Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells

The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness (G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ((t)) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18(t) + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.     

Effect of the cytoskeletal prestress on the mechanical impedance of cultured airway smooth muscle cells.

We investigated the effect of the cytoskeletal prestress (P) on the elastic and frictional properties of cultured human airway smooth muscle cells during oscillatory loading; P is preexisting tensile stress in the actin cytoskeleton generated by the cell contractile apparatus. We oscillated (0.1 Hz, 6 Pa peak to peak) small ferromagnetic beads bound to integrin receptors and computed the storage (elastic) modulus (G') and the loss (frictional) modulus (G") from the applied torque and the corresponding bead rotation. All measurements were done at baseline and after cells were treated with graded doses of either histamine (0.1, 1, 10 microM) or isoproterenol (0.01, 0.1, 1, 10 microM). Values for P for these concentrations were taken from a previous study (Wang et al., Am J Physiol Cell Physiol, in press). It was found that G' and G", as well as P, increased/decreased with increasing doses of histamine/isoproterenol. Both G' and G" exhibited linear dependences on P: G'(Pa) = 0.20P + 82 and G"(Pa) = 0.05P + 32. The dependence of G' on P is consistent with our previous findings and with the behavior of stress-supported structures. The dependence of G" on P is a novel finding. It could be attributed to a variety of mechanisms. Some of those mechanisms are discussed in detail. We concluded that, in addition to the central mechanisms by which stress-supported structures develop mechanical stresses, other mechanisms might need to be invoked to fully explain the observed dependence of the cell mechanical properties on the state of cell contractility.     

Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor

Analysis of cell regulation requires methods for perturbing molecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.     

From filaments to function: The role of the plant actin cytoskeleton in pathogen perception,signaling and immunity

The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. In recent years,research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens,including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activation of specific signaling responses following pathogen perception. B4 ased on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens.     

Tensegrity behaviour of cortical and cytosolic cytoskeletal components in twisted living adherent cells

The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure - evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.     

Mechanical assessment by magnetocytometry of the cytosolic and cortical cytoskeletal compartments in adherent epithelial cells

This study aims at quantifying the cellular mechanical properties based on a partitioning of the cytoskeleton in a cortical and a cytosolic compartments. The mechanical response of epithelial cells obtained by magnetocytometry - a micromanipulation technique which uses twisted ferromagnetic beads specifically linked to integrin receptors - was purposely analysed using a series of two Voigt bodies. Results showed that the cortical cytoskeleton has a faster response ( approximately 1 s) than the cytosolic compartment ( approximately 30 s). Moreover, the two cytoskeletal compartments have specific mechanical properties, i.e., the cortical (resp. cytosolic) cytoskeleton has a rigidity in the range: 49-85 Pa (resp.: 74-159 Pa) and a viscosity in the range 5-14 Pa.s (resp.: 593-1534 Pa.s), depending on the level of applied stress. Depolymerising actin-filaments strongly modified these values and especially those of the cytosolic compartment. The structural relevance of this two-compartment partitioning was supported by images of F-actin structure obtained on the same cells.     

The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.     

The role of adherent cells in the secondary cell-mediated response in vitro to a natural poxvirus pathogen.

The role of adherent cells in an in vitro secondary response to ectromelia virus infection was investigated. Spleen cells from ectromelia-primed mice ("responder" cells) depleted of adherent cells by either carbonyl iron treatment, adherence to plastic or passage through cotton wool columns had a markedly decreased capacity to produce a secondary response, as indicated by decreased T cell-mediated cytotoxicity against virus-infected target cells, when cultured with virus-infected "stimulator" cells. The secondary response was restored by the addition of peritoneal cells from either normal or ectromelia-immune mice. Small numbers of peritoneal cells completely reconstituted the response within a certain dose range but larger numbers produced a marked inhibition of the response. Spleen cells were less effective in restoring the response. The peritoneal cells were not merely acting as additional, infected "stimulator" or antigen-presenting cells, since they could be added as late as 3 days after culture. Reconstituting activity was not affected by pretreatment with anti-theta serum and complement and cell separation studies showed that the activity was associated mainly with Ig-negative cells and that the active cell probably bears Ia antigens on its surface. These results indicate that the adherent cells involved are probably macrophages and that they act non-specifically to produce optimum conditions for the specific response of T cells.     

