells. Furthermore, non-linear responses of cells may be present due to the high average pressures applied by sharp probes, which would lead to an overestimation of the Young's modulus. Pressure versus contact radius simulations for the different tip geometries for a 0.5 kPa sample suggested similar average pressure for Bio-MLCTs, PFQNM and cut tips, except spherical tips that showed much lower average pressure at the same 400 nm indentation. However, real data of the cells suggested different results. Using the same indentation depth (400 nm), PFQNM and Bio-MLCTs showed similar average pressure and it decreased for cut and spherical tips. The calculated contact area at 400 nm cell indentation, using the obtained apparent Young's modulus for each tip geometry, showed the following distribution: Bio-MLCTs < PFQNM < cut << spherical. In summary, tip geometry as well as average pressure and tip-sample contact area are important parameters to take into account when measuring mechanical properties of soft samples. The larger the tip radius, the larger the contact area that will lead to a more evenly distribution of the applied pressure.

Comparison of Rheological Properties of Healthy versus Dupuytren Fibroblasts When Treated with a Cell Contraction Inhibitor by Atomic Force Microscope.

Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.

Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP.

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force-distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell-Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.