Cytoplasmic Stiffness Research Articles

β-II tubulin isotype directs stiffness and differentiation of neuroblastoma SH-SY5Y cells.

β-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression, varying due to physical and biochemical cues. In this work, we investigated the effect of transient heat shock at 42°C on the nuclear and cytoplasmic stiffness of SH-SY5Y neuroblastoma cells through atomic force microscopy. Moreover, the variations in the expression of β-tubulin isotypes as a heat shock response were also monitored. The heat-exposed cells endured a recovery at 37°C for 24h and they manifested an increase of cytoplasmic stiffness by 130 ± 25% with respect to untreated controls. The expression of β-II tubulin isotype in heat-recovered cells is augmented by 51 ± 5% whereas the levels of total tubulin and β-III tubulin isotype remain unaltered. Upon depletion of β-II tubulin isotype using shRNA, the increase in cytoplasmic stiffness was dampened. However, it remained unaffected upon depletion with β-III tubulin isotype shRNA. This features the role of the β-II tubulin isotype in regulating cellular stiffness. In addition, neuroblastoma SH-SY5Y cells undergo differentiation by initiating neuritogenesis and prior evidence suggests the indispensable role of β-II tubulin isotype in this process. The heat-recovered cells which expressed higher levels of β-II tubulin isotype expedited the differentiation process in 3-day which was around 5-day for control cells, however, upon depletion of β-II tubulin isotype, the cells almost lost their differentiation potential. Altogether, this work highlights the role of β-II tubulin isotype as a biomarker for cellular stiffness.

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole-cell mechanics

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the lamin–LINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics.

The role of linc complexes in regulating the intracellular concentration of ribosomes and cytoplasmic stiffness in C. elegans

The mechanical deformability of the cytoplasm is a key determinant of several fundamental cellular processes, including mechanotransduction, migration, and organelle positioning. Consequently, changes in the cytoplasmic mechanical properties are correlated with diseases, such as cancer, muscular dystrophy, and premature aging. While the mechanical deformability of the cytoplasm has been well-studied in vitro using tissue culture cells, much less is known about the in vivo biophysical properties of the cytoplasm.

3D Computational Modeling of Bleb Initiation Dynamics

Blebbing occurs in cells under high cortical tension when the membrane locally detaches from the actin cortex, resulting in pressure-driven flow of the cytosol and membrane expansion. Some cells use blebs as leading edge protrusions during cell migration, particularly in 3D environments such as a collagen matrix. Blebs can be initiated through either a localized loss of membrane-cortex adhesion or ablation of the cortex in a region. Bleb morphologies resulting from different initiation mechanisms have not been studied in detail, either experimentally or with theoretical models. Additionally, material properties of the cytoplasm, such as elasticity, have been shown to be important for limiting bleb size. A 3D dynamic computational model of the cell is presented that includes mechanics and the interactions of the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model is used to quantify bleb expansion dynamics and shapes that result from simulations using different initiation mechanisms. The cytoplasm is modeled as a both viscous fluid and as a poroelastic material. Results from model simulations with a viscous fluid cytoplasm model show much broader blebs that expand faster when they are initiated via cortical ablation than when they are initiated by removing only membrane-cortex adhesion. Simulation results using the poroelastic model of the cytoplasm provide qualitatively similar bleb morphologies regardless of the initiation mechanism. Parameter studies on bleb expansion time, cytoplasmic stiffness, and permeability reveal different scaling properties, namely a smaller power-law exponent, in 3D simulations compared to 2D ones.

Junctional and cytoplasmic contributions in wound healing.

