Intracellular stress transmission through actin stress fiber network in adherent vascular cells

Intracellular stress transmission through subcellular structural components has been proposed to affect activation of localized mechano-sensing sites such as focal adhesions in adherent cells. Previous studies reported that physiological extracellular forces produced heterogeneous spatial distributions of cytoplasmic strain. However, mechanical signaling pathway involved in intracellular force transmission through basal actin stress fibers (SFs), a mechano-responsive cytoskeletal structure, remains elusive. In the present study, we investigated force balance within the basal SFs of cultured smooth muscle cells and endothelial cells by (i) removing the cell membrane and cytoplasmic constituents except for materials physically attaching to the substrate (i.e., SF-focal adhesion complexities) or (ii) dislodging either mechanically or chemically the cell processes of the cells expressing fluorescent proteins-labeled actin and focal adhesions in order, to examine stress-release-induced deformation of the basal SFs. The result showed that a removal of mechanical restrictions for SFs resulted in a decrease in the length of the remaining SFs, which means SFs bear tension. In addition, a release of the preexisting tension in a single SF was transmitted to another SF physically linked to the former, but not transmitted to the other ones physically independent of the former, suggesting that the prestress is balanced in tensed SF networks. These results support a hypothesis regarding cell structural architecture that physiological extracellular forces can produce in the basal SF network a directional intracellular stress or strain distribution. Therefore, consideration of the coexistence of the directional stretching strain along the axial direction of SFs and the heterogeneous strain in the other cytoplasmic region will be essential for understanding intracellular stress transmission in the adherent cells.     

Movement of stress fibers away from focal adhesions identifies focal adhesions as sites of stress fiber assembly in stationary cells

Force generated in contractile actin filament bundles (stress fibers-SFs) is transmitted to the extracellular matrix (ECM) via linker proteins and transmembrane integrins at focal adhesions (FAs). Though it has long been known that actin is rapidly exchanged in FAs, the connection between SFs and FAs has not been studied in detail. We introduced fiduciary marks on SFs by expressing GFP-palladin or GFP-alpha-actinin-1, which are both FA and dense body proteins, and by pattern bleaching of GFP-actin. Following fiduciary marks on SFs over time by time-lapse fluorescence microscopy, we detected assembly of SFs at FAs in stationary cells resulting in movement of SFs away from FAs with a velocity of 0.2-0.4 microm/min. Visualization of FAs in GFP-palladin/DsRed-paxillin double transfected cells showed that SF elongation was not accompanied by a change in FA length. SF elongation at FAs depended on actin polymerization and force as demonstrated by inhibitors of actin polymerization (cytochalasin D, jasplakinolide) and inhibitors of myosin-dependent contraction (blebbistatin, Y-27632), respectively. Our finding of SF assembly at FAs has important implications for SF formation, force transmission, and tension distribution within the actin cytoskeletal network of stationary cells.     

Buckling of actin stress fibers: a new wrinkle in the cytoskeletal tapestry

Intracellular tension is considered an important determinant of cytoskeletal architecture and cell function. However, many details about cytoskeletal tension remain poorly understood because these forces cannot be directly measured in living cells. Therefore, we have developed a method to characterize the magnitude and distribution of pre-extension of actin stress fibers (SFs) due to resting tension in the cytoskeleton. Using a custom apparatus, human aortic endothelial cells (HAECs) were cultured on a pre-stretched silicone substrate coated with a fibronectin-like polymer. Release of the substrate caused SFs aligned in the shortening direction in adhered cells to buckle when compressed rapidly (5% shortening per second or greater) beyond their unloaded slack length. Subsequently, the actin cytoskeleton completely disassembled in 5 sec and reassembled within 60 sec. Quantification of buckling in digital fluorescent micrographs of cells fixed and stained with rhodamine phalloidin indicated a nonuniform distribution of 0-26% pre-extension of SFs in non-locomoting HAECs. Local variability suggests heterogeneity of cytoskeletal tension and/or stiffness within individual cells. These findings provide new information about the magnitude and distribution of cytoskeletal tension and the dynamics of actin stress fibers, and the approach offers a novel method to elucidate the role of specific cytoskeletal elements and crosslinking proteins in the force generating apparatus of non-muscle cells.     

