Conformational states during vinculin unlocking differentially regulate focal adhesion properties.

Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the detailed molecular mechanisms underlying FA mechanosensitivity are still unclear. Besides talin, another key candidate for regulating FA-associated mechanosensing, is vinculin, a prominent FA component, which possesses either closed ("auto-inhibited") or open ("active") conformation. A direct experimental demonstration, however, of the conformational transition between the two states is still absent. In this study, we combined multiple structural and biological approaches to probe the transition from the auto-inhibited to the active conformation, and determine its effects on FA structure and dynamics. We further show that the transition from a closed to an open conformation requires two sequential steps that can differentially regulate FA growth and stability.

Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin.

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.

The fidelity of stochastic single-molecule super-resolution reconstructions critically depends upon robust background estimation.

The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.

Phosphorylation primes vinculin for activation.

Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues-Y100, S1033, S1045, and Y1065-are phosphorylated by kinases during focal adhesion maturation. In this study, phosphorylation at each of these residues is simulated using molecular dynamics models. The simulations demonstrate that once each phosphorylated vinculin structure is at equilibrium, significant local conformational changes result that may impact either vinculin activation or vinculin binding to actin and PIP2. Simulation of vinculin activation after phosphorylation shows that the added phosphoryl groups can prime vinculin for activation. It remains to be seen if vinculin can be phosphorylated at S1033 in vivo, but these simulations highlight that in the event of a S1033 phophorylation vinculin will likely be primed for activation.

Activation and differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells and exhibit two main characteristics that define stem cells: self-renewal and differentiation. MSCs can migrate to sites of injury, inflammation, and tumor. Moreover, MSCs undergo myofibroblast-like differentiation, including increased production of alpha smooth muscle actin (α-SMA) in response to transforming growth factor-ß (TGF-ß), a growth factor commonly secreted by tumor cells to evade immune surveillance. Based on our previous finding, hMSCs become activated and resemble carcinoma-associated myofibroblasts upon prolonged exposure to conditioned medium from MDAMB231 human breast cancer cells. Here, we show that keratinocyte-conditioned medium (KCM) induces differentiation of MSCs to resemble dermal myofibroblast-like cells using immunofluorescence techniques demonstrating punctate vinculin staining, and F-actin filaments.

Molecular dynamics study of talin-vinculin binding.

Cells can sense mechanical force in regulating focal adhesion assembly. One vivid example is the force-induced recruitment of vinculin to reinforce initial contacts between a cell and the extracellular matrix. Crystal structures of the unbound proteins and bound complex between the vinculin head subdomain (Vh1) and the talin vinculin binding site 1 (VBS1) indicate that vinculin undergoes a conformational change upon binding to talin. However, the molecular basis for this event and the precise nature of the binding pathway remain elusive. In this article, molecular dynamics is used to investigate the binding mechanism of Vh1 and VBS1 under minimal constraints to facilitate binding. One simulation demonstrates binding of the two molecules in the complete absence of external force. VBS1 makes early hydrophobic contact with Vh1 by positioning the critical hydrophobic residues (L608, L615, and L622) in the groove formed by helices 1 and 2 of Vh1. The solvent-exposed hydrophobic residues (V619 and L623) then gradually penetrate the hydrophobic core of Vh1, thus further separating helix 1 from helix 2. These critical residues are highly conserved as large hydrophobic side groups in other vinculin binding sites; studies also have demonstrated that these residues are essential in Vh1-VBS1 binding. Similar binding mechanisms are also demonstrated in separate molecular dynamics simulations of Vh1 binding to other vinculin binding sites both in talin and alpha-actinin.

How force might activate talin's vinculin binding sites: SMD reveals a structural mechanism.

Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486-889 constituting helices H1-H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1-H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated alpha-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds.

Solution structure of the first SH3 domain of human vinexin and its interaction with vinculin peptides.

