From mechanical stimulation to biological pathways in the regulation of stem cell fate

Mechanical stimuli are important in directing the fate of stem cells; the effects of mechanical stimuli reported in recent research are reviewed here. Stem cells normally undergo two fundamental processes: proliferation, in which their numbers multiply, and differentiation, in which they transform into the specialized cells needed by the adult organism. Mechanical stimuli are well known to affect both processes of proliferation and differentiation, although the complete pathways relating specific mechanical stimuli to stem cell fate remain to be elucidated. We identified two broad classes of research findings and organized them according to the type of mechanical stress (compressive, tensile or shear) of the stimulus. Firstly, mechanical stress of any type activates stretch‐activated channels (SACs) on the cell membrane. Activation of SACs leads to cytoskeletal remodelling and to the expression of genes that regulate the basic growth, survival or apoptosis of the cells and thus regulates proliferation. Secondly, mechanical stress on cells that are physically attached to an extracellular matrix (ECM) initiates remodelling of cell membrane structures called integrins. This second process is highly dependent on the type of mechanical stress applied and result into various biological responses. A further process, the Wnt pathway, is also implicated: crosstalk between the integrin and Wnt pathways regulates the switch from proliferation to differentiation and finally regulates the type of differentiation. Therefore, the stem cell differentiation process involves different signalling molecules and their pathways and most likely depends upon the applied mechanical stimulation. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of hydroperoxides on red blood cell membrane mechanical properties

We investigate the effect of oxidative stress on red blood cell membrane mechanical properties in vitro using detailed analysis of the membrane thermal fluctuation spectrum. Two different oxidants, the cytosol-soluble hydrogen peroxide and the membrane-soluble cumene hydroperoxide, are used, and their effects on the membrane bending elastic modulus, surface tension, strength of confinement due to the membrane skeleton, and 2D shear elastic modulus are measured. We find that both oxidants alter significantly the membrane elastic properties, but their effects differ qualitatively and quantitatively. While hydrogen peroxide mainly affects the elasticity of the membrane protein skeleton (increasing the membrane shear modulus), cumene hydroperoxide has an impact on both membrane skeleton and lipid bilayer mechanical properties, as can be seen from the increased values of the shear and bending elastic moduli. The biologically important implication of these results is that the effects of oxidative stress on the biophysical properties, and hence the physiological functions, of the cell membrane depend on the nature of the oxidative agent. Thermal fluctuation spectroscopy provides a means of characterizing these different effects, potentially in a clinical milieu.     

Effect of stress on the membrane capacitance of the auditory outer hair cell.

The membrane capacitance of the outer hair cell, which has unique membrane potential-dependent motility, was monitored during application of membrane tension. It was found that the membrane capacitance of the cell decreased when stress was applied to the membrane. This result is the opposite of stretching the lipid bilayer in the plasma membrane. It thus indicates the importance of some other capacitance component that decreases on stretching. It has been known that charge movement across the membrane can appear to be a nonlinear capacitance. If membrane stress at the resting potential restricts the movement of the charge associated with force generation, the nonlinear capacitance will decrease. Furthermore, less capacitance reduction by membrane stretching is expected when the membrane is already extended by the (hyperpolarizing) membrane potential. Indeed, it was found that at hyperpolarized potentials, the reduction of the membrane capacitance due to stretching is less. The capacitance change can be described by a two state model of a force-producing unit in which the free energy difference between the contracted and stretched states has both electrical and mechanical components. From the measured change in capacitance, the estimated difference in the membrane area of the unit between the two states is about 2 nm2.     

Viscoelastic relaxation in the membrane of the auditory outer hair cell.

The outer hair cell (OHC) in the mammalian ear has a unique membrane potential-dependent motility, which is considered to be important for frequency discrimination (tuning). The OHC motile mechanism is located at the cell membrane and is strongly influenced by its passive mechanical properties. To study the viscoelastic properties of OHCs, we exposed cells to a hypoosmotic solution for varying durations and then punctured them, to immediately release the osmotic stress. Using video records of the cells, we determined both the imposed strain and the strain after puncturing, when stress was reset to zero. The strain data were described by a simple rheological model consisting of two springs and a dashpot, and the fit to this model gave a time constant of 40 +/- 19 s for the relaxation (reduction) of tension during prolonged strain. For time scales much shorter or longer than this, we would expect essentially elastic behavior. This relaxation process affects the membrane tension of the cell, and because it has been shown that membrane tension has a modulatory role in the OHC's motility, this relaxation process could be part of an adaptation mechanism, with which the motility system of the OHC can adjust to changing conditions and maintain optimum membrane tension.     

