Interaction Of Alpha-actinin Research Articles

Phosphoinositide Binding Regulates α-Actinin Dynamics: MECHANISM FOR MODULATING CYTOSKELETAL REMODELING

The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of alpha-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-alpha-actinin and a GFP-alpha-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039-24045). In fibroblasts, recovery of the mutant alpha-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, alpha-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of alpha-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant alpha-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the alpha-actinin mutant inhibited actin reorganization with the appearance of enlarged alpha-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of alpha-actinin with actin filaments and integrin adhesion receptors and that the dynamics of alpha-actinin is important for PI 3-kinase-induced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of alpha-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.

Phosphoinositide Binding Inhibits α-Actinin Bundling Activity

alpha-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to alpha-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627- 642). In this study, phosphoinositide binding and regulation of alpha-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that alpha-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of alpha-actinin. Phosphoinositide binding inhibited the bundling activity of alpha-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments was observed in fibroblasts expressing an alpha-actinin mutant with decreased phosphoinositide affinity. We conclude that the interaction of alpha-actinin with phosphoinositides regulates actin stress fibers in the cell by controlling the extent to which microfilaments are bundled.

The interaction of titin and alpha-actinin is controlled by a phospholipid-regulated intramolecular pseudoligand mechanism.

The assembly of stable cytoskeletal structures from dynamically recycled molecules requires developmental and spatial regulation of protein interactions. In muscle, titin acts as a molecular ruler organizing the actin cytoskeleton via interactions with many sarcomeric proteins, including the crosslinking protein alpha-actinin. An interaction between the C-terminal domain of alpha-actinin and titin Z-repeat motifs targets alpha-actinin to the Z-disk. Here we investigate the cellular regulation of this interaction. alpha-actinin is a rod shaped head-to-tail homodimer. In contrast to C-terminal fragments, full-length alpha-actinin does not bind Z-repeats. We identify a 30-residue Z-repeat homologous sequence between the actin-binding and rod regions of alpha-actinin that binds the C-terminal domain with nanomolar affinity. Thus, Z-repeat binding is prevented by this 'pseudoligand' interaction between the subunits of the alpha-actinin dimer. This autoinhibition is relieved upon binding of the Z-disk lipid phosphatidylinositol-bisphosphate to the actin-binding domain. We suggest that this novel mechanism is relevant to control the site-specific interactions of alpha-actinin during sarcomere assembly and turnover. The intramolecular contacts defined here also constrain a structural model for intrasterical regulation of all alpha-actinin isoforms.

Exchange of alpha-actinin in isolated rigor myofibrils.

In the current study, the process of alpha-actinin binding to the myofibrillar Z-line was investigated to determine its mechanism. Pretreatment of rigor myofibrils with unlabeled alpha-actinin did not prevent or slow the incorporation of fluorescein skeletal alpha-actinin into myofibrils suggesting that incorporation was not the filling of empty binding sites but rather an exchange reaction. Further support for this was obtained using quantitative measures of labeled alpha-actinin incorporation and measures of total myofibrillar alpha-actinin. These results showed that there was no change in myofibrillar alpha-actinin content when up to 15% of the total alpha-actinin was the labeled protein. Measurement of the time-course of fluorescein alpha-actinin incorporation by quantitative fluorescence microscopy showed that the increase in Z-line fluorescence was well described by a rapid (unresolved) incorporation of fluorescence followed by a much slower phase. The slower phase was independent of fluorescein alpha-actinin concentration (2.5-160 nM) and had an apparent rate of 0.008-0.016 min(-1). Pretreatment of myofibrils with fluorescein alpha-actinin followed by incubation with unlabeled alpha-actinin resulted in a decrease in Z-line fluorescence with an apparent rate of 0.021 min(-1). The slow phase was interpreted as representing the dissociation rate of intrinsic Z-line alpha-actinin. Thus, the dissociation rate for the in situ interaction of alpha-actinin with actin appears to be three orders of magnitude slower than that determined from solution studies.

Evidence for an alpha-granular pool of the cytoskeletal protein alpha-actinin in human platelets that redistributes with the adhesive glycoprotein thrombospondin-1 during the exocytotic process.

In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein alpha-actinin in a solid-phase binding assay. Stored in the alpha-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. alpha-Actinin is an actin-binding and actinin-cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of alpha-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence. alpha-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of alpha-actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as alpha-granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both alpha-actinin and TSP-1 were seen redistributing from the alpha-granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic alpha-actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of 125I-surface-labeled, thrombin-activated platelets further demonstrated that alpha-actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could from molecular complexes with TSP-1 on activated platelets. These results suggest that alpha-actinin found to be present on the platelet surface together with TSP-1 originates in the alpha-granules by fusion of the alpha-granules with the plasma membrane during platelet exocytosis.