Solution structure of the first Src homology (SH) 3 domain of human vinexin (V_SH3_1) was determined using nuclear magnetic resonance (NMR) method and revealed that it was a canonical SH3 domain, which has a typical beta-beta-beta-beta-alpha-beta fold. Using chemical shift perturbation and surface plasmon resonance experiments, we studied the binding properties of the SH3 domain with two different peptides from vinculin hinge regions: P856 and P868. The observations illustrated slightly different affinities of the two peptides binding to V_SH3_1. The interaction between P868 and V_SH3_1 belonged to intermediate exchange with a modest binding affinity, while the interaction between P856 and V_SH3_1 had a low binding affinity. The structure and ligand-binding interface of V_SH3_1 provide a structural basis for the further functional study of this important molecule.

The effect of combined hypergravity and micro-grooved surface topography on the behaviour of fibroblasts.

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and micro-grooved substrata (groove depth: 1 mum, width: 1, 2, 5, 10 microm), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and alpha(1)- and beta(1)-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces.

Microfabricated discontinuous-edge surface topographies influence osteoblast adhesion, migration, cytoskeletal organization, and proliferation and enhance matrix and mineral deposition in vitro.

The fabrication of surfaces that stimulate increased adhesion, migration, and differentiated function of osteoblasts has been viewed as being desirable for many orthopedic applications. Previous studies have shown that microfabricated pits and grooves alter adhesion, spreading, matrix secretion, and production of mineral by rat calvarial osteoblasts (RCOs). The mechanisms underlying these effects are unknown, although microenvironment and cell alignment are considered to play a role. The aim of this work was to investigate the behavior of RCOs on microfabricated discontinuous-edge surfaces (DESs), which could provide an alternative means to control both the microenvironment and cellular alignment. Two types of discontinuous-type structures were employed, gap-cornered boxes and micron scale pillars. DES gap-cornered boxes and the pillars influenced the arrangement of F-actin, microtubules, and vinculin. Osteoblasts were guided in their direction of migration on both types of substrata. Both box DESs and pillars altered the staining intensity and localization pattern of phosphotyrosine and src-activated FAK localization. Cell multilayering, matrix deposition, and mineralization were enhanced on both discontinuous topographies when compared with smooth controls. This study shows that DESs alter adhesion, migration, and proliferative responses from osteoblasts at early time points (<1 week) and promote multilayering, matrix deposition, and mineral deposition at later times (2-6 weeks). Such topographical patterns could potentially be employed as effective surface features on bone-contacting implants or in membrane-based periodontal applications.

Three-dimensional structure of vinculin bound to actin filaments.

Vinculin plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors, integrin and cadherin. We used a combination of electron microscopy, computational docking, and biochemistry to provide an atomic model of how the vinculin tail binds actin filaments. The vinculin tail actin binding site comprises two distinct regions. One of these regions is exposed in the full-length autoinhibited conformation of vinculin, whereas the second site is sterically occluded by vinculin's N-terminal domain. The partial accessibility of the F-actin binding site in the autoinhibited full-length vinculin structure suggests that F-actin can act as part of a combinatorial input framework with other binding partners such as alpha-catenin or talin to induce vinculin head-tail dissociation, thus promoting vinculin activation. Furthermore, binding to F-actin potentiates a local rearrangement in the vinculin tail that in turn promotes vinculin dimerization and, hence, formation of actin bundles.

Effects of two-step freezing on the ultra-structural components of murine osteoblast cultures.

Understanding the ultra-structural response of cells to the cryopreservation process is important for designing cryopreservation strategies for cells and tissues. Cell-cell interaction and cell-scaffold interactions alter cryopreservation response and, in turn, the cellular structures involved in adhesion and intercellular contact are possible targets of cryopreservation-induced damage. Immuno-fluorescence was used to assess the status of the actin filaments (F-actin), focal adhesions (vinculin) and gap junctions (connexin-43) of murine osteoblasts attached to hydroxyapatite (HA) discs and plastic coverslips for a two-step freezing process. The freezing process de-polymerized and distorted the actin filaments of dead cells, while those of live cells experienced little change. Vinculin and connexin-43 structures were rarely seen in dead cells, while a portion of vinculin (8.14+/-2.27 percent) and connexin-43 (21.7+/-4.7 percent) structures remained in live cells. These results suggest that focal adhesions and gap junctions may support cell robustness during cryopreservation. The present study contributes to our knowledge of the damage mechanisms associated with attached cells during a freezing process.