A physical basis for red cell membrane elasticity

An estimate is made of the effect of lipid-water interactions at the membrane surface on observed membrane elasticity in the red blood cell. It is shown that elastic effects are expected to result from these interactions even in a completely fluid membrane. A simple statistical model is described and used to estimate the magnitude of the resultant elasticity. The estimate thus derived is compared with results of experiments during which a biaxial stress is applied to the membrane. The derived elasticity is sufficient to account for the results of these studies. Other properties of the red cell membrane are discussed in the light of this result.     

Effects of cell swelling on intracellular calcium and membrane currents in bovine articular chondrocytes

Chondrocytes experience a dynamic extracellular osmotic environment during normal joint loading when fluid is forced from the matrix, increasing the local proteoglycan concentration and therefore the ionic strength and osmolarity. To exist in such a challenging environment, chondrocytes must possess mechanisms by which cell volume can be regulated. In this study, we investigated the ability of bovine articular chondrocytes (BAC) to regulate cell volume during a hypo-osmotic challenge. We also examined the effect of hypo-osmotic stress on early signaling events including [Ca2+](i) and membrane currents. Changes in cell volume were measured by monitoring the fluorescence of calcein-loaded cells. [Ca2+](i) was quantified using fura-2, and membrane currents were recorded using patch clamp. BAC exhibited regulated volume decrease (RVD) when exposed to hypo-osmotic saline which was inhibited by Gd3+. Swelling stimulated [Ca2+](i) transients in BAC which were dependent on swelling magnitude. Gd3+, zero [Ca2+](o), and thapsigargin all attenuated the [Ca2+](i) response, suggesting roles for Ca2+ influx through stretch activated channels, and Ca2+ release from intracellular stores. Inward and outward membrane currents significantly increased during cell swelling and were inhibited by Gd3+. These results indicate that RVD in BAC may involve [Ca2+](i) and ion channel activation, both of which play pivotal roles in RVD in other cell types. These signaling pathways are also similar to those activated in chondrocytes subjected to other biophysical signals. It is possible, then, that these signaling events may also be involved in a mechanism by which mechanical loads are transduced into appropriate cellular responses by chondrocytes.     

Cells respond to mechanical stress by rapid disassembly of caveolae

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.     

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways

To investigate the mechanism of theregulation of human red blood cell deformability, we examined thedeformability under mechanical stress. Washed human red blood cellswere rapidly injected through a fine needle, and their filterabilitywas measured using a nickel mesh filter. The decrease in filterabilityshowed a V-shaped curve depending on the extracellularCa²⁺ concentration; the maximumdecrease was achieved at ~50 µM. The decreased filterability wasaccompanied by no change in cell morphology and cell volume, indicatingthat the decrease in filterability can be ascribed to alterations ofthe membrane properties. Ca²⁺entry blockers (nifedipine and felodipine) inhibited the impairment offilterability under mechanical stress. ProstaglandinsE₁ and E₂, epinephrine, andpentoxifylline, which are thought to modulate the intracellularadenosine 3',5'-cyclic monophosphate (cAMP) level of redblood cells, improved or worsened the impaired filterability accordingto their expected actions on the cAMP level of the cells. These resultsstrongly suggest that the membrane properties regulating red blood celldeformability are affected by the signal transduction system, includingCa²⁺-dependent and cAMP-mediatedsignaling pathways.

