Cells sense stiffness of surrounding tissues and adapt their activity, proliferation, motility and mechanical properties based on such interactions. Cells probe the stiffness of the substrate by anchoring and pulling to their surroundings, transmitting force to the extracellular matrix and other cells, and respond to the resistance they sense, mainly through changes in their cytoskeleton. Cancer and other diseases alter stiffness of tissues, and the response of cancer cells to this stiffness can also be affected. In the present study we show that MCF-7 breast cancer cells seeded on polyacrylamide gels have the ability to detect the stiffness of the substrate and alter their mechanical properties in response. MCF-7 cells plated on soft substrates display lower stiffness and viscosity when compared to those seeded on stiffer gels or glass. These differences can be associated with differences in the morphology and cytoskeleton organisation, since cells seeded on soft substrates have a round morphology, while cells seeded on stiffer substrates acquire a flat and spread morphology with formation of actin filaments, similar to that observed when seeded on glass. These findings show that MCF-7 cells can detect the stiffness of the surrounding microenvironment and thus, modify their mechanical properties.

Infection of humans by many viruses is typically initiated by the internalization of a single virion in each of a few susceptible cells. Thus, the outcome of the infection process may depend on stochastic single‐molecule events. A crucial process for viral infection, and thus a target for developing antiviral drugs, is the uncoating of the viral genome. Here a force spectroscopy procedure using an atomic force microscope is implemented to study uncoating for individual human rhinovirus particles. Application of an increasing mechanical force on a virion led to a high force‐induced structural transition that facilitated extrusion of the viral RNA molecule without loss of capsid integrity. Application of force to virions that h ad previously extruded the RNA, or to RNA‐free capsids, led to a lower force‐induced event associated with capsid disruption. The kinetic parameters are determined for each reaction. The high‐force event is a stochastic process governed by a moderate free energy barrier (≈20 kcal mol⁻¹), which results in a heterogeneous population of structurally weakened virions in which different fractions of the RNA molecule are externalized. The effects of antiviral compounds or capsid mutation on the kinetics of this reaction reveal a correlation between the reaction rate and virus infectivity.

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y₁ and P2Y₂ receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young’s modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y₂ receptor stimulation with UTP. Such exposure to UTP doubled the Young’s modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y₁ and P2Y₁₃ receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y₁₃ receptor, although they were negatively modulated by P2Y₁ receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

Biopolymers, such as polynucleotides, polypeptides and polysaccharides, are macromolecules that direct most of the functions in living beings. Studying the mechanical unfolding of biopolymers provides important information about their molecular elasticity and mechanical stability, as well as their energy landscape, which is especially important in proteins, since their three‐dimensional structure is essential for their correct activity. In this primer, we present how to study the mechanical properties of proteins with atomic force microscopy and how to obtain information about their stability and energetic landscape. In particular, we discuss the preparation of polyprotein constructs suitable for AFM single molecule force spectroscopy (SMFS), describe the parameters used in our force‐extension SMFS experiments and the models and equations employed in the analysis of the data. As a practical example, we show the effect of the temperature on the unfolding force, the distance to the transition state, the unfolding rate at zero force, the height of the transition state barrier, and the spring constant of the protein for a construct containing nine repeats of the I27 domain from the muscle protein titin.Highlights: 1. Atomic force microscopy (AFM) can be used to study the mechanical unfolding of polymers.2. AFM provides a direct measurement of unfolding (unbinding) forces.3. Force measurements for different rates provide information about the distance to the transition state and the unfolding rate at zero force.