Interaction of talin with actin: sensitive modulation of filament crosslinking activity.

Talin is an adhesion plaque protein believed important in linking actin filaments to the plasma membrane. The nature of a direct talin-actin interaction, however, is complex and has remained unclear. We have systematically characterized the effects of pH, ionic strength, temperature, and protein molar ratio on the interaction between highly purified talin and actin. The ability of talin to increase viscosity of F-actin at 25 degrees C and low ionic strength increased with decreasing pH from 7.3 to 6.4 and increasing molar ratio of talin to actin. At pH 6.4 and low ionic strength, talin could extensively crosslink actin filaments into ordered bundles as shown by negative staining and could cosediment with F-actin at molar ratios as high as one talin to two actin monomers. Talin crosslinked prepolymerized actin filaments to a similar extent as actin filaments polymerized in its presence. The 190-kDa calpain-generated proteolytic fragment of talin bound poorly to actin under conditions favorable for intact talin, but was able to crosslink actin filaments at a lower pH. Increasing the ionic strength within a relatively narrow range significantly decreased ability of talin to bind to actin, regardless of pH. The effects of pH and ionic strength on the talin-actin interaction were rapid and reversible. Low-shear-viscosity studies revealed a strong temperature dependence in the talin-actin interaction with significant crosslinking activity at physiological-like ionic conditions and temperature (37 degrees C). Our results consistently demonstrated that talin crosslinks actin filaments and that this direct interaction is highly sensitive to, and dependent upon, ionic conditions and temperature.     

Characterization of an actin-binding site within the talin FERM domain

Talin is a large cytoskeletal protein that couples integrins to F-actin. Three actin-binding sites (ABS1-3) have been reported: one in the N-terminal head, and two in the C-terminal rod domain. Although the C-terminal ABS3 has been partially characterized, the presence and properties of ABS1 within the talin head are less well defined. We show here that the talin head binds F-actin in vitro and in vivo at a specific site within the actin filament. Thus, purified talin head liberated from gizzard talin by calpain cleavage cosediments with F-actin in a low salt buffer at pH 6.4 (conditions that are optimal for binding intact talin), and using recombinant polypeptides, we have mapped ABS1 to the FERM domain within the talin head. Both the F2 and F3 FERM subdomains contribute to binding, and EGFP-tagged FERM subdomains colocalize with actin stress fibers when expressed in COS cells. High-resolution electron microscopy of actin filaments decorated with F2F3 localizes binding to a site that is distinct from that recognized by members of the calponin-homology superfamily. Finally, we show that the FERM domain can couple F-actin to PIPkin, and by inference to integrins, since they bind to the same pocket in the F3 subdomain. This suggests that the talin FERM domain functions as a linker between PIPkin or integrins and F-actin at sites of cell-matrix adhesions.     

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.     

Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

Background  

Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene.     

Talin rod domain–containing protein 1 (TLNRD1) is a novel actin-bundling protein which promotes filopodia formation

Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.     

A tal(in) of cell spreading

Talin can activate integrins to bind the extracellular matrix and also connect matrix-engaged integrins to the actin cytoskeleton. New work shows that cell spreading can be dissected into three distinct phases according to their differential requirements for talin function.     

Accumulation of talin in nodes at the edge of the lamellipodium and separate incorporation into adhesion plaques at focal contacts in fibroblasts

The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F-actin at the membrane.     

Coincidence of actin filaments and talin is required to activate vinculin

Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to the head domain of vinculin and F-actin to its tail domain is a potential mechanism for this function, but vinculin is autoinhibited by intramolecular interactions between its head and tail domain and must be activated to bind talin and actin. Because autoinhibition of vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea we use a fluorescence resonance energy transfer probe that reports directly on activation of vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin alone can activate vinculin. But in the presence of F-actin either talin rod or VBS3 induces dose-dependent activation of vinculin. The activation data are supported by solution phase binding studies, which show that talin rod or VBS3 fails to bind vinculin, whereas the same two ligands bind tightly to vinculin head domain (K(d) approximately 100 nM). These data strongly support a combinatorial mechanism of vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate vinculin. Combinatorial activation implies that at cell adhesion sites vinculin is a coincidence detector awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity.     

Regulation of endothelial nitric oxide synthase by the actin cytoskeleton

In the present study, the association ofendothelial nitric oxide synthase (eNOS) with the actin cytoskeleton inpulmonary artery endothelial cells (PAEC) was examined. We found thatthe protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAECresulted in the coprecipitation of actin, and immunoprecipitation ofactin from lysates of caveolar fractions resulted in thecoprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS waslabeled with fluorescein, F-actin was labeled with Texasred-phalloidin, and G-actin was labeled with deoxyribonuclease Iconjugated with Texas red, also demonstrated an association betweeneNOS and F-actin or G-actin. Incubation of purified eNOS with purifiedF-actin and G-actin resulted in an increase in eNOS activity. Theincrease in eNOS activity caused by G-actin was much higher than thatcaused by F-actin. Incubation of PAEC with swinholide A, an actinfilament disruptor, resulted in an increase in eNOS activity, eNOSprotein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activitywas higher than that in eNOS protein content in swinholide A-treatedcells. In contrast, exposure of PAEC to phalloidin, an actin filamentstabilizer, caused decreases in eNOS activity and association of eNOSwith G-actin and increases in association of eNOS with F-actin. Theseresults suggest that eNOS is associated with actin in PAEC and thatactin and its polymerization state play an important role in theregulation of eNOS activity.