Wound healing is characterized by the re-epitheliation of a tissue through the activation of contractile forces concentrated mainly at the wound edge. While the formation of an actin purse string has been identified as one of the main mechanisms, far less is known about the effects of the viscoelastic properties of the surrounding cells, and the different contribution of the junctional and cytoplasmic contractilities. In this paper, we simulate the wound healing process, resorting to a hybrid vertex model that includes cell boundary and cytoplasmic contractilities explicitly, together with a differentiated viscoelastic rheology based on an adaptive rest-length. From experimental measurements of the recoil and closure phases of wounds in the Drosophila wing disc epithelium, we fit tissue viscoelastic properties. We then analyse in terms of closure rate and energy requirements the contributions of junctional and cytoplasmic contractilities. Our results suggest that reduction of junctional stiffness rather than cytoplasmic stiffness has a more pronounced effect on shortening closure times, and that intercalation rate has a minor effect on the stored energy, but contributes significantly to shortening the healing duration, mostly in the later stages.

Tracking intracellular forces and mechanical property changes in mouse one-cell embryo development.

Cells comprise mechanically active matter that governs their functionality, but intracellular mechanics are difficult to study directly and are poorly understood. However, injected nanodevices open up opportunities to analyse intracellular mechanobiology. Here, we identify a programme of forces and changes to the cytoplasmic mechanical properties required for mouse embryo development from fertilization to the first cell division. Injected, fully internalized nanodevices responded to sperm decondensation and recondensation, and subsequent device behaviour suggested a model for pronuclear convergence based on a gradient of effective cytoplasmic stiffness. The nanodevices reported reduced cytoplasmic mechanical activity during chromosome alignment and indicated that cytoplasmic stiffening occurred during embryo elongation, followed by rapid cytoplasmic softening during cytokinesis (cell division). Forces greater than those inside muscle cells were detected within embryos. These results suggest that intracellular forces are part of a concerted programme that is necessary for development at the origin of a new embryonic life.

Reactive oxygen species-mediated cytoplasmic stiffening impairs the phagocytic ability of the macrophage.

The phagocytic ability of macrophages empowers them to enforce innate immunity. RAW264.7, THP-1 and peripheral blood mononuclear cell-derived macrophages display considerable variability with regards to their phagocytic ability. We identify the underlying causes that attenuate the phagocytic abilities of a macrophage. Deformability of the cytoplasm and cortex influences the macrophage's phagocytic ability, and macrophages use the large cell-to-cell variability of their cytoplasmic stiffness to modulate their phagocytic ability. We find that the more-deformable macrophages have a higher phagocytic ability than those that are less deformable. Further, the subcellular spatial variability of cortex stiffness gives rise to more-deformable subdomains on the membrane for pathogen ingestion. We report a previously unknown negative-feedback loop that is triggered by the phagocytic oxidative burst. Macrophages utilize the excess reactive oxygen species to stiffen the cytoplasm, reducing their phagocytic propensity. In organisms, ageing or pathological conditions impair the phagocytic ability of macrophages. Our findings identify the targets that could potentially be utilized for restoring the phagocytic ability of the defunct macrophages.

Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs

The large length scale of Xenopus laevis eggs facilitates observation of bulk cytoplasm dynamics far from the cortex during cytokinesis. The first furrow ingresses through the egg midplane, which is demarcated by chromosomal passenger complex (CPC) localized on microtubule bundles at the boundary between asters. Using an extract system, we found that local kinase activity of the Aurora B kinase (AURKB) subunit of the CPC caused disassembly ofF-actin and keratin between asters and local softening of the cytoplasm as assayed by flow patterns. Beads coated with active CPC mimicked aster boundaries and caused AURKB-dependent disassembly of F-actin and keratin that propagated ∼40μm without microtubules and much farther with microtubules present. Consistent with extract observations, we observed disassembly of the keratin network between asters in zygotes fixed before and during 1st cytokinesis. We propose that active CPC at aster boundaries locally reduces cytoplasmic stiffness by disassembling actin and keratin networks. Possible functions of this local disassembly include helping sister centrosomes move apart after mitosis, preparing a soft path for furrow ingression, and releasing G-actin from internal networks to build cortical networks that support furrow ingression.

Measuring Cytoplasmic Stiffness of Fibroblasts as a Function of Location and Substrate Rigidity Using Atomic Force Microscopy.

