Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.     

Force probing cell shape changes to molecular resolution

Atomic force microscopy (AFM) is a force sensing nanoscopic tool that can be used to undertake a multiscale approach to understand the mechanisms that underlie cell shape change, ranging from the cellular to molecular scale. In this review paper, we discuss the use of AFM to characterize the dramatic shape changes of mitotic cells. AFM-based mechanical assays can be applied to measure the considerable rounding force and hydrostatic pressure generated by mitotic cells. A complementary AFM technique, single-molecule force spectroscopy, is able to quantify the interactions and mechanisms that functionally regulate individual proteins. Future developments of these nanomechanical methods, together with advances in light microscopy imaging and cell biological and genetic tools, should provide further insight into the biochemical, cellular and mechanical processes that govern mitosis and other cell shape change phenomena.     

Prestress and Adhesion Site Dynamics Control Cell Sensitivity to Extracellular Stiffness

This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.     

Unified multiscale theory of cellular mechanical adaptations to substrate stiffness

Rigidity of the extracellular matrix markedly regulates many cellular processes. However, how cells detect and respond to matrix rigidity remains incompletely understood. Here, we propose a unified two-dimensional multiscale framework accounting for the chemomechanical feedback to explore the interrelated cellular mechanosensing, polarization, and migration, which constitute the dynamic cascade in cellular response to matrix stiffness but are often modeled separately in previous theories. By combining integrin dynamics and intracellular force transduction, we show that substrate stiffness can act as a switch to activate or deactivate cell polarization. Our theory quantitatively reproduces rich stiffness-dependent cellular dynamics, including spreading, polarity selection, migration pattern, durotaxis, and even negative durotaxis, reported in a wide spectrum of cell types, and reconciles some inconsistent experimental observations. We find that a specific bipolarized mode can determine the optimal substrate stiffness, which enables the fastest cell migration rather than the largest traction forces that cells apply on the substrate. We identify that such a mechanical adaptation stems from the force balance across the whole cell. These findings could yield universal insights into various stiffness-mediated cellular processes within the context of tissue morphogenesis, wound healing, and cancer invasion.     

In situ measurement of cell stiffness of Arabidopsis roots growing on a glass micropillar support by atomic force microscopy

Atomic force microscopy (AFM) can measure the mechanical properties of plant tissue at the cellular level, but for in situ observations, the sample must be held in place on a rigid support and it is difficult to obtain accurate data for living plants without inhibiting their growth. To investigate the dynamics of root cell stiffness during seedling growth, we circumvented these problems by using an array of glass micropillars as a support to hold an Arabidopsis thaliana root for AFM measurements without inhibiting root growth. The root elongated in the gaps between the pillars and was supported by the pillars. The AFM cantilever could contact the root for repeated measurements over the course of root growth. The elasticity of the root epidermal cells was used as an index of the stiffness. By contrast, we were not able to reliably observe roots on a smooth glass substrate because it was difficult to retain contact between the root and the cantilever without the support of the pillars. Using adhesive to fix the root on the smooth glass plane overcame this issue, but prevented root growth. The glass micropillar support allowed reproducible measurement of the spatial and temporal changes in root cell elasticity, making it possible to perform detailed AFM observations of the dynamics of root cell stiffness.     

A hybrid computational model for collective cell durotaxis

Collective cell migration is regulated by a complex set of mechanical interactions and cellular mechanisms. Collective migration emerges from mechanisms occurring at single cell level, involving processes like contraction, polymerization and depolymerization, of cell–cell interactions and of cell–substrate adhesion. Here, we present a computational framework which simulates the dynamics of this emergent behavior conditioned by substrates with stiffness gradients. The computational model reproduces the cell’s ability to move toward the stiffer part of the substrate, process known as durotaxis. It combines the continuous formulation of truss elements and a particle-based approach to simulate the dynamics of cell–matrix adhesions and cell–cell interactions. Using this hybrid approach, researchers can quickly create a quantitative model to understand the regulatory role of different mechanical conditions on the dynamics of collective cell migration. Our model shows that durotaxis occurs due to the ability of cells to deform the substrate more in the part of lower stiffness than in the stiffer part. This effect explains why cell collective movement is more effective than single cell movement in stiffness gradient conditions. In addition, we numerically evaluate how gradient stiffness properties, cell monolayer size and force transmission between cells and extracellular matrix are crucial in regulating durotaxis.     

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.     

An AFM-based stiffness clamp for dynamic control of rigidity

Atomic force microscopy (AFM) has become a powerful tool for measuring material properties in biology and imposing mechanical boundary conditions on samples from single molecules to cells and tissues. Constant force or constant height can be maintained in an AFM experiment through feedback control of cantilever deflection, known respectively as a ‘force clamp’ or ‘position clamp’. However, stiffness, the third variable in the Hookean relation F = kx that describes AFM cantilever deflection, has not been dynamically controllable in the same way. Here we present and demonstrate a ‘stiffness clamp’ that can vary the apparent stiffness of an AFM cantilever. This method, employable on any AFM system by modifying feedback control of the cantilever, allows rapid and reversible tuning of the stiffness exposed to the sample in a way that can decouple the role of stiffness from force and deformation. We demonstrated the AFM stiffness clamp on two different samples: a contracting fibroblast cell and an expanding polyacrylamide hydrogel. We found that the fibroblast, a cell type that secretes and organizes the extracellular matrix, exhibited a rapid, sub-second change in traction rate (dF/dt) and contraction velocity (dx/dt) in response to step changes in stiffness between 1–100 nN/µm. This response was independent of the absolute contractile force and cell height, demonstrating that cells can react directly to changes in stiffness alone. In contrast, the hydrogel used in our experiment maintained a constant expansion velocity (dx/dt) over this range of stiffness, while the traction rate (dF/dt) changed with stiffness, showing that passive materials can also behave differently in different stiffness environments. The AFM stiffness clamp presented here, which is applicable to mechanical measurements on both biological and non-biological samples, may be used to investigate cellular mechanotransduction under a wide range of controlled mechanical boundary conditions.     

