The role of extracellular matrix stiffness in regulating cytoskeletal remodeling via vinculin in synthetic smooth muscle cells

Vinculin is a key player in sensing and responding to external mechanical cues such as extracellular matrix stiffness. Increased matrix stiffness is often associated with certain pathological conditions including hypertension induced cellular cytoskeleton changes in vascular smooth muscle (VSM) cells. However, little is known on how stiffness affects cytoskeletal remodeling via vinculin in VSM cells. Thus, we utilized matrices with elastic moduli that simulate vascular stiffness in different stages of hypertension to investigate how matrix stiffness regulates cell cytoskeleton via vinculin in synthetic VSM cells. Through selecting a suitable reference gene, we found that an increase in physiologically relevant extracellular matrix stiffness (2–50?kPa) downregulates vinculin gene expression but upregulates vinculin protein expression. This discrepancy, which was not observed previously for non-muscle cells, suggests that the vinculin-mediated mecahnotransduction mechanism in synthetic VSM cells may be more complex than those proposed for non-muscle cells. Also adding to previous findings, we found that VSM cell growth may be impeded by substrates that are either too soft or too rigid.     

A constitutive formulation of arterial mechanics including vascular smooth muscle tone

A pseudo-strain energy function (pseudo-SEF) describing the biomechanical properties of large conduit arteries under the influence of vascular smooth muscle (VSM) tone is proposed. In contrast to previous models that include the effects of smooth muscle contraction through generation of an active stress, in this study we consider the vascular muscle as a structural element whose contribution to load bearing is modulated by the contraction. This novel pseudo-SEF models not only arterial mechanics at maximal VSM contraction but also the myogenic contraction of the VSM in response to local increases in stretch. The proposed pseudo-SEF was verified with experimentally obtained pressure-radius curves and zero-stress state configurations from rat carotid arteries displaying distinct differences in VSM tone: arteries from normotensive rats displaying minimal VSM tone and arteries from hypertensive rats exhibiting significant VSM tone. The pressure-radius curves were measured in three different VSM states: fully relaxed, maximally contracted, and normal VSM tone. The model fitted the experimental data very well (r2 > 0.99) in both the normo- and hypertensive groups for all three states of VSM activation. The pseudo-SEF was used to illustrate the localized reduction of circumferential stress in the arterial wall due to normal VSM tone, suggesting that the proposed pseudo-SEF can be of general utility for describing stress distribution not only under passive VSM conditions, as most SEFs proposed so far, but also under physiological and pathological conditions with varying levels of VSM tone.     

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells

ANa⁺ current is present in human jejunal circular smoothmuscle cells. The aim of the present study was to determine the role ofthe cytoskeleton in the regulation of the Na⁺ current.Whole cell currents were recorded by using standard patch-clamptechniques with Cs⁺ in the pipette to block K⁺currents. Cytochalasin D and gelsolin were used to disrupt the actincytoskeleton and phalloidin to stabilize it. Colchicine was used todisassemble the microtubule cytoskeleton (and intermediate filaments)and paclitaxel to stabilize it. Acrylamide was used to disrupt theintermediate filament cytoskeleton. Perfusion of the recording chamberat 10 ml/min increased peak Na⁺ current recorded fromjejunal smooth muscle cells by 27 ± 3%. Cytochalasin D andgelsolin abolished the perfusion-induced increase in Na⁺current, whereas incubation with phalloidin, colchicine, paclitaxel, oracrylamide had no effect. In conclusion, the Na⁺ currentexpressed in human jejunal circular smooth muscle cells appears to beregulated by the cytoskeleton. An intact actin cytoskeleton is requiredfor perfusion-induced activation of the Na⁺ current.

     

Cytochalasin B induced changes in cytoplasmic Na+/K+ balance in 2-cell mouse embryo

This work was performed to study changes in intracellular elemental (Na/K) concentrations caused by Cytochalasin B in two-cell mouse embryo using Electron Probe Microanalysis. The presence of Cytochalasin B is required to transfer a somatic cell nuclear into an early embryo cell. The direct effect of this chemical is cytoskeleton transformation, which would be able to cause the increase of potassium channel activity resulting in cytoplasmic Na/K imbalance. In our study Cytochalasin B was shown to decrease the intracellular sodium concentration. The Na/K balance in the cytoplasm of mouse embryos reverted to its intact level after treatment them with Cytochalasin B free Dulbecco's solution. Possible mechanisms responsible for the changes in the intracellular sodium concentration observed in the embryo cells are discussed.     