Strain waveform dependence of stress fiber reorientation in cyclically stretched osteoblastic cells: effects of viscoelastic compression of stress fibers

Actin stress fibers (SFs) of cells cultured on cyclically stretched substrate tend to reorient in the direction in which a normal strain of substrate becomes zero. However, little is known about the mechanism of this reorientation. Here we investigated the effects of cyclic stretch waveform on SF reorientation in osteoblastic cells. Cells adhering to silicone membranes were subjected to cyclic uniaxial stretch, having one of the following waveforms with an amplitude of 8% for 24 h: triangular, trapezoid, bottom hold, or peak hold. SF reorientation of these cells was then analyzed. No preferential orientation was observed for the triangular and the peak-hold waveforms, whereas SFs aligned mostly in the direction with zero normal strain (~55°) with other waveforms, especially the trapezoid waveform, which had a hold time both at loaded and unloaded states. Viscoelastic properties of SFs were estimated in a quasi-in situ stress relaxation test using intact and SF-disrupted cells that maintained their shape on the substrate. The dynamics of tension F(SFs) acting on SFs during cyclic stretching were simulated using these properties. The simulation demonstrated that F(SFs) decreased gradually during cyclic stretching and exhibited a compressive value (F(SFs) < 0). The magnitude and duration time of the compressive forces were relatively larger in the group with a trapezoid waveform. The frequency of SF orientation had a significant negative correlation with the applied compressive forces integrated with time in a strain cycle, and the integrated value was largest with the trapezoid waveform. These results may indicate that the applied compressive forces on SFs have a significant effect on the stretch-induced reorientation of SFs, and that SFs realigned to avoid their compression. Stress relaxation of SFs might be facilitated during the holding period in the trapezoid waveform, and depolymerization and reorientation of SFs were significantly accelerated by their viscoelastic compression.     

Estimation of the mechanical connection between apical stress fibers and the nucleus in vascular smooth muscle cells cultured on a substrate

Actin stress fibers (SFs) generate intercellular tension and play important roles in cellular mechanotransduction processes and the regulation of various cellular functions. We recently found, in vascular smooth muscle cells (SMCs) cultured on a substrate, that the apical SFs running across the top surface of the nucleus have a mechanical connection with the cell nucleus and that their internal tension is transmitted directly to the nucleus. However, the effects of the connecting conditions and binding forces between SFs and the nucleus on force transmission processes are unclear at this stage. Here, we estimated the mechanical connection between apical SFs and the nucleus in SMCs, taking into account differences in the contractility of individual SFs, using experimental and numerical approaches. First, we classified apical SFs in SMCs according to their morphological characteristics: one subset appeared pressed onto the apical surface of the nucleus (pressed SFs), and the other appeared to be smoothly attached to the nuclear surface (attached SFs). We then dissected these SFs by laser irradiation to release the pretension, observed the dynamic behavior of the dissected SFs and the nucleus, and estimated the pretension of the SFs and the connection strength between the SFs and the nucleus by using a simple viscoelastic model. We found that pressed SFs generated greater contractile force and were more firmly connected to the nuclear surface than were attached SFs. We also observed line-like concentration of the nuclear membrane protein nesprin 1 and perinuclear DNA that was significantly located along the pressed SFs. These results indicate that the internal tension of pressed SFs is transmitted to the nucleus more efficiently than that of attached SFs, and that pressed SFs have significant roles in the regulation of the nuclear morphology and rearrangement of intranuclear DNA.     

Image Analysis for the Quantitative Comparison of Stress Fibers and Focal Adhesions

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.     