Exogenous and endogenous force regulation of endothelial cell behavior

Endothelial cells live in a dynamic environment where they are constantly exposed to external hemodynamic forces and generate cytoskeletal-based endogenous forces. These exogenous and endogenous forces are critical regulators of endothelial cell health and blood vessel maintenance at all generations of the vascular system, from large arteries to capillary beds. The first part of this review highlights the role of the primary exogenous hemodynamic forces of shear, cyclic strain, and pressure forces in mediating endothelial cell response. We then discuss the emergent role of the mechanical properties of the extracellular matrix and of cellular endogenous force generation on endothelial cell function, implicating substrate stiffness and cellular traction stresses as important mediators of endothelial cell health. The intersection of exogenous and endogenous forces on endothelial cell function is discussed, suggesting some of the many remaining questions in the field of endothelial mechanobiology.     

Cellular Pressure and Volume Regulation and Implications for Cell Mechanics

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death, and migration. Volume and shape regulation also directly impacts the mechanics of cells and tissues. Here, we develop a mathematical model of cellular volume and pressure regulation, incorporating essential elements such as water permeation, mechanosensitive channels, active ion pumps, and active stresses in the cortex. The model can fully explain recent experimental data, and it predicts cellular volume and pressure for several models of cell cortical mechanics. Moreover, we show that when cells are subjected to an externally applied load, such as in an atomic force microscopy indentation experiment, active regulation of volume and pressure leads to a complex cellular response. Instead of the passive mechanics of the cortex, the observed cell stiffness depends on several factors working together. This provides a mathematical explanation of rate-dependent response of cells under force.     

Cellular Pressure and Volume Regulation and Implications for Cell Mechanics

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death, and migration. Volume and shape regulation also directly impacts the mechanics of cells and tissues. Here, we develop a mathematical model of cellular volume and pressure regulation, incorporating essential elements such as water permeation, mechanosensitive channels, active ion pumps, and active stresses in the cortex. The model can fully explain recent experimental data, and it predicts cellular volume and pressure for several models of cell cortical mechanics. Moreover, we show that when cells are subjected to an externally applied load, such as in an atomic force microscopy indentation experiment, active regulation of volume and pressure leads to a complex cellular response. Instead of the passive mechanics of the cortex, the observed cell stiffness depends on several factors working together. This provides a mathematical explanation of rate-dependent response of cells under force.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Eigenstrain as a mechanical set-point of cells

Cell contraction regulates how cells sense their mechanical environment. We sought to identify the set-point of cell contraction, also referred to as tensional homeostasis. In this work, bovine aortic endothelial cells (BAECs), cultured on substrates with different stiffness, were characterized using traction force microscopy (TFM). Numerical models were developed to provide insights into the mechanics of cell–substrate interactions. Cell contraction was modeled as eigenstrain which could induce isometric cell contraction without external forces. The predicted traction stresses matched well with TFM measurements. Furthermore, our numerical model provided cell stress and displacement maps for inspecting the fundamental regulating mechanism of cell mechanosensing. We showed that cell spread area, traction force on a substrate, as well as the average stress of a cell were increased in response to a stiffer substrate. However, the cell average strain, which is cell type-specific, was kept at the same level regardless of the substrate stiffness. This indicated that the cell average strain is the tensional homeostasis that each type of cell tries to maintain. Furthermore, cell contraction in terms of eigenstrain was found to be the same for both BAECs and fibroblast cells in different mechanical environments. This implied a potential mechanical set-point across different cell types. Our results suggest that additional measurements of contractility might be useful for monitoring cell mechanosensing as well as dynamic remodeling of the extracellular matrix (ECM). This work could help to advance the understanding of the cell-ECM relationship, leading to better regenerative strategies.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

Architecture of tissue cells the structural basis which determines shape and locomotion of cells

Shape and locomotion of tissue cells depend on the interaction of elements of the cytoskeleton, adhesion to the substrate and an intracellular hydrostatic pressure. The existence of this pressure becomes obvious from increase in cell volume on cessation of contractile forces and from observations with ultrasound acoustic microscopy. Wherever such an internal pressure is established, it is involved in generation of shape and driving force of cell locomotion. Therefore each hypothesis on cell shape and locomotion must consider this property of a living cell. Apparently different types of locomotion depend on differences in substrate adhesion and/or cytoskeleton organisation.     

Controlling Cellular Volume via Mechanical and Physical Properties of Substrate

The mechanical and physical properties of substrate play a crucial role in regulating many cell functions and behaviors. However, how these properties affect cell volume is still unclear. Here, we show that an increase in substrate stiffness, available spread area, or effective adhesion energy density results in a remarkable cell volume decrease (up to 50%), and the dynamic cell spreading process is also accompanied by dramatic cell volume decrease. Further, studies of ion channel inhibition and osmotic shock suggest that these volume decreases are due to the efflux of water and ions. We also show that disrupting cortex contractility leads to bigger cell volume. Collectively, these results reveal the “mechanism of adhesion-induced compression of cells,” i.e., stronger interaction between cell and substrate leads to higher actomyosin contractility, expels water and ions, and thus decreases cell volume.     

Probing Cellular Mechanoadaptation Using Cell-Substrate De-Adhesion Dynamics: Experiments and Model

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.