A role for the cytoskeleton in renal vitamin D metabolism

Conversion of circulating 25-hydroxyvitamin D3 (25(OH)D3) to its active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) occurs in the renal tubule mitochondrion. Recent reports have implicated the cytoskeleton in certain other steroid metabolizing cells as a mediator of a rate-limiting mitochondrial transport step. Whilst the activity of the renal converting enzyme, a typical steroid hydroxylase, is known to be regulated closely by a number of well studied factors, no information is available to indicate whether an analogous transport step is relevant to the regulation of vitamin D metabolism. Cytochalasin B and vinblastine were used as chemical antagonists of the microfilamentous and microtubular elements of the cytoskeleton. Both agents inhibited the conversion of 25(OH)D3 to 1,25(OH)2D3 by isolated vitamin D-deficient chick renal tubules in a dose-dependent manner. At the concentrations required to inhibit 25(OH)D3-1 alpha-hydroxylase activity in whole cells, these agents inhibited neither isolated mitochondrial 1,25(OH)2D3 production, nor 24,25(OH)2D3 synthesis by vitamin D-replete tubules. The cytoskeletal antagonists were found to increase the content of labelled 1,25(OH)2D3 and 25(OH)D3 in a mitochondrial fraction prepared by Percoll fractionation of tubule cells pre-exposed to the antagonists and labelled 25(OH)D3 substrate. The data suggest that disruption of the cytoskeleton may result in inhibition of transport of newly synthesised 1,25(OH)2D3 out of the mitochondrion and through the cell, and accumulating 1,25(OH)2D3 may oppose its further synthesis. This is consistent with a transport process mediated by the cytoskeleton being involved in the regulation of renal vitamin D metabolism.     

Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.     

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.     

Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.     

Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth,Galleria mellonella

The effects of Metarhizium anisopliae infection and three different secondary metabolites released by the fungus, destruxin A and E and cytochalasin D, on the morphology and cytoskeleton of plasmatocytes of the greater wax moth Galleria mellonella were studied. Plasmatocytes isolated from M. anisopliae infected larvae exhibited impairment of attachment, spreading and cytoskeleton formation accompanied with the occurrence of blebbing and pycnotic nuclei. Plasmatocytes treated with destruxin in vitro exhibited similar morphological and cytoskeleton alterations. The corresponding effects were characterized by inhibition of attachment, spreading and filopodia formation as well as by impaired formation of actin filaments and microtubules. Cytochalasin was shown to affect plasmatocytes in vitro in a different manner than destruxin A and E. The results of our comparative study strongly suggested that the morphology and cytoskeleton alterations of plasmatocytes observed in M. anisopliae infected larvae were predominantly caused by destruxins released by the fungus during mycosis. Its mode of action is discussed with regard to present knowledge about its effects on target cells.     

Cellular control of connective tissue matrix tension

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occurs during viscoelastic relaxation. We propose that this response of fibroblasts plays a role in regulating extracellular fluid flow into the tissue, and protects against swelling when the matrix is stretched. This article reviews the evidence supporting possible mechanisms underlying this response including autocrine purinergic signaling. We also discuss fibroblast regulation of connective tissue tension with respect to lymphatic flow, immune function, and cancer. J. Cell. Biochem. 114: 1714–1719, 2013. © 2013 Wiley Periodicals, Inc.     

A biophysical model of the contractile activity of muscle cells

Muscle cells have a distinctive structure, a developed cytoskeleton, which occupies most of the cell’s volume and forms, among other things, the contractile apparatus. A mathematical model of the biomechanical behavior of the cell as a whole was suggested based on the equations of continuum mechanics, which was next modified to describe the contractile activity of a muscle cell as an elastic rod. The model considers the result of the transduction of external effects that are manifested as an internal deformation, which allows the evaluation of the mobility and/or the emerging tension in muscle cells under the effects of external factors.     

Modeling the Mechanosensitivity of Neutrophils Passing through a?Narrow?Channel

Recent experiments have found that neutrophils may be activated after passing through microfluidic channels and filters. Mechanical deformation causes disassembly of the cytoskeleton and a sudden drop of the elastic modulus of the neutrophil. This fluidization is followed by either activation of the neutrophil with protrusion of pseudopods or a uniform recovery of the cytoskeleton network with no pseudopod. The former occurs if the neutrophil traverses the narrow channel at a slower rate. We propose a chemo-mechanical model for the fluidization and activation processes. Fluidization is treated as mechanical destruction of the cytoskeleton by sufficiently rapid bending. Loss of the cytoskeleton removes a pathway by which cortical tension inhibits the Rac protein. As a result, Rac rises and polarizes through a wave-pinning mechanism if the chemical reaction rate is fast enough. This leads to recovery and reinforcement of the cytoskeleton at the front of the neutrophil, and hence protrusion and activation. Otherwise the Rac signal returns to a uniform pre-deformation state and no activation occurs. Thus, mechanically induced neutrophil activation is understood as the competition between two timescales: that of chemical reaction and that of mechanical deformation. The model captures the main features of the experimental observation.     

Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts

Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole-mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity.     