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells.

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high-resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non-polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR-FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP-vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR-FM and wide-field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR-FSM affords major improvements in these parameters compared with wide-field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low-level GFP-fusion protein expression is a useful tool for FSM imaging. When used in time-lapse mode, TIR-FSM of actin and GFP-conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.

The beta1 integrin subunit is not a specific component of the costamere domain in human myocardial cells.

Studies on altered integrin receptor expression during cardiac hypertrophy and heart failure requires accurate knowledge of the distributional pattern of integrins in myocardial cells. At present the general consensus is that in cardiac muscle the beta1 integrin receptor is mainly localized to the same sarcolemmal domain as vinculin at Z-band levels ('costamere'). Since most previous studies have been focusing on myocardial integrin distribution in lower mammals, the myocardial localization of the beta1 integrin subunit was investigated in biopsies collected from the auricle of patients undergoing a coronary bypass operation. Non-invasive serial optical sectioning was carried out by immuno-laser scanning confocal microscopy. Double-labelling for vinculin/alpha-actinin, and the cytoplasmic domain for the beta1 integrin subunit, showed that beta1 integrin is deposited throughout both the vinculin/alpha-actinin domains and the non-vinculin/alpha-actinin domains. These results were supported by a semi-quantitative analysis in extended focus images of the latter preparations. Higher magnification views at the electron microscopical levels of the large, extracellular domain of the beta1 integrin subunit disclosed a pronounced labelling in the form of a dense, irregular punctuate pattern that was distributed at Z-disc domains as well as along the entire sarcolemmal area between Z-discs. Our findings show that in human, myocardial cells, the beta1 integrin receptor does not only localize to the surface membrane at the Z-disc level ('costamere' in cardiac muscle), but has a widespread distribution along the sarcolemma.

Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy.

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.

The cytoskeleton and related proteins in the human failing heart.

In addition to functional alterations, heart failure has a structural basis as well. This concerns all components of the cardiac myocytes as well as the extracellular space. Proteins of the cardiomyocyte can be subdivided in 5 different categories: 1) Contractile proteins including myosin, actin, tropomyosin and the troponins. 2) Sarcomeric skeleton: titin, myosin binding protein C, alpha-actinin, myomesin, and M-protein. 3) True 'cytoskeletal' proteins: tubulin, desmin and actin. 4) Membrane-associated proteins: dystrophin, spectrin, talin, vinculin, ankyrin and others. 5) Proteins of the intercalated disc: desmosomes consisting of desmoplakin, desmocollin, desmoglein and desmin; adherens junctions with N-cadherin, the catenins and vinculin, and gap junctions with connexin. Failing myocardium obtained from patients undergoing cardiac transplantation exhibits ultrastuctural degeneration and an altered nucleus/cytoplasm relationship. The contractile proteins and those of the sarcomeric skeleton, especially titin, are downregulated, the cytoskeletal proteins desmin and tubulin and membrane-associated proteins such as vinculin and dystrophin are upregulated and those of the intercalated disc are irregularly arranged. Elevation of cytoskeletal proteins correlates well with diastolic and contractile dysfunction in these patients. The enlarged interstitial space contains fibrosis, i.e. accumulations of fibroblasts and extracellular matrix components, in addition to macrophages and microvascular elements. Loss of the contractile machinery and related proteins such as titin and alpha-actinin may be the first and decisive event initiating an adaptive increase in cytoskeleton and membrane associated components. Fibrosis may be stimulated by subcellular degeneration. The hypothesis is put forward that all proteins of the different myocardial compartments contribute to the deterioration of cardiac function in heart failure.

Signaling of de-adhesion in cellular regulation and motility.