     

Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses

In order to develop successful cryopreservation protocols a better understanding of the freeze- and dehydration-induced changes occurring in the cell membrane and its underlying support, the actin cytoskeleton, is required. In this study, we compared the biophysical response of model mammalian cells (human foreskin fibroblasts) to hyperosmotic stress and freeze/thaw. Transmitted light, infrared spectroscopy, fluorescence- and cryo-microscopy were used to investigate the changes in the cell membrane and the actin cytoskeleton. We observed that a purely hyperosmotic challenge at room temperature resulted in bleb formation. A decrease in temperature abrogated the blebbing behavior, but was accompanied by a decrease in viability. These results suggested that cell survival depended on the availability of the membrane material to accommodate the volumetric expansion back to the original cell volume at isotonic conditions. Our data also showed that freeze/thaw stresses altered the cell membrane morphology resulting in a loss of membrane material. There was also a significantly lower incidence of blebbing after freeze/thaw as compared to isothermal osmotic stress experiments at room temperature. Significant depolymerization of the actin cytoskeleton was seen in cells whose membranes had been compromised by freeze/thaw stresses. Actin depolymerization using cytochalasin D affected the stability of the membrane against mechanical stress at isothermal conditions. This study shows that both the membrane and cytoskeleton, as a system, are involved in the osmotic and freeze/thaw-induced responses of the mammalian cells.     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

Osmomechanical regulation of membrane trafficking in polarized cells

The regulation of membrane trafficking is thought to be predominantly under the control of agonist-receptor transduction pathways. In the present study, osmomechanical stress due to swelling, a condition often accompanying cell activation, was shown to induce multiple membrane trafficking pathways in polarized absorptive epithelial cells in the absence of agonists. Osmomechanical stress activated rapidly (seconds) pathways of calcium-dependent membrane insertion into the basolateral domain, pathways of calcium-independent membrane retrieval from the basolateral domain, and a novel pathway of transcytosis (transcellular) between basolateral and apical cell domains. These pathways appear to underlie the transfer and regulation of transport proteins amongst cell compartments. This broad affect of osmomechanical stress on trafficking pathways may reflect a global mechanism for redistribution of transport proteins and other membrane components amongst cell compartments during states of mechanical stress.     

Effect of lanthanides on red blood cell deformability and response to mechanical stress: role of lanthanide ionic radius

Prior studies exploring the effects of lanthanides (Ln) on red blood cells (RBC) have primarily focused on ion transport, cell fusion, and membrane protein structure. Our previous report [Biorheology 44 (2007), 361-373] dealt only with lanthanum (La) and cell rigidity; the present study extends these observations to other lanthanides (Nd, Sm, Eu, Dy, Er) and to RBC response to mechanical shear. Deformation-shear stress behavior of normal human RBC was measured at Ln concentrations up to 200 μM. In another series of experiments, RBC were exposed to mechanical stress (190 Pa, 300 s) at 50 μM Ln and deformation-stress data obtained prior to and after this stress. Data were fitted to a Lineweaver-Burke model to obtain the shear stress at one-half maximum deformation (SS1/2). Our results include: (1) lanthanides cause decreased cell deformability with the magnitude of the decrease dependent on concentration and shear stress; (2) this decrease of deformability is affected by Ln ionic radius such that La>Nd>Sm>Eu>Dy>Er and is reversible for cells in Ln-free media; (3) mechanical stress decreases deformability (i.e., increases SS1/2) such that compared to control, La and Sm reduce and Dy and Er enhance the mechanical stress effect; (4) the decrease of deformability consequent to mechanical stress scales inversely with Ln ionic radius. These results indicate a reciprocal relation between cell rigidity and sensitivity to mechanical stress that is mediated by Ln ionic radius. Additional studies are clearly warranted, particularly those that explore membrane-glycocalyx and intracellular mechanisms.     

An elasto-visco-plastic model of cell aggregates

Concentrated cell suspensions exhibit different mechanical behavior depending on the mechanical stress or deformation they undergo. They have a mixed rheological nature: cells behave elastically or viscoelastically, they can adhere to each other whereas the carrying fluid is usually Newtonian. We report here on a new elasto-visco-plastic model which is able to describe the mechanical properties of a concentrated cell suspension or aggregate. It is based on the idea that the rearrangement of adhesion bonds during the deformation of the aggregate is related to the existence of a yield stress in the macroscopic constitutive equation. We compare the predictions of this new model with five experimental tests: steady shear rate, oscillatory shearing tests, stress relaxation, elastic recovery after steady prescribed deformation, and uniaxial compression tests. All of the predictions of the model are shown to agree with these experiments.     