Involvement of calcium signaling and the actin cytoskeleton in the membrane block to polyspermy in mouse eggs

This study examines the effects of actin microfilament-disrupting drugs on events of fertilization, with emphasis on gamete membrane interactions. Mouse eggs, freed of their zonae pellucidae, were treated with drugs that perturb the actin cytoskeleton by different mechanisms (cytochalasin B, cytochalasin D, jasplakinolide, latrunculin B) and then inseminated. Cytochalasin B, jasplakinolide, and latrunculin B treatments resulted in a decrease in the percentage of eggs fertilized and the average number of sperm fused per egg. However, cytochalasin D treatment resulted in an increase in the average number of sperm fused per egg and the percentage of polyspermic eggs. This increase in polyspermy occurred despite the observation that cytochalasin D treatment caused a decrease in sperm-egg binding and did not affect spontaneous acrosome reactions or sperm motility. This suggested that cytochalasin D-treated eggs had an impaired ability to establish a block to polyspermy at the level of the plasma membrane. The effect of cytochalasin D on the block to polyspermy was not due to a general disruption of egg activation because sperm-induced calcium oscillations and cortical granule exocytosis were similar in cytochalasin D-treated and control eggs. However, buffering of intracellular calcium levels with the calcium chelator BAPTA-AM resulted in an increase in polyspermy. Together, these data suggest that a postfertilization decrease in egg membrane receptivity to sperm requires functions of the egg actin cytoskeleton that are disrupted by cytochalasin D. Furthermore, egg activation-associated increased intracellular calcium levels are necessary but not sufficient to affect postfertilization membrane dynamics that contribute to a membrane block to polyspermy.     

Endoplasmic reticulum calcium release is modulated by actin polymerization

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.     

Deconvolution of the cellular force-generating subsystems that govern cytokinesis furrow ingression

Cytokinesis occurs through the coordinated action of several biochemically-mediated stresses acting on the cytoskeleton. Here, we develop a computational model of cellular mechanics, and using a large number of experimentally measured biophysical parameters, we simulate cell division under a number of different scenarios. We demonstrate that traction-mediated protrusive forces or contractile forces due to myosin II are sufficient to initiate furrow ingression. Furthermore, we show that passive forces due to the cell's cortical tension and surface curvature allow the furrow to complete ingression. We compare quantitatively the furrow thinning trajectories obtained from simulation with those observed experimentally in both wild-type and myosin II null Dictyostelium cells. Our simulations highlight the relative contributions of different biomechanical subsystems to cell shape progression during cell division.     

A cytoskeletal structure with associated polyribosomes obtained from HeLa cells

A method is described by which HeLa cells can be fractionated to reveal a skeletal-like structure in the cytoplasm. This cytoskeleton has many of the cell's ultrastructural features, such as 100Å filaments, microfilaments, centrioles, and microspikes, although most of the cellular protein, membranes, and microtubules have been extracted. Associated with the cytoskeleton are most of the polysomal, but not the monomeric, ribosomes. These polysomes are distributed throughout the cytoskeleton except in the region of the 100Å filaments, which resembles the distribution in intact cells. Degradation of mRNA with low levels of ribonuclease releases most ribosomes from the cytoskeleton. Prior disaggregation of polyribosomes in vivo releases ribosomes but not mRNA. Cytochalasin B administered in vivo releases the mRNA from the cytoskeleton. These results suggest an attachment of polyribosomes to the cytoskeleton via mRNA.     

Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells

Calponin is an actin filament-associated regulatory protein, and its h2 isoform is expressed in lung alveolar epithelial cells under postnatal upregulation during lung development corresponding to the commencement of respiratory expansion. Consistent with this correlation to mechanical tension, the expression of h2-calponin in alveolar cells is dependent on substrate stiffness and cytoskeleton tension. The function of h2-calponin in the stability of actin cytoskeleton implicates a role in balancing the strength and compliance of alveoli. An interesting finding is a rapid degradation of h2-calponin in lung after prolonged deflation, which is prevented by inflation of the lung to the in situ expanded volume. Decreasing mechanical tension in cultured alveolar cells by reducing the dimension of culture matrix reproduced the degradation of h2-calponin. Inhibition of myosin II ATPase also resulted in the degradation of h2-calponin in alveolar cells, showing a determining role of the tension in the actin cytoskeleton. Alveolar cells statically cultured on silicon rubber membrane build high tension in the cytoskeleton corresponding to a high expression of h2-calponin. Chronic cyclic stretching of cells on the membrane did not increase but decreased the expression of h2-calponin. This finding suggests that when cellular structure adapts to the stretched dimension, cyclic relaxations periodically release cytoskeleton tension and lower the total amount of tension that the cell senses over time. Therefore, the isometric tension, other than tension dynamics, determines the expression of h2-calponin. The tension regulation of h2-calponin synthesis and degradation demonstrates a novel mechanical regulation of cellular biochemistry.