     

The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo ¹H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β‐integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F‐actin through the agency of its five‐helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α‐helices coined vinculin binding sites (VBSs) that reside within numerous four‐ and five‐helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N‐terminal seven‐helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head‐tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five‐helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2‐vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo 1H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ～5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Augmentation of alpha-actinin-induced gelation of actin by talin.

Interactions among the three major constituents of focal adhesions, talin, actin, and alpha-actinin, were studied. No evidence was obtained for the direct interaction between talin and alpha-actinin. Both talin and alpha-actinin increased the rate and extent of polymerization of actin, and their effects were additive. Whereas talin alone exhibited very little actin-gelating activity, it potentiated markedly the gelation in the presence of alpha-actinin and lowered the concentration of alpha-actinin necessary for the gel formation. Its gelation-potentiating activity on prepolymerized actin was much smaller than observed on G-actin. Treatment of talin with a cross-linking reagent, 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide or dimethyl suberimidate, resulted in the formation of its oligomeric polypeptides. The complexes of talin and G-actin were also demonstrated with the cross-linking reagents and fluorescence-labeled actin. These results indicate that talin is able to cross-link some limited regions of actin filaments.     

Kinetic determination of talin-actin binding

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.     

Talin2 is induced during striated muscle differentiation and is targeted to stable adhesion complexes in mature muscle

The cytoskeletal protein talin serves as an essential link between integrins and the actin cytoskeleton in several similar, but functionally distinct, adhesion complexes, including focal adhesions, costameres, and intercalated disks. Vertebrates contain two talin genes, TLN1 and TLN2, but the different roles of Talin1 and Talin2 in cell adhesion are unclear. In this report we have analyzed Talin1 and Talin2 in striated muscle. Using isoform-specific antibodies, we found that Talin2 is highly expressed in mature striated muscle. Using mouse C2C12 cells and primary human skeletal muscle myoblasts as models of muscle differentiation, we show that Talin1 is expressed in undifferentiated myoblasts and that Talin2 expression is upregulated during muscle differentiation at both the mRNA and protein levels. We have also identified regulatory sequences that may be responsible for the differential expression of Talin1 and Talin2. Using GFP-tagged Talin1 and Talin2 constructs, we found that GFP-Talin1 targets to focal adhesions while GFP-Talin2 targets to abnormally large adhesions in myoblasts. We also found that ectopic expression of Talin2 in myoblasts, which do not contain appreciable levels of Talin2, dysregulates the actin cytoskeleton. Finally we demonstrate that Talin2, but not Talin1, localizes to costameres and intercalated disks, which are stable adhesions required for the assembly of mature striated muscle. Our results suggest that Talin1 is the primary link between integrins and actin in dynamic focal adhesions in undifferentiated, motile cells, but that Talin2 may serve as the link between integrins and the sarcomeric cytoskeletonin stable adhesion complexes in mature striated muscle.     

The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments

Previously, we have shown that the V-ATPase holoenzyme as well as the V1 complex isolated from the midgut of the tobacco hornworm (Manduca sexta) exhibits the ability of binding to actin filaments via the V1 subunits B and C (Vitavska, O., Wieczorek, H., and Merzendorfer,H. (2003) J. Biol. Chem. 278, 18499-18505). Since the recombinant subunit C not only enhances actin binding of the V1 complex but also can bind separately to F-actin, we analyzed the interaction of recombinant subunit C with actin. We demonstrate that it binds not only to F-actin but also to monomeric G-actin. With dissociation constants of approximately 50 nm, the interaction exhibits a high affinity, and no difference could be observed between binding to ATP-G-actin or ADP-G-actin, respectively. Unlike other proteins such as members of the ADF/cofilin family, which also bind to G- as well as to F-actin, subunit C does not destabilize actin filaments. On the contrary, under conditions where the disassembly of F-actin into G-actin usually occurred, subunit C stabilized F-actin. In addition, it increased the initial rate of actin polymerization in a concentration-dependent manner and was shown to cross-link actin filaments to bundles of varying thickness. Apparently bundling is enabled by the existence of at least two actin-binding sites present in the N- and in the C-terminal halves of subunits C, respectively. Since subunit C has the possibility to dimerize or even to oligomerize, spacing between actin filaments could be variable in size.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

The structure of the C-terminal actin-binding domain of talin

Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.     

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.