Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.     

Cytoskeletal organization affects cellular responses to cytochalasins: comparison of a normal line and its transformant

The relationships between cytoskeletal network organization and cellular response to cytochalasin D (CD) in a normal rat fibroblast cell line (Hmf-n) and its spontaneous transformant (tHmf-e), with markedly different cytoskeletal phenotypes, were compared (using immunofluorescence, electron microscopy, and DNAse I assay for actin content). Hmf-n have prominent, polar stress fiber (SF) arrays terminating in vinculin adhesion plaques whereas tHmf-e, which are apolar, epithelioid cells with dense plasma membrane-associated actin networks, lack SF and adhesion plaques. Hmf-n exposed to CD become markedly retracted and dendritic, SF-derived actin aggregates form large endoplasmic masses, and discrete tabular aggregates at the distal ends of retraction processes. Prolonged exposure leads to recession of process, cellular rounding, and development of large cystic vacuoles. tHmf-e cells exposed to similar doses of CD display a diagnostically different response; retraction is less drastic, cells retain broad processes containing scattered actin aggregates in discrete foci often associated with plasma membrane, large tabular aggregates are never found and processes persist throughout long exposure, vacuolation is uncommon. The CD-induced microfilamentous aggregates in Hmf-n are composed of short, kinky filament fragments forming a felt-like skein, often aggregates contain a more ordered array of roughly parallel fragments, while those of tHmf-e are very short, kinky, randomly orientated filaments imparting a distinctly granular nature to the mass. Total actin content and the amount of actin associated with detergent-resistant cytoskeletons increase following CD exposure in both cell types. Throughout exposure to CD, the actin-associated contractile proteins tropomyosin, myosin, and alpha-actinin co-localize within the actin aggregates in both cell types. Fodrin, the protein linking cortical actin to membrane, co-localizes with actin aggregates in tHmf-e cells and most, but not all, such aggregates in Hmf-n cells, consistent with their stress fiber derivation. Vinculin is lost from the tabular aggregates at the distal ends of retraction processes in Hmf-n cells concomitant with the fragmentation and contraction of SF. The aborized processes in both cells types contain strikingly similar axial cores of bundled vimentin filaments associated with passively compressed microtubules. The characteristic CD-induced distribution of actin filament aggregates and redistribution of vimentin in these cell types also occur when cells are allowed to respread from the rounded state in the presence of CD.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Intermediate filaments exchange subunits along their length and elongate by end-to-end annealing

Actin filaments and microtubules lengthen and shorten by addition and loss of subunits at their ends, but it is not known whether this is also true for intermediate filaments. In fact, several studies suggest that in vivo, intermediate filaments may lengthen by end-to-end annealing and that addition and loss of subunits is not confined to the filament ends. To test these hypotheses, we investigated the assembly dynamics of neurofilament and vimentin intermediate filament proteins in cultured cells using cell fusion, photobleaching, and photoactivation strategies in combination with conventional and photoactivatable fluorescent fusion proteins. We show that neurofilaments and vimentin filaments lengthen by end-to-end annealing of assembled filaments. We also show that neurofilaments and vimentin filaments incorporate subunits along their length by intercalation into the filament wall with no preferential addition of subunits to the filament ends, a process which we term intercalary subunit exchange.     

A direct interaction between actin and vimentin filaments mediated by the tail domain of vimentin

The assembly and organization of the three major eukaryotic cytoskeleton proteins, actin, microtubules, and intermediate filaments, are highly interdependent. Through evolution, cells have developed specialized multifunctional proteins that mediate the cross-linking of these cytoskeleton filament networks. Here we test the hypothesis that two of these filamentous proteins, F-actin and vimentin filament, can interact directly, i.e. in the absence of auxiliary proteins. Through quantitative rheological studies, we find that a mixture of vimentin/actin filament network features a significantly higher stiffness than that of networks containing only actin filaments or only vimentin filaments. Maximum inter-filament interaction occurs at a vimentin/actin molar ratio of 3 to 1. Mixed networks of actin and tailless vimentin filaments show low mechanical stiffness and much weaker inter-filament interactions. Together with the fact that cells featuring prominent vimentin and actin networks are much stiffer than their counterparts lacking an organized actin or vimentin network, these results suggest that actin and vimentin filaments can interact directly through the tail domain of vimentin and that these inter-filament interactions may contribute to the overall mechanical integrity of cells and mediate cytoskeletal cross-talk.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Actin-Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co-align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co-align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin-binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly-merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross-linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.     