Mechanical properties of actin stress fibers in living cells

Actin stress fibers (SFs) play an important role in many cellular functions, including morphological stability, adhesion, and motility. Because of their central role in force transmission, it is important to characterize the mechanical properties of SFs. However, most of the existing studies focus on properties of whole cells or of actin filaments isolated outside cells. In this study, we explored the mechanical properties of individual SFs in living endothelial cells by nanoindentation using an atomic force microscope. Our results demonstrate the pivotal role of SF actomyosin contractile level on mechanical properties. In the same SF, decreasing contractile level with 10 μM blebbistatin decreased stiffness, whereas increasing contractile level with 2 nM calyculin A increased stiffness. Incrementally stretching and indenting SFs made it possible to determine stiffness as a function of strain level and demonstrated that SFs have nearly linear stress-stain properties in the baseline state but nonlinear properties at a lower contractile level. The stiffnesses of peripheral and central portions of the same SF, which were nearly the same in the baseline state, became markedly different after contractile level was increased with calyculin A. Because these results pertain to effects of interventions in the same SF in a living cell, they provide important new understanding about cell mechanics.     

Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant k(OFF) of individual GFP-labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques--chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices--increases the k(OFF) of the focal adhesion protein zyxin. In contrast, the k(OFF) of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force-dependent increases in zyxin's k(OFF) that occur over seconds are sufficient to quantitatively predict large-scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force-dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells.     

Estimation of single stress fiber stiffness in cultured aortic smooth muscle cells under relaxed and contracted states: Its relation to dynamic rearrangement of stress fibers

For a quantitative analysis of intracellular mechanotransduction, it is crucial to know the mechanical properties of actin stress fibers in situ. Here we measured tensile properties of cultured aortic smooth muscle cells (SMCs) in a quasi-in situ tensile test in relaxed and activated states to estimate stiffness of their single stress fibers (SFs). An SMC cultured on substrates was held using a pair of micropipettes and detached from the substrate while maintaining its in situ cell shape and cytoskeletal integrity. Stretching up to ～15% followed by unloading was repeated three times to stabilize their tension–strain curves in the untreated (relaxed) and 10 μM-serotonin-treated (activated) condition. Cell stiffness defined as the average slope of the loading limb of the stable loops was ～25 and ～40 nN/% in relaxed and activated states, respectively. It decreased to ～10 nN/% following SF disruption with cytochalasin D in both states. The number of SFs in each cell measured with confocal microscopy decreased significantly upon serotonin activation from 21.5±3.8 (mean±SD, n=80) to 17.5±3.9 (n=77). The dynamics of focal adhesions (FAs) were observed in adherent cells using surface reflective interference contrast microscopy. FAs aligned and elongated along the cell major axis following activation and then merged with each other, suggesting that the decrease in SFs was caused by their fusion. Average stiffness of single SFs estimated by the average decrease in whole-cell stiffness following SF disruption divided by the average number of SFs in each cell was ～0.7 and ～1.6 nN/% in the relaxed and activated states, respectively. Stiffening of single SFs following SF activation was remarkably higher than stiffening at the whole-cell level. Results indicate that SFs stiffen not only due to activation of the actomyosin interaction, but also due to their fusion, a finding which would not be obtained from analysis of isolated SFs.     

Role of marginal stress fibers formed in the rat vascular endothelial cells

Fluorescence cytochemistry using en face preparations of rat vascular endothelial cells (ECs) revealed the localization of actin, fibronectin (FN) and fibronectin receptor (FNR) along not only central stress fibers (SFs) but also the cell margins. Electron microscopy showed very close proximity between the topographical distribution of intracellular microfilament bundles and that of subendothelial FN in the EC margins. Therefore, these basal and marginal actin cables may be comparable to the well-established central SFs present in ECs. Formation of the central SFs was induced in ECs or mesothelial cells in response to tension, by which their cellular integrity seems to be effectively maintained. However, even when central SF formation was inhibited by cytochalasin D, the ECs with marginal SFs showed high resistance to mechanical tension, whereas mesenteric mesothelial cells having no such fibers easily lost their integrity. Thus, together with central SFs, the marginal SFs characteristic of rat vascular ECs may play an essential role in strengthening cell-matrix adhesion.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid cytoskeletal response of epithelial cells to force generation by type IV pili