Adhesion is a process that can be divided into three separate stages: (1) cell attachment, (2) cell spreading, and (3) the formation of focal adhesions and stress fibers. With each stage the adhesive strength of the cell increases. De-adhesion can be defined as the process involving the transition of the cell from a strongly adherent state, characterized by focal adhesions and stress fibers, to a state of intermediate adherence, represented by a cell that is spread, but that lacks stress fibers terminating at adhesion plaques. We propose that this modification of the structural link between the actin cytoskeleton and the extracellular matrix results in a more malleable cellular state conducive for dynamic processes such as cytokinesis, mitogenesis, and motility. Anti-adhesive proteins, including thrombospondin, tenascin, and SPARC, rapidly signal de-adhesion, potentially mediating proliferation and migration during development and wound healing. Intracellular signaling molecules involved in the regulation of de-adhesion are only beginning to be identified. Interestingly, many of the same signaling proteins recognized to play important roles during the process of adhesion have also been found to act during de-adhesion. Characterization of the precise mechanisms by which these signals modulate adhesive structures and the cytoskeleton will further our understanding of the regulation of adhesive strength and its function in cellular physiology.

Differential protein localization in sarcomeric and nonsarcomeric contractile structures of cultured cardiomyocytes.

The use of cardiomyocyte cell culture models allows the identification of various cell mediators that bring about changes in subcellular structures and gene expression associated with hypertrophy. The effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), and triiodothyronine (T3) on gene expression and on the structural organization of myofibrillar and cytoskeletal proteins were compared in adult atrial (aARC) and ventricular (vARC) as well as in neonatal ventricular rat cardiomyocytes (vNRC) in long-term culture. Structural changes were evaluated by confocal microscopy and correlated to biochemical alterations. In vARC, IGF-I enhanced myofibrillar growth, whereas bFGF or T3 restricted sarcomere assembly to the central cell area, forming a sharp boundary in more than 50% of the cells. However, myosin occurred both in the cross-striated myofibrillar structures and in patches running along the nonsarcomeric fibrillar structures (also called stress fiber-like structures) in the cell periphery. In cells treated with either bFGF or T3, the expression of alpha-smooth muscle actin (alpha-sm actin) was greatly increased. This actin isoform was incorporated mainly into the nonsarcomeric contractile structures outside the area where myofibrils ended abruptly. alpha-sm actin protein increased up to 14- to 17-fold while the mRNA showed a moderate increase of 2- to 4-fold. This suggests that alpha-sm actin is mainly regulated at the translational or posttranslational level. In contrast, the cytoskeletal proteins alpha-actinin and vinculin increased only moderately (less than 2-fold) but also showed a relocalization in cells with restricted myofibrils. In aARC and in vNRC, alpha-sm actin was only moderately upregulated by bFGF or T3 and no drastic morphological changes were observed. In conclusion, IGF-I, bFGF, and T3 induced characteristic structural phenotypes depending on the type of cardiomyocyte. Large amounts of alpha-sm actin as expressed in bFGF and T3 treated vARC seem to be incompatible with sarcomere assembly.

Determining data independence on a digitized membrane in three dimensions.

A method for determining whether structures distributed along a cell's membrane represent a random spatial distribution is presented in this paper. Two three-dimensional (3-D) images are acquired from one cell by wide-field digital imaging of cells which have been labeled with two different fluorescent antibodies. Prior to spatial analysis, a constrained regularized least squares restoration of the images is performed. This is followed by registration via fiducial markers (dual-labeled beads). A deformable model is then used to map data near the surface to the surface. Finally, each resulting data set is analyzed to determine whether it is spatially random. To do this, we generalize the test for complete spatial randomness of points in a plane, to test voxels distributed along a voxelized membrane in three dimensions. We also test whether the distribution of one protein is independent of the distribution of a second protein. The method is applied to compare the distribution of the protein kinase C to that of vinculin. Vinculin is a protein which anchors intracellular filaments to the cell's plasma membrane. It is also used as a (sparse) membrane marker for the deformable model. Protein kinase C facilitates molecular motors inside the cell. These may be associated with actin and myosin filaments.

Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation.

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.