The cytoplasmic stiffness of cells plays a significant role during cell migration. As a cell migrates, differences in cytoplasmic properties occur that subsequently modulate migratory behavior. The properties of the substrate to which cells are adherent also play a role. To accurately measure the cytoplasmic stiffness of cells, we provide detailed instructions on how to assemble hydrogels that exhibit different elastic moduli, culturing cells on these substrates followed by a step-by-step process to measure and analyze the cytoplasmic properties of fibroblasts. In this study, we have measured the elastic moduli of cells at different locations to demonstrate how this property varies as a function of where the measurement is performed. The degree of anisotropy measured by the difference between cytoplasmic stiffness at the two edges of the cell also varied as a function of the elasticity of their underlying substrates. Larger differences in cytoplasmic stiffness between the leading and trailing edges were observed on substrates with a higher elastic modulus. The methods reported in this study can provide information on cellular properties, specifically, how the elastic modulus of cells can be probed and analyzed in vitro.

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts.

The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament (IF) networks. Indeed the contribution of IFs, such as vimentin, keratins and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an IF specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. In this study, using three types of cell rheometers, we assess the consequences of expressing wild-type (WT) or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of WT or mutated desmin polymerised in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm viscoelasticity. Desminopathies are associated with muscular weaknesses attributed to a disorganisation of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue.

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.

Cyclic stretch of alveolar epithelial cells alters cytoskeletal micromechanics

Cytoplasmic transport of large molecules such as plasmid DNA (pDNA) has been shown to increase when cells are subjected to mild levels of cyclic stretch for brief periods. In the case of pDNA, this is in part due to the increased active transport of pDNA along stabilized, acetylated microtubules in the cytoplasm, whose levels are increased in response to stretch. It also has been shown that disruption of the dense actin network leads to increased pDNA and macromolecule diffusion as well. We hypothesize that stretch not only increases active transport of pDNA but also, similar to actin disrupting drugs, decreases cytoplasmic stiffness leading to a less restive pathway for macromolecules to diffuse. To test this we used particle tracking microrheology to measure cytoplasmic mechanics. We conclude that while cyclic stretch transiently decreases cytoplasmic stiffness and increases diffusivity, stretch-independent modulation of the levels of acetylated, stable microtubules has no effect on cytoplasmic stiffness. Furthermore, stretching cells that have maximally acetylated microtubules increases cytoplasmic trafficking of pDNA, without increasing levels of acetylated microtubules. These findings suggest that stretch-enhanced gene transfer may occur by two independent mechanisms: increased levels of acetylated microtubules for directed active transport, and reduced cytoplasmic stiffness for increased diffusion.

22. Cyclic Stretch-Induced Gene Delivery May Result from Decreased Cytoplasmic Stiffness

22. Cyclic Stretch-Induced Gene Delivery May Result from Decreased Cytoplasmic Stiffness

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.

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.

Sound attenuation of polymerizing actin reflects supramolecular structures: viscoelastic properties of actin gels modified by cytochalasin D, profilin and alpha-actinin.

Polymerization and depolymerization of cytoskeletal elements maintaining cytoplasmic stiffness are key factors in the control of cell crawling. Rheometry is a significant tool in determining the mechanical properties of the single elements in vitro. Viscoelasticity of gels formed by these polymers strongly depends on both the length and the associations of the filaments (e.g. entanglements, annealings and side-by-side associations). Ultrasound attenuation is related to viscosity, sound velocity and supramolecular structures in the sample. In combination with a small glass fibre (2 mm x 50 microm), serving as a viscosity sensor, an acoustic microscope was used to measure the elasticity and acoustic attenuation of actin solutions. Changes in acoustic attenuation of polymerizing actin by far exceed the values expected from calculations based on changes in viscosity and sound velocity. During the lag-phase of actin polymerization, attenuation slightly decreases, depending on actin concentration. After the half-maximum viscosity is accomplished and elasticity turns into steady state, attenuation distinctly rises. Changes in ultrasound attenuation depend on actin concentration, and they are modulated by the addition of alpha-actinin, cytochalasin D and profilin. Thus absorption and scattering of sound on the polymerization of actin is related to the packing density of the actin net, entanglements and the length of the actin filaments. Shortening of actin filaments by cytochalasin D was also confirmed by electron micrographs and falling-ball viscosimetry. In addition to viscosity and elasticity, the attenuation of sound proved to be a valuable parameter in characterizing actin polymerization and the supramolecular associations of F-actin.