Visualization of the cytoskeleton in Leydig cells in vitro. Effect of luteinizing hormone and cytoskeletal disrupting drugs

Effect of LH, vinblastine and cytochalasin B on the cytoskeleton of cultured Leydig cells was investigated using monoclonal antibodies and fluorescence microscopy. After LH addition and treatment with cytoskeletal disrupting drugs, three main effects were observed: 1) increase of androgen level secreted by cultured mouse Leydig cells, 2) changes of cell-shape towards regular and rounded, 3) increase of delta 5,3 beta-HSD activity. The results are discussed in respect to possible involvement of cytoskeleton in the regulation of steroidogenesis.     

The role of adherent accessory cells in the generation of effector suppressor T cells

The role of accessory cell populations in the generation of effector suppressor (Ts3) cells was studied. By using an in vitro culture system, it was previously determined that the induction of NP-specific effector suppressor activity requires T cells, antigen, and an anti-idiotypic B cell population. We now demonstrate that the generation of Ts3 cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen-presenting accessory cells are required early in the induction phase of Ts3 generation. These accessory cells can present NP coupled to immunogenic or non-immunogenic polypeptide carriers, including polymers of L-amino acids. However, NP coupled to polymers of poorly metabolized D-amino acids fail to induce suppressor T cell generation. Furthermore, the data demonstrate that an H-2 homology must exist between the Ts3 precursors and the antigen-presenting cell population if suppressor activity is to be generated. We also characterize the differential genetic restrictions that govern the induction of Ts3 cells that control suppression of either T cell or B cell responses. The data suggest that although I-J region encoded gene products control the induction and effector phases of suppressor cell activity as measured on T cell responses, the suppression of B cell responses appear to be controlled by I-A gene products. Possible cellular mechanisms that might explain these findings are discussed.     

The role of adherent cells in supporting natural cytotoxicity reactions

The cytotoxic activity of natural killer (NK) cells and interferon-alpha (IFN-alpha) production by NK cells were studied. It was shown that in healthy donors adherent cells (AC) play an important role in reactions of natural cytotoxicity: they provide the main IFN production in the NK system. AC suppresses the cytotoxic activity of NK cells in patients with scleroderma and AC has a high cytotoxic activity in patients with multiple sclerosis. The role of AC in appearance and development of NK immunodeficiency in these pathologies was discussed.     

Early spindle assembly in Drosophila embryos: role of a force balance involving cytoskeletal dynamics and nuclear mechanics

Mitotic spindle morphogenesis depends upon the action of microtubules (MTs), motors and the cell cortex. Previously, we proposed that cortical- and MT-based motors acting alone can coordinate early spindle assembly in Drosophila embryos. Here, we tested this model using microscopy of living embryos to analyze spindle pole separation, cortical reorganization, and nuclear dynamics in interphase-prophase of cycles 11-13. We observe that actin caps remain flat as they expand and that furrows do not ingress. As centrosomes separate, they follow a linear trajectory, maintaining a constant pole-to-furrow distance while the nucleus progressively deforms along the elongating pole-pole axis. These observations are incorporated into a model in which outward forces generated by zones of active cortical dynein are balanced by inward forces produced by nuclear elasticity and during cycle 13, by Ncd, which localizes to interpolar MTs. Thus, the force-balance driving early spindle morphogenesis depends upon MT-based motors acting in concert with the cortex and nucleus.     

Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton.

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44-specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-alpha converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-alpha from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab- or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-alpha. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab- and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.     

The function of intermediate filaments in cell shape and cytoskeletal integrity

This study describes the development and use of a specific method for disassembling intermediate filament (IF) networks in living cells. It takes advantage of the disruptive effects of mimetic peptides derived from the amino acid sequence of the helix initiation 1A domain of IF protein chains. The results demonstrate that at 1:1 molar ratios, these peptides disassemble vimentin IF into small oligomeric complexes and monomers within 30 min at room temperature in vitro. Upon microinjection into cultured fibroblasts, these same peptides induce the rapid disassembly of IF networks. The disassembly process is accompanied by a dramatic alteration in cell shape and the destabilization of microtubule and actin-stress fiber networks. These changes in cell shape and IF assembly states are reversible. The results are discussed with respect to the roles of IF in cell shape and the maintenance of the integrity and mechanical properties of the cytoplasm, as well as the stability of the other major cytoskeletal systems.     