Biorheological views of endothelial cell responses to mechanical stimuli

Vascular endothelial cells are located at the innermost layer of the blood vessel wall and are always exposed to three different mechanical forces: shear stress due to blood flow, hydrostatic pressure due to blood pressure and cyclic stretch due to vessel deformation. It is well known that endothelial cells respond to these mechanical forces and change their shapes, cytoskeletal structures and functions. In this review, we would like to mainly focus on the effects of shear stress and hydrostatic pressure on endothelial cell morphology. After applying fluid shear stress, cultured endothelial cells show marked elongation and orientation in the flow direction. In addition, thick stress fibers of actin filaments appear and align along the cell long axis. Thus, endothelial cell morphology is closely related to the cytoskeletal structure. Further, the dynamic course of the morphological changes is shown and the related events such as changes in mechanical stiffness and functions are also summarized. When endothelial cells were exposed to hydrostatic pressure, they exhibited a marked elongation and orientation in a random direction, together with development of centrally located, thick stress fibers. Pressured endothelial cells also exhibited a multilayered structure with less expression of VE-cadherin unlike under control conditions. Simultaneous loading of hydrostatic pressure and shear stress inhibited endothelial cell multilayering and induced elongation and orientation of endothelial cells with well-developed VE-cadherin in a monolayer, which suggests that for a better understanding of vascular endothelial cell responses one has to take into consideration the combination of the different mechanical forces such as exist under in vivo mechanical conditions.     

Viscoelastic properties of the cell nucleus

Mechanical factors play an important role in the regulation of cell physiology. One pathway by which mechanical stress may influence gene expression is through a direct physical connection from the extracellular matrix across the plasma membrane and to the nucleus. However, little is known of the mechanical properties or deformation behavior of the nucleus. The goal of this study was to quantify the viscoelastic properties of mechanically and chemically isolated nuclei of articular chondrocytes using micropipet aspiration in conjunction theoretical viscoelastic model. Isolated nuclei behaved as viscoelastic solid materials similar to the cytoplasm, but were 3-4 times stiffer and nearly twice as viscous as the cytoplasm. Quantitative information of the biophysical properties and deformation behavior of the nucleus may provide further insight on the relationships between the stress-strain state of the nucleus and that of the extracellular matrix, as well as potential mechanisms of mechanical signal transduction.     

Spatial patterning of cell proliferation and differentiation depends on mechanical stress magnitude

Mechanical stress has been proposed as a major regulator of tissue morphogenesis; however, it remains unclear what is the exact mechanical signal that leads to local tissue pattern formation. We explored this question by using a micropatterned cell aggregate model in which NIH 3T3 fibroblasts were cultured on micropatterned adhesive islands and formed cell aggregates (or “cell islands”) of triangular, square, and circular shapes. We found that the cell islands generated high levels of mechanical stresses at their perimeters compared to their inner regions. Regardless of the shape of cell islands, the mechanical stress patterns corresponded to both cell proliferation and differentiation patterns, meaning that high level of cell proliferation and differentiation occurred at the locations where mechanical stresses were also high. When mechanical stretching was applied to cell islands to elevate overall mechanical stress magnitudes, cell proliferation and differentiation generally increased with the relatively higher mechanical stresses, but neither cell proliferation nor differentiation patterns followed the new mechanical stress pattern. Thus, our findings indicate that a certain range of mechanical stress magnitudes, termed window stress threshold, drives formation of cell proliferation and differentiation patterns and hence possibly functions as a morphogenetic cue for local tissue pattern formation in vivo.     

A three-dimensional viscoelastic model for cell deformation with experimental verification

A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry. The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate. Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry. Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work. The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing. Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending <10 microm from the bead. Modulation of cell height has little effect on the results, provided the monolayer is >5 micro m thick. NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.     

A two-state piezoelectric model for outer hair cell motility

Recent studies have revealed that voltage-dependent length changes of the outer hair cell are based on charge transfer across the membrane. Such a motility can be explained by an "area motor" model, which assumes two states in the motor and that conformational transitions involve transfer of motor charge across the membrane and mechanical displacements of the membrane. Here it is shown that the area motor is piezoelectric and that the hair cell that incorporates such a motor in its lateral membrane is also piezoelectric. Distinctive features of the outer hair cell are its exceptionally large piezoelectric coefficient, which exceeds the best known piezoelectric material by four orders of magnitude, and its prominent nonlinearity due to the discreteness of motor states.