Wolfgang H. Goldmann

Mechano-coupling and regulation of contractility by the vinculin tail domain.

Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site.

Vinculin Facilitates Cell Invasion into Three-dimensional Collagen Matrices

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

Talin binds to actin and promotes filament nucleation

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non‐interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase‐I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G‐buffer conditions. The overall binding constant was ≈ 0.25 μM.

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine—phalloidin labelling of actin filaments, Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein–lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of artin filament growth at lipid interfaces have been visualized.

Becoming stable and strong: the interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation.

The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.

Focal Adhesion Kinase Stabilizes the Cytoskeleton

Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK-/-), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK-/- cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.

Analysis of filamin and α-actinin binding to actin by the stopped flow method

We ascertained by the stopped flow method the overall association rate constant, k +1, of filamin and α‐actinin to fluorescently labelled filamentous actin of ~ 1.3 × 106M−1 · s−1 and ~ 1.0 × 106M−1 · s−1 as well as the overall dissociation rate constant,k −1 of ~ 0.6s−1 and ~ 0.4s−1, respectively. The overall equilibrium constant, K, for filamin and α‐actinin to actin deduced from the relation K = agree well with published data.

Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle

The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD.

Vinculin, cell mechanics and tumour cell invasion

The focal adhesion protein, vinculin, is important for transmitting mechanical forces and orchestrating mechanical signalling events. Deregulation of vinculin results in altered cell adhesion, contractility, motility and growth, all of which are important processes in cancer metastasis. This review summarises recent reports on the role of vinculin in cellular force generation and signalling, and discusses implications for a role of vinculin in promoting cancer cell migration in 3D environments.