Characterization of cytoskeleton mechanical properties and 3D-actin structure in twisted adherent epithelial cells

Evaluation of the cytoskeleton mechanical properties requires specific micromanipulation techniques such as the magnetic twisting cytometry technique, in which microbeads are specifically linked to the cytoskeleton via transmembrane receptors. The aim of the study was to assess the structural relationship between the bead and the cytoskeleton structure. The spatial arrangement of the CSK network was therefore studied in fixed cells probed by beads and stained for F-actin by rhodamined phallo?dine. The spatial character of the actin CSK network, both in the bead neighborhood and at the cell scale, could then be studied for various degrees of fluorescent intensity from 3D-images of the actin structure, reconstructed from z-stack views obtained by confocal microscopy. Results show the feasibility of the staining/reconstruction technique which allows to reveal the three-dimensional organization of the cytoskeleton structure including an internal cytosolic structure with a high fluorescent F-actin intensity, and a sub-membranous cortical structure with a low fluorescent F-actin intensity.     

Phenotype modulation in non-adherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure

We characterised two sublines of Walker carcinosarcoma cells generated by epigenetic changes. Subline 1 cells were mostly polarised and made no or only non-adhesive cell-substratum contacts. Subline 2 cells were spread, adhesive and mainly non-polar. Subline 1 cells migrate in a non-adhesive mode which is very efficient but operates only in a 3D environment, whereas subline 2 cells migrate in an adhesive mode, which is less efficient but works on 2D and 3D substrata. Nocodazole had little or no effect on shape, polarity and locomotion of subline 1 cells. In glass-adherent subline 2 cells, 10(-6)M nocodazole increased the proportion of polarised cells migrating in an adhesive mode and decreased adhesion to the substratum, whereas 10(-5)M nocodazole further reduced the contacts and the cells reverted to a non-adhesive mode of locomotion. When non-polar subline 2 cells were detached mechanically or by nocodazole, they became polarised and morphologically indistinguishable from non-adherent subline 1 cells. On more adhesive plastic substrata, subline 2 cells produced heterogeneous responses to nocodazole including loss of polarity. The phenotypes of Walker carcinosarcoma sublines have similarities with a broad range of cell types ranging from leucocytes to fibroblast-like cells, suggesting that these phenotypic differences can be controlled by the adhesive and contractile state rather than the cell type. Adhesion modulates contractility (isometric or isotonic contraction) and vice versa and this determines morphology (shape, F-actin, myosin and alpha-actinin), locomotion and responses to microtubule-disassembly. The model may be applied to analyse the mechanisms controlling the phenotype of cells in general.     

The role of deformability of endothelial cells in the arterial response to shear tension

Treatment of the femoral artery luminal surface with glutaraldehyde dimere or dithiosuccinimidyl propionate reduced or eliminated flow-induced dilation, the responses to acetylcholine and the ATP being preserved. The findings suggest that the endothelial cells perceive changes in shear stress and that the cell stiffness is a factor subject to the influence of the magnitude of flow-induced arterial dilation.     

Gravisensitivity of endothelial cells: the role of cytoskeleton and adhesion molecules

The review addresses the effect of microgravity on the endothelial cells, an important mechanosensory element of the cardiovascular system that is known to undergo functional changes in space flight. The chalanges that arise in performing space flight experiments are presented, as well as approaches used to simulate microgravity effects in vitro. The role of cytoskeletal elements as the putative gravity sensors in the cells is demonstrated. The changes in the expression of adhesion molecules that may underlie the mechanisms of gravity sensing by endothelial cells are described. The possible reasons for the discrepancies between the results obtained, such as the differences between the cell lines and experimental design, the variation in time of cultivation, and the specific spaceflight related factors, are analyzed.     

Allograft cytotoxicity: critical role for adherent T cells in effector mechanism of the peritoneal population.

C57BL10 (H-2^b) mice were immunized intraperitoneally with the P815 (H-2^d) mastocytoma. Cytolytic activity measured by the ⁵¹Cr-release assay and cytostatic activity measured by ¹²⁵I-labeled UdR incorporation were assessed in the peritoneal population 12 days after tumor inoculation. Both antitumor effects were immunologically specific and totally dependent on T cells. In the adherent fraction of the immune peritoneal population cytostasis was quantitatively of greater significance than cytolysis. Despite the predominance of macrophages as identified functionally and morphologically, cytostatic activity of the adherent fraction appeared to depend on a small proportion (<13%) of adherent T cells. In addition, a macrophage of highly characteristic morphology was identified in the adherent population. This cell was relatively large and packed with large osmophilic granules.