Many bacterial pathogens interfere with cellular functions including phagocytosis and barrier integrity. The human pathogen Neissieria gonorrhoeae generates grappling hooks for adhesion, spreading, and induction of signal cascades that lead to formation cortical plaques containing f-actin and ezrin. It is unclear whether high mechanical forces generated by type IV pili (T4P) are a direct signal that leads to cytoskeletal rearrangements and at which time scale the cytoskeletal response occurs. Here we used laser tweezers to mimic type IV pilus mediated force generation by T4P-coated beads on the order of 100 pN. We found that actin-EGFP and ezrin-EGFP accumulated below pilus-coated beads when force was applied. Within 2 min, accumulation significantly exceeded controls without force or without pili, demonstrating that T4P-generated force rapidly induces accumulation of plaque proteins. This finding adds mechanical force to the many strategies by which bacteria modulate the host cell cytoskeleton.     

Finger joint force minimization in pianists using optimization techniques

A numerical optimization procedure was used to determine finger positions that minimize and maximize finger tendon and joint force objective functions during piano play. A biomechanical finger model for sagittal plane motion, based on finger anatomy, was used to investigate finger tendon tensions and joint reaction forces for finger positions used in playing the piano. For commonly used piano key strike positions, flexor and intrinsic muscle tendon tensions ranged from 0.7 to 3.2 times the fingertip key strike force, while resultant inter-joint compressive forces ranged from 2 to 7 times the magnitude of the fingertip force. In general, use of a curved finger position, with a large metacarpophalangeal joint flexion angle and a small proximal interphalangeal joint flexion angle, reduces flexor tendon tension and resultant finger joint force.     

Estimating intercellular surface tension by laser-induced cell fusion

Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30-90 μN m(-1) range. Our estimate was in close agreement with cell-medium surface tensions measured at single-cell resolution.     

Stress fibers of the aortic smooth muscle cells in tissues do not align with the principal strain direction during intraluminal pressurization

Stress fibers (SFs) in cells transmit external forces to cell nuclei, altering the DNA structure, gene expression, and cell activity. To determine whether SFs are involved in mechanosignal transduction upon intraluminal pressure, this study investigated the SF direction in smooth muscle cells (SMCs) in aortic tissue and strain in the SF direction. Aortic tissues were fixed under physiological pressure of 120 mmHg. First, we observed fluorescently labeled SFs using two-photon microscopy. It was revealed that SFs in the same smooth muscle layers were aligned in almost the same direction, and the absolute value of the alignment angle from the circumferential direction was 16.8° ± 5.2° (n = 96, mean ± SD). Second, we quantified the strain field in the aortic tissue in reference to photo-bleached markers. It was found in the radial-circumferential plane that the largest strain direction was − 21.3° ± 11.1°, and the zero normal strain direction was 28.1° ± 10.2°. Thus, the SFs in aortic SMCs were not in line with neither the largest strain direction nor the zero strain direction, although their orientation was relatively close to the zero strain direction. These results suggest that SFs in aortic SMCs undergo stretch, but not maximal and transmit the force to nuclei under intraluminal pressure.