Subcellular tension fields and mechanical resistance of the lamella front related to the direction of locomotion.

Keratocytes derived from the epidermis of aquatic vertebrates are now widely used for investigation of the mechanism of cell locomotion. One of the main topics under discussion is the question of driving force development and concomitantly subcellular force distribution. Do cells move by actin polymerization-driven extension of the lamella, or is the lamella edge extended at regions of weakness by a flow of cytoplasm generated by hydrostatic pressure? Thus, elasticity changes were followed and the stiffness of the leading front of the lamella was manipulated by local application of phalloidin and cytochalasin D (CD). In scanning acoustic microscopy (SAM), elasticity is revealed from the propagation velocity of longitudinal sound waves (1 GHz). The lateral resolution of SAM is in the micrometer range. Using this method, subcellular tension fields with different stiffnesses (elasticity) can be determined. A typical pattern of subcellular stiffness distribution is related to the direction of migration. Cells forced to change their direction of movement by exposure to DC electric fields of varying polarity alter their pattern of subcellular stiffness in relationship to the new direction. The cells spread into the direction of low stiffness and retract at zones of high stiffness. The pattern of subcellular stiffness distribution reveals force distribution in migrating cells; i.e., if a cell moves exactly in a direction perpendicular to its long axis, then the contractile forces are largest along the long axis and decrease toward the short axis. Locomotion in any angle oblique to this axis requires an asymmetric stiffness distribution. Inhibition of actomyosin contractions by La3+ (2 mM), which inhibits Ca2+ influx, reduces cytoplasmic stiffness accompanied by an immediate cessation of locomotion and a change of cell shape. Local release of CD in front of a progressing lamella activates a cell to follow the CD gradient: The lamella thickens locally and is extended toward the tip of the microcapillary. Release of phalloidin stops extension of the lamella, and the cell turns away from the releasing microcapillary. The response to CD is assumed to be the result of local weakening of the cytoplasm due to severing of the actin fibrils. Phalloidin is supposed to stabilize the leading front by inhibition of F-actin depolymerization. These observations are in favor of the assumption that migration is due to an extension of the cell into the direction of minimum stiffness, and they are consistent with the hypothesis that local release of hydrostatic pressure provides the driving force for the flux of cytoplasm.

The leukocyte response to fluid stress.

Leukocyte migration from a hemopoietic pool across marrow endothelium requires active pseudopod formation and adhesion. Leukocytes rarely show pseudopod formation while in circulation. At question then is the mechanism that serves to minimize leukocyte pseudopod formation in the circulation. We tested the hypothesis that fluid shear stress acts to prevent pseudopod formation. When individual human leukocytes (neutrophils, monocytes) spreading on glass surfaces in vitro were subjected to fluid shear stress ( approximately 1 dyn/cm2), an instantaneous retraction of pseudopods was observed. Removal of the fluid shear stress in turn led to the return of pseudopod projection and cell spreading. When steady shear stress was prolonged over several minutes, leukocyte swelling occurs together with an enhanced random motion of cytoplasmic granules and a reduction of cytoplasmic stiffness. The response to shear stress could be suppressed by K+ channel blockers and chelation of external Ca2+. In rat mesentery microvessels after occlusion, circulating leukocytes project pseudopods in free suspension or when attached to the endothelium, even though immediately after occlusion only few pseudopods were present. When flow was restored, pseudopods on adhering leukocytes were retracted and then the cells began to roll and detach from the endothelium. In conclusion, plasma shear stress in the circulation serves to reduce pseudopod projection and adhesion of circulating leukocytes and vice versa reduction of shear stress leads to pseudopod projection and spreading of leukocytes on the endothelium.