High-affinity interaction of vinculin with actin filaments in vitro

Immunofluorescence and microinjection experiments have shown that vinculin (molecular weight 130,000) is localized at adhesion plaques of fibroblasts spread on a solid substrate. We found that this protein affects actin filament assembly and interactions in vitro at substoichiometric levels. Vinculin inhibits the rate of actin polymerization under conditions that limit nuclei formation, indicating an effect on the filament elongation step of the reaction. Vinculin also reduces actin filament-filament interaction measured with a low-shear viscometer. Scatchard plot analysis of the binding of ³H-labeled vinculin to actin filaments showed that there is one high-affinity binding site (dissociation constant = 20 nM) for every 1,500–2,000 actin monomers. These results suggest that vinculin interacts with a specific site located at the growing ends of actin filaments in a cytochalasin-like manner, a property consistent with its proposed function as a linkage protein between filaments and the plasma membrane.     

Actin-capping protein promotes microtubule stability by antagonizing the actin activity of mDia1

In migrating fibroblasts, RhoA and its effector mDia1 regulate the selective stabilization of microtubules (MTs) polarized in the direction of migration. The conserved formin homology 2 domain of mDia1 is involved both in actin polymerization and MT stabilization, and the relationship between these two activities is unknown. We found that latrunculin A (LatA) and jasplakinolide, actin drugs that release mDia1 from actin filament barbed ends, stimulated stable MT formation in serum-starved fibroblasts and caused a redistribution of mDia1 onto MTs. Knockdown of mDia1 by small interfering RNA (siRNA) prevented stable MT induction by LatA, whereas blocking upstream Rho or integrin signaling had no effect. In search of physiological regulators of mDia1, we found that actin-capping protein induced stable MTs in an mDia1-dependent manner and inhibited the translocation of mDia on the ends of growing actin filaments. Knockdown of capping protein by siRNA reduced stable MT levels in proliferating cells and in starved cells stimulated with lysophosphatidic acid. These results show that actin-capping protein is a novel regulator of MT stability that functions by antagonizing mDia1 activity toward actin filaments and suggest a novel form of actin–MT cross-talk in which a single factor acts sequentially on actin and MTs.     

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.     

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.     

Secondary lysosomes as an integral part of the cytoskeleton: a morphological study in rat Kupffer cells

Rat Kupffer cells contain the three major cytoskeletal components: microfilaments (MF), microtubules (MT), and intermediate filaments (IF) of the vimentin type. Previous cytomagnetometric data obtained from alveolar macrophages and rat Kupffer cells in culture provided evidence that actin filaments contribute to the movements of lysosomes. The lysosomal transport in living cells was affected, when the MFs were selectively disturbed, whereas the depolymerization of the MTs had no effect on the lysosomal movement measured by cytomagnetometric means. Immunofluorescence and ultrastructural studies of isolated and cultured rat Kupffer cells, presented in this paper, will investigate the relationship between lysosomes and the cytoskeleton. The principal filamentous structure in the peripheral cytoplasm of Kupffer cells in a dense meshwork of actin filaments. The dimension of the meshes combined with the dimensions of lysosomes implies the necessity of either (i) disintegration of the actin filament cross-links, (ii) depolarymerization and redistribution of MF's, or (iii) a displacement of actin filaments by the lysosomes during the organelle transport. The presence of microtubules in cytoplasmic protrusions and their track from the periphery to the perinuclear region during interphase might play a role in the transport mechanism of lysosomes, the more so because microtubules could often be demonstrated in closest association with lysosomes even in the first phase of endocytosis. The distribution pattern of vimentin, found as a dense interconnected framework surrounding the lysosomes like a basket, could play a role in positioning the organelles. The dynamic functions of MF's and MT's and their multifunctionality led to an adaptive and flexible organization of these filaments which may both be involved in lysosomal motion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking

Gelsolin is a calcium binding protein that shortens actin filaments. This effect occurs in the presence but not in the absence of micromolar calcium ion concentrations and is partially reversed following removal of calcium ions. Once two actin molecules have bound to gelsolin in solutions containing Ca2+, one of the actins remains bound following chelation of calcium, so that the reversal of gelsolin's effect cannot be accounted for simply by its dissociation from the ends of the shortened filaments to allow for elongation. In this paper, the interactions with actin of the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stable 1:1 gelsolin-actin complexes are compared with those of free gelsolin. The abilities of free or complexed gelsolin to sever actin filaments, nucleate filament assembly, bind to the fast growing (+) filament ends, and lower the filament size distribution in the presence of either Ca2+ or EGTA were examined. The results show that both free gelsolin and gelsolin-actin complexes are highly dependent on Ca2+ concentration when present in a molar ratio to actin less than 1:50. The gelsolin-actin complexes, however, differ from free gelsolin in that they have a higher affinity for (+) filament ends in EGTA and they cannot sever filaments in calcium. The limited reversal of actin-gelsolin binding following removal of calcium and the calcium sensitivity of nucleation by complexes suggest an alternative to reannealing of shortened filaments that involves redistribution of actin monomers and may account for the calcium-sensitive functional reversibility of the solation of actin by gelsolin.     

Viscoelastic properties of vimentin compared with other filamentous biopolymer networks

The cytoplasm of vertebrate cells contains three distinct filamentous biopolymers, the microtubules, microfilaments, and intermediate filaments. The basic structural elements of these three filaments are linear polymers of the proteins tubulin, actin, and vimentin or another related intermediate filament protein, respectively. The viscoelastic properties of cytoplasmic filaments are likely to be relevant to their biologic function, because their extreme length and rodlike structure dominate the rheologic behavior of cytoplasm, and changes in their structure may cause gel-sol transitions observed when cells are activated or begin to move. This paper describes parallel measurements of the viscoelasticity of tubulin, actin, and vimentin polymers. The rheologic differences among the three types of cytoplasmic polymers suggest possible specialized roles for the different classes of filaments in vivo. Actin forms networks of highest rigidity that fluidize at high strains, consistent with a role in cell motility in which stable protrusions can deform rapidly in response to controlled filament rupture. Vimentin networks, which have not previously been studied by rheologic methods, exhibit some unusual viscoelastic properties not shared by actin or tubulin. They are less rigid (have lower shear moduli) at low strain but harden at high strains and resist breakage, suggesting they maintain cell integrity. The differences between F-actin and vimentin are optimal for the formation of a composite material with a range of properties that cannot be achieved by either polymer alone. Microtubules are unlikely to contribute significantly to interphase cell rheology alone, but may help stabilize the other networks.     

Organization and expression of Drosophila tropomyosin genes

It has been shown (Jockusch &; Isenberg, 1981) that vinculin (130K protein) binds to actin and induces actin filaments to form bundles even at low ionic strength. Here, we present structural details on the vinculin molecule itself and on its interaction with actin. In negatively stained preparations, vinculin appeared as a globular protein with an average diameter of 85 Å. The ability of vinculin to form actin filament bundles was confirmed using shadowing techniques and gel analysis of sedimented material. Analysis of vinculin-induced paracrystals by optical diffraction and computer processing revealed their structural similarity to Mg-induced paracrystals. The lateral position of vinculin on surface-exposed actin filaments of such paracrystals was demonstrated directly in electron micrographs and indirectly by labelling vinculin with ferritin-coupled anti-vinculin F(ab′) fragments. Polymerization of actin in the presence of vinculin-coated polystyrene beads did not result in an “end-on” binding of filaments to the beads. Rather, actin bundles were laterally associated with the whole surface of the beads, from where they radiated in a star-like pattern. The growth of actin filaments onto myosin subfragment-I decorated, vinculin-incubated. fixed filament fragments was not inhibited, as was shown directly by electron microscopy and monitored viscometrically in a nucleation assay. These results suggest that in vivo at the site of an adhesion plaque vinculin may link actin filaments together into a suitable configuration to interact with the plasma membrane.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodamine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. The enhancement of binding of the beads by TPA was suppressed by addition of an adhesion-inhibitory peptide (Gly-Arg-Gly-Asp-Ser-Pro). Treatment with TPA did not enhance nonspecific binding of beads coated with heat-denatured bovine serum albumin. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.     

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules

We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.     

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.