Mutated lamin A modulates stiffness in muscle cells.

The cytoskeleton is a complex network interlinking filaments that extend throughout the cytoplasm from the nucleus to the plasma membrane. Three major types of filaments are found in the cytoskeleton: actin filaments, microtubules, and intermediate filaments. They play a key role in the ability of cells to both resist mechanical stress and generate force. However, the precise involvement of intermediate filament proteins in these processes remains unclear. Here, we focused on nuclear A-type lamins, which are connected to the cytoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Using micro-constriction rheology, we investigated the impact of A-type lamins (p.H222P) mutation on the mechanical properties of muscle cells. We demonstrate that the expression of point mutation of lamin A in muscle cells increases cellular stiffness compared with cells expressing wild type lamin A and that the chemical agent selumetinib, an inhibitor of the ERK1/2 signaling, reversed the mechanical alterations in mutated cells. These results highlight the interplay between A-type lamins and mechano-signaling, which are supported by cell biology measurements.

αvß3 integrin expression increases elasticity in human melanoma cells.

Living cells interact with the extracellular matrix (ECM) transducing biochemical signals into mechanical cues and vice versa. Thanks to this mechano-transduction process, cells modify their internal organization and upregulate their physiological functions differently. In this complex mechanism integrins play a fundamental role, connecting the extracellular matrix with the cytoskeleton. Cytoskeletal rearrangements, such as the increase of the overall contractility, impact cell mechanical properties, the entire cell stiffness, and cell deformability. How cell mechanics is influenced via different integrins and their interaction with ECM in health and disease is still unclear. Here, we investigated the influence of αvß3 integrin expression on the mechanics of human melanoma M21 cells using atomic force microscopy and micro-constriction. Evidence is provided that (i) αvß3 integrin expression in human melanoma cells increases cell stiffness in both adherent and non-adherent conditions; (ii) replacing αvß3 with αIIbß3 integrin in melanoma cells, cell stiffness is increased under adherent, while decreased under non-adherent conditions; (iii) αvß3 integrin cell stiffening is also maintained when cells adhere to fibronectin, but this phenomenon does not strongly depend on the fibronectin concentration. In all, this study sheds light on the role of αvß3 in regulating cellular mechanics.

Unbiased High-Precision Cell Mechanical Measurements with Microconstrictions.

We describe a quantitative, high-precision, high-throughput method for measuring the mechanical properties of cells in suspension with a microfluidic device, and for relating cell mechanical responses to protein expression levels. Using a high-speed (750 fps) charge-coupled device camera, we measure the driving pressure Δp, maximum cell deformation εmax, and entry time tentry of cells in an array of microconstrictions. From these measurements, we estimate population averages of elastic modulus E and fluidity ß (the power-law exponent of the cell deformation in response to a step change in pressure). We find that cell elasticity increases with increasing strain εmax according to E ∼ εmax, and with increasing pressure according to E ∼ Δp. Variable cell stress due to driving pressure fluctuations and variable cell strain due to cell size fluctuations therefore cause significant variability between measurements. To reduce measurement variability, we use a histogram matching method that selects and analyzes only those cells from different measurements that have experienced the same pressure and strain. With this method, we investigate the influence of measurement parameters on the resulting cell elastic modulus and fluidity. We find a small but significant softening of cells with increasing time after cell harvesting. Cells harvested from confluent cultures are softer compared to cells harvested from subconfluent cultures. Moreover, cell elastic modulus increases with decreasing concentration of the adhesion-reducing surfactant pluronic. Lastly, we simultaneously measure cell mechanics and fluorescence signals of cells that overexpress the GFP-tagged nuclear envelope protein lamin A. We find a dose-dependent increase in cell elastic modulus and decrease in cell fluidity with increasing lamin A levels. Together, our findings demonstrate that histogram matching of pressure, strain, and protein expression levels greatly reduces the variability between measurements and enables us to reproducibly detect small differences in cell mechanics.

Influence of αvß3 integrin on the mechanical properties and the morphology of M21 and K562 cells.

Integrins play an important role in cell adhesion, morphology, migration, and many other physiological processes. The role of αvß3 integrin has been intensively investigated in the past. However, much is still unclear about its selective role in cell contractility, adhesion, and mechanics. We looked at the influence of αvß3 integrin on the cell mechanics of adherent M21 and suspended K562 cells with a microconstriction assay and found that the expression of αvß3 integrin leads to higher cell stiffness and decreased fluidity in both cell lines. The disruption of the actin cytoskeleton decreased cellular stiffness in M21 (expressing α5ß1 and αvß3 integrins) and M21L (expressing only α5ß1 integrin) cell lines in a similar way, but did not lead to the same baseline stiffness. The activation of integrins after the addition of Mn(2+) led to higher stiffness in all observed cell lines, independent of αvß3 integrin expression and disruption of the actin cytoskeleton. In summary, these results show that differences in stiffness/fluidity due to αvß3 integrin expression or integrin activation by Mn(2+) might not simply be explained by the coupling of integrins to actin via focal adhesions, which in turn induces changes in the actin cytoskeleton, but also by other cellular components such as the cell nucleus, intermediate filaments, or microtubules.

Microconstriction arrays for high-throughput quantitative measurements of cell mechanical properties.

We describe a method for quantifying the mechanical properties of cells in suspension with a microfluidic device consisting of a parallel array of micron-sized constrictions. Using a high-speed charge-coupled device camera, we measure the flow speed, cell deformation, and entry time into the constrictions of several hundred cells per minute during their passage through the device. From the flow speed and the occupation state of the microconstriction array with cells, the driving pressure across each constriction is continuously computed. Cell entry times into microconstrictions decrease with increased driving pressure and decreased cell size according to a power law. From this power-law relationship, the cell elasticity and fluidity can be estimated. When cells are treated with drugs that depolymerize or stabilize the cytoskeleton or the nucleus, elasticity and fluidity data from all treatments collapse onto a master curve. Power-law rheology and collapse onto a master curve are predicted by the theory of soft glassy materials and have been previously shown to describe the mechanical behavior of cells adhering to a substrate. Our finding that this theory also applies to cells in suspension provides the foundation for a quantitative high-throughput measurement of cell mechanical properties with microfluidic devices.

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness, Nuclear Volume, Contractility, and Cell Stiffness.

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 µm length, 3.7 µm height, and a decreasing width from 11.2 to 1.7 µm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 µm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 µm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.

Cell and tissue mechanics in cell migration.

Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies.

Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solution.

Precise nanometre-scale imaging of soft structures at room temperature poses a major challenge to any type of microscopy because fast thermal fluctuations lead to significant motion blur if the position of the structure is measured with insufficient bandwidth. Moreover, precise localization is also affected by optical heterogeneities, which lead to deformations in the imaged local geometry, the severity depending on the sample and its thickness. Here we introduce quantitative thermal noise imaging, a three-dimensional scanning probe technique, as a method for imaging soft, optically heterogeneous and porous matter with submicroscopic spatial resolution in aqueous solution. By imaging both individual microtubules and collagen fibrils in a network, we demonstrate that structures can be localized with a precision of ∼10 nm and that their local dynamics can be quantified with 50 kHz bandwidth and subnanometre amplitudes. Furthermore, we show how image distortions caused by optically dense structures can be corrected for.