     

Relationship between Apical Membrane Elasticity and Stress Fiber Organization in Fibroblasts Analyzed by Fluorescence and Atomic Force Microscopy

To investigate the relationship between cellular microelasticity and the structural features of cytoskeletons (CSKs), a microindentation test for apical cell membranes and observation of the spatio-distribution of actin CSKs of fibroblasts were performed by fluorescence and atomic force microscopy (FM/AFM). The indentation depths of apical cell membranes were measured from AFM force–indentation (f–i) curves under equal final loads and mapped two-dimensionally to show the relative distribution of local microelasticity on cell membranes. Intracellular spatial distribution of actin CSKs was visualized fluorescently by high Z-resolution cross-sectional observation of a cell on which indentation mapping analysis had been performed in advance. Structural features of stress fibers (SFs) were observed as three typical patterns of dense SF, sparse SF and sparser SF cell groups, which were quantitated using the degree of orientation in apical SFs (ASFs) that had been defined using two-dimensional Fourier analysis. In indentation depth maps, the upper nuclear region was markedly softer than the pseudopodium region. The mean indentation depth of the upper nuclear region decreased with increased SF density in whole cells and the degree of orientation of ASF, although the pseudopodium region did not exhibit such a trend. The apical membrane of adhered cells was found to tend to stiffen with the increase in both density and degree of orientation of SFs.     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

Finite-element analysis of the adhesion-cytoskeleton-nucleus mechanotransduction pathway during endothelial cell rounding: axisymmetric model

Endothelial cells possess a mechanical network connecting adhesions on the basal surface, the cytoskeleton, and the nucleus. Transmission of force at adhesions via this pathway can deform the nucleus, ultimately resulting in an alteration of gene expression and other cellular changes (mechanotransduction). Previously, we measured cell adhesion area and apparent nuclear stretch during endothelial cell rounding. Here, we reconstruct the stress map of the nucleus from the observed strains using finite-element modeling. To simulate the disruption of adhesions, we prescribe displacement boundary conditions at the basal surface of the axisymmetric model cell. We consider different scenarios of the cytoskeletal arrangement, and represent the cytoskeleton as either discrete fibers or as an effective homogeneous layer When the nucleus is in the initial (spread) state, cytoskeletal tension holds the nucleus in an elongated, ellipsoidal configuration. Loss of cytoskeletal tension during cell rounding is represented by reactive forces acting on the nucleus in the model. In our simulations of cell rounding, we found that, for both representations of the cytoskeleton, the loss of cytoskeletal tension contributed more to the observed nuclear deformation than passive properties. Since the simulations make no assumption about the heterogeneity of the nucleus, the stress components both within and on the surface of the nucleus were calculated. The nuclear stress map showed that the nucleus experiences stress on the order of magnitude that can be significant for the function of DNA molecules and chromatin fibers. This study of endothelial cell mechanobiology suggests the possibility that mechanotransduction could result, in part, from nuclear deformation, and may be relevant to angiogenesis, wound healing, and endothelial barrier dysfunction.     

Molecular Tension Probes to Quantify Cell-Generated Mechanical Forces

Living cells generate, sense, and respond to mechanical forces through their interaction with neighboring cells or extracellular matrix, thereby regulating diverse cellular processes such as growth, motility, differentiation, and immune responses. Dysregulation of mechanosensitive signaling pathways is found associated with the development and progression of various diseases such as cancer. Yet, little is known about the mechanisms behind mechano-regulation, largely due to the limited availability of tools to study it at the molecular level. The recent development of molecular tension probes allows measurement of cellular forces exerted by single ligand-receptor interaction, which has helped in revealing the hitherto unknown mechanistic details of various mechanosensitive processes in living cells. Here, we provide an introductory overview of two methods based on molecular tension probes, tension gauge tether (TGT), and molecular tension fluorescence microscopy (MTFM). TGT utilizes the irreversible rupture of double-stranded DNA tether upon application of force in the piconewton (pN) range, whereas MTFM utilizes the reversible extension of molecular springs such as polymer or single-stranded DNA hairpin under applied pN forces. Specifically, the underlying principle of how molecular tension probes measure cell-generated mechanical forces and their applications to mechanosensitive biological processes are described.     

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, C(m)) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, C(m), used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce C(m) and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.