The Role of Vimentin Intermediate Filaments in Cortical and Cytoplasmic Mechanics

The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin^−/− mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.     

The Mechanical Role of Microtubules in Tissue Remodeling

During morphogenesis, tissues undergo extensive remodeling to get their final shape. Such precise sculpting requires the application of forces generated within cells by the cytoskeleton and transmission of these forces through adhesion molecules within and between neighboring cells. Within individual cells, microtubules together with actomyosin filaments and intermediate filaments form the composite cytoskeleton that controls cell mechanics during tissue rearrangements. While studies have established the importance of actin-based mechanical forces that are coupled via intercellular junctions, relatively little is known about the contribution of other cytoskeletal components such as microtubules to cell mechanics during morphogenesis. In this review the focus is on recent findings, highlighting the direct mechanical role of microtubules beyond its well-established role in trafficking and signaling during tissue formation.     

Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics.     

Focal adhesion kinase stabilizes the cytoskeleton

Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK−/−), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK−/− cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.     

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.     

Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics

We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 μm, corresponding to a flexural rigidity of 0.040 pN μm². Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 μm and the filament flexural rigidity to 0.0091 pN μm². That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity.     

Measurement of intracellular strain on deformable substrates with texture correlation

Mechanical stimuli are important factors that regulate cell proliferation, survival, metabolism and motility in a variety of cell types. The relationship between mechanical deformation of the extracellular matrix and intracellular deformation of cellular sub-regions and organelles has not been fully elucidated, but may provide new insight into the mechanisms involved in transducing mechanical stimuli to biological responses. In this study, a novel fluorescence microscopy and image analysis method was applied to examine the hypothesis that mechanical strains are fully transferred from a planar, deformable substrate to cytoplasmic and intranuclear regions within attached cells. Intracellular strains were measured in cells derived from the anulus fibrosus of the intervertebral disc when attached to an elastic silicone membrane that was subjected to tensile stretch. Measurements indicated cytoplasmic strains were similar to those of the underlying substrate, with a strain transfer ratio (STR) of 0.79. In contrast, nuclear strains were much smaller than those of the substrate, with an STR of 0.17. These findings are consistent with previous studies indicating nuclear stiffness is significantly greater than cytoplasmic stiffness, as measured using other methods. This study provides a novel method for the study of cellular mechanics, including a new technique for measuring intranuclear deformations, with evidence of differential magnitudes and patterns of strain transferred from the substrate to cell cytoplasm and nucleus.     

Dissecting the contribution of actin and vimentin intermediate filaments to mechanical phenotype of suspended cells using high-throughput deformability measurements and computational modeling

Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently observed overexpression of vimentin in diverse types of cancer and underline the role of vimentin targeting drugs, such as Withaferin-A, as a potent cancerostatic supplement.     

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.     

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.     

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.     

Depth-sensing analysis of cytoskeleton organization based on AFM data

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young’s modulus, which is typically determined for indentation depths within the range 300–500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young’s moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young’s modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.     

Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

Cell division is inherently mechanical, with cell mechanics being a critical determinant governing the cell shape changes that accompany progression through the cell cycle. The mechanical properties of symmetrically dividing mitotic cells have been well characterized, whereas the contribution of cellular mechanics to the strikingly asymmetric divisions of female meiosis is very poorly understood. Progression of the mammalian oocyte through meiosis involves remodeling of the cortex and proper orientation of the meiotic spindle, and thus we hypothesized that cortical tension and stiffness would change through meiotic maturation and fertilization to facilitate and/or direct cellular remodeling. This work shows that tension in mouse oocytes drops about sixfold during meiotic maturation from prophase I to metaphase II and then increases ∼1.6-fold upon fertilization. The metaphase II egg is polarized, with tension differing ∼2.5-fold between the cortex over the meiotic spindle and the opposite cortex, suggesting that meiotic maturation is accompanied by assembly of a cortical domain with stiffer mechanics as part of the process to achieve asymmetric cytokinesis. We further demonstrate that actin, myosin-II, and the ERM (Ezrin/Radixin/Moesin) family of proteins are enriched in complementary cortical domains and mediate cellular mechanics in mammalian eggs. Manipulation of actin, myosin-II, and ERM function alters tension levels and also is associated with dramatic spindle abnormalities with completion of meiosis II after fertilization. Thus, myosin-II and ERM proteins modulate mechanical properties in oocytes, contributing to cell polarity and to completion of meiosis.     

The role of F-actin and myosin in epithelial cell rheology

Although actin and myosin are important contributors to cell-force generation, shape change, and motility, their contributions to cell stiffness and frequency-dependent rheology have not been conclusively determined. We apply several pharmacological interventions to cultured epithelial cells to elucidate the roles of actin and myosin in the mechanical response of cells and intracellular fluctuations. A suite of different methods is used to separately examine the mechanics of the deep cell interior and cortex, in response to depletion of intracellular ATP, depolymerization of F-actin, and inhibition of myosin II. Comparison of these results shows that F-actin plays a significant role in the mechanics of the cortical region of epithelial cells, but its disruption has no discernable effect on the rheology of the deeper interior. Moreover, we find that myosins do not contribute significantly to the rheology or ATP-dependent, non-Brownian motion in the cell interior. Finally, we investigate the broad distribution of apparent stiffness values reported by some microrheology methods, which are not observed with two-point microrheology. Based on our findings and a simple model, we conclude that heterogeneity of the tracer-cytoskeleton contacts, rather than the network itself, can explain the broad distribution of apparent stiffnesses.     

TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion

Recent studies implicate a role for cell mechanics in cancer progression. The epithelial-to-mesenchymal transition (EMT) regulates the detachment of cancer cells from the epithelium and facilitates their invasion into stromal tissue. Although classic EMT hallmarks include loss of cell–cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response. Here we demonstrate that transforming growth factor β (TGF-β)–induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins. We find that suppression of the RhoA guanine nucleotide exchange factors (GEFs) LARG and GEF-H1 through TGF-β/ALK5–enhanced proteasomal degradation mediates these changes in cell mechanics and affects EMT-associated invasion. Taken together, our results reveal a functional connection between attenuated stiffness and stiffening response and the increased invasion capacity acquired after TGF-β–induced EMT.     

Regulation of stretch-activated intracellular calcium transients by actin filaments.

Stretch activation of cation-permeable channels may be an important proximal sensory mechanism in mechanotransduction. As actin filaments may mediate cellular responses to changes of the mechanical properties of the substrate and regulate stretch-induced calcium transients, we examined the role of actin filaments and substrate flexibility in modulating the amplitude of stretch-activated intracellular calcium transients. Human gingival fibroblasts were subjected to mechanical stretch through integrins by magnetic force acting on collagen-coated ferric oxide beads. Intracellular calcium concentration was measured in fura-2-loaded cells by ratio fluorimetry. Cytochalasin D-treatment greatly increased (3-fold) the amplitude of stretch-activated calcium transients in well-spread cells grown on glass coverslips while phalloidin, colchicine or taxol exerted no signficant effects, indicating that actin filaments but not microtubules modulate stretch-activated calcium transients. In freshly plated cells with rounded shapes and poorly developed cortical actin filaments, stretch-induced calcium transients were of 3-fold higher amplitude than well-spread cells plated for 6-24 hrs and with well developed actin filaments. Cells plated on soft collagen-polyacrylamide gels showed round morphology but exhibited <50% of the response to stretch of well-spread cells on inflexible gels. Notably, cells on soft gels showed very heavy phalloidin staining for cortical actin filaments compared with cells on more inflexible surfaces which showed only light staining for cortical actin. While cell shape may have some effect on responsiveness to mechanical stretch, the rigidity of the cell membrane mediated by the extensive cortical actin network appears to be a central determinant in the regulation of stretch-induced calcium signals.     

Structure and Mechanics of Supporting Cells in the Guinea Pig Organ of Corti

The mechanical properties of the mammalian organ of Corti determine its sensitivity to sound frequency and intensity, and the structure of supporting cells changes progressively with frequency along the cochlea. From the apex (low frequency) to the base (high frequency) of the guinea pig cochlea inner pillar cells decrease in length incrementally from 75–55 µm whilst the number of axial microtubules increases from 1,300–2,100. The respective values for outer pillar cells are 120–65 µm and 1,500–3,000. This correlates with a progressive decrease in the length of the outer hair cells from >100 µm to 20 µm. Deiters''cell bodies vary from 60–50 µm long with relatively little change in microtubule number. Their phalangeal processes reflect the lengths of outer hair cells but their microtubule numbers do not change systematically. Correlations between cell length, microtubule number and cochlear location are poor below 1 kHz. Cell stiffness was estimated from direct mechanical measurements made previously from isolated inner and outer pillar cells. We estimate that between 200 Hz and 20 kHz axial stiffness, bending stiffness and buckling limits increase, respectively,∼3, 6 and 4 fold for outer pillar cells, ∼2, 3 and 2.5 fold for inner pillar cells and ∼7, 20 and 24 fold for the phalangeal processes of Deiters''cells. There was little change in the Deiters''cell bodies for any parameter. Compensating for effective cell length the pillar cells are likely to be considerably stiffer than Deiters''cells with buckling limits 10–40 times greater. These data show a clear relationship between cell mechanics and frequency. However, measurements from single cells alone are insufficient and they must be combined with more accurate details of how the multicellular architecture influences the mechanical properties of the whole organ.     

Mechano-coupling and regulation of contractility by the vinculin tail domain

Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site.     

Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress

Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin’s mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vimentin-null (vim^−/−) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim^−/−mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin’s enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim^−/−mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim^−/−mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of vim^−/−mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young’s moduli of normal and vim^−/−mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.     

Cytoplasmic fibrils in living cultured cells

Summary By a combined light and electron microscopic study, the structure and behavior of the stress fibers of cultured rat embryo cells are described. From an analysis of movie records of living cells it is seen that the stress fibers are in a state of flux, continually altering their dimensions and dispositions within the cell. However, compared to most other cellular movements, these rates of change are slow. By electron microscopy it is shown that the stress fibers consist of bundles of close packed elongate 75 Å filaments, arranged just beneath the plasma membrane adjacent to the cell's plane of attachment and that similar filaments, forming a loose framework, permeate the cytoplasmic matrix. On the basis of careful light and electron microscopic comparisons, it is concluded that the filamentous structure shown within the cytoplasm of glutaraldehyde/osmium-fixed cells is a generally accurate representation of the structure of the living cell cytoplasm. It seems likely that the stress fibers are concerned in stabilizing areas of cellular attachment as well as with resisting forces that stretch the cell. The suggestion is made that, by controlling cytoplasmic viscosity and responding to cytoplasmic microtubules, the diffuse framework of filaments helps to determine the form of the cell and that, by a coordinate dynamic activity of its filaments, it provides the motive power for the various forms of cellular and intracellular movements.