Interactions between smooth muscle alpha-actinin and lipid bilayers.

alpha-Actinin has been proposed to be the actin-plasma membrane linker. This assumption is based on the discovery of direct interaction of alpha-actinin with two specific lipids, diacylglycerol and palmitic acid [Burn, P. (1988) Trends Biochem. Sci. 13, 79-83]. In our study, the binding of alpha-actinin with vesicles containing negatively charged phospholipids was measured by the method of 90 degrees light-scattering. Our results show that alpha-actinin is able to bind membranes containing negatively charged phospholipids, but not to bind membranes composed of neutral lipids only. Diacylglycerol and palmitic acid, on the other hand, have little effect on the binding of alpha-actinin to lipid vesicles. Analysis of binding isotherms in terms of a membrane binding model gave apparent dissociation constants which varied between 0.2 and 3 microM over a range of 5-20 mol % negatively charged phospholipid. Comparing the kinetics of alpha-chymotrypsin digestion of alpha-actinin in solution to those of vesicle-bound alpha-actinin, it can be seen that the cleavage site at the junction between the C-terminal and the central rod domain of alpha-actinin and another cleavage site on the C-terminal domain can be most effectively protected by its membrane binding. Analysis of the amide I and II regions of Fourier-transform infrared spectra of alpha-actinin revealed that the association of alpha-actinin with negatively charged phospholipid vesicles resulted in some perturbation of the protein secondary structure. Monolayers containing negatively charged phospholipid were layered and incubated on the surface of a polymerization solution of actin and alpha-actinin, and observed with an electron microscope. The results show that the bundle structure of actin filaments can be formed if diacylglycerol and palmitic acid are present in lipid layers.

Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin.

Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E-, P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta-catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha-actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin-independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin.

Actin binding to lipid-inserted alpha-actinin

The interaction of alpha-actinin with lipid films and actin filaments was investigated. First alpha-actinin was incorporated in lipid films at the air/water interface. Injection of alpha-actinin into the subphase of a lipid monolayer led to a significant increase of the surface pressure only for lipid films consisting of a mixture of a negatively charged lipid with a high proportion of diacylglycerol. These alpha-actinin-containing films were transferred onto silanized quartz slides. Photobleaching experiments in the evanescent field allowed quantification of the lateral number density of the lipid-bound alpha-actinin. In combination with the area increase from the monolayer experiments, the photobleaching measurements suggest that alpha-actinin is incorporated into the lipid film in such a way that actin binding sites are accessible from the bulk phase. Binding experiments confirmed that the alpha-actinin selectively binds actin filaments in this configuration. We also showed that, in contrast to actin filaments which are adsorbed directly onto planar surfaces, the alpha-actinin-bound actin filaments are recognized and cleaved by the actin-severing protein gelsolin. Thus we have constructed an in vitro system which opens new ways for investigations of membrane-associated actin-binding proteins and of the physical behavior of actin filaments in the close neighborhood to membranes.

Diffusion hindrance and geometry of filament crossings account for the complex interactions of F-actin with alpha-actinin from chicken gizzard.

The interaction of alpha-actinin from chicken gizzard with F-actin is quite complex. The apparent dissociation constant, C, increases with the increase of actin concentration according to the following expression: C = Ko + a[actin] - c[actin]5/2. At pH 7.5 and 37 degrees C, in the presence of 0.1 M KCl and 2 mM MgCl2, the dissociation constant at infinite actin dilution, Ko, is 2.17 microM. The binding of alpha-actinin to actin is related by the term a[actin] to the diffusion of actin filaments and by the term c[actin]5/2 to the crossing number concentration of the F-actin network. Especially at low actin concentration, the binding of alpha-actinin to actin is increased by gelsolin, which fragments actin filaments and increases their diffusion. The different binding isotherms of alpha-actinin to actin filaments and to actin bundles are discussed.

Alpha-actinin and vinculin in human neutrophils: reorganization during adhesion and relation to the actin network.

We have studied the reorganization of vinculin and alpha-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twelve minutes after addition of cells to glass, vinculin was found to become concentrated in small patches at the cell periphery, partially colocalizing with the black IRM areas and with small F-actin-containing adherent protrusions. In contrast, vinculin was not significantly enriched in the less adherent F-actin-rich large pseudopods. alpha-Actinin became enriched during cell adhesion in retraction fibers and, in 40-50% of the inspected cells, also in large less adherent pseudopods where it colocalized with F-actin. The latter finding suggests a continuous dynamic reorganization of pseudopods, with incorporation of alpha-actinin at a certain stage. Disruption of the actin network with cytochalasin D revealed a differential interaction of alpha-actinin and vinculin with the actin network. alpha-Actinin was strongly influenced by cytochalasin D, comparable to F-actin, and both proteins formed colocalizing peripheral caps in 10(-5) M of the drug. Vinculin organization in contrast was not affected by up to 10(-6) M cytochalasin. At 10(-5) M of the drug, however, the patches disappeared completely, vinculin now assuming a diffuse cytoplasmic location. Our results suggest a specialized function of vinculin in adhesion sites of human neutrophils, whereas alpha-actinin may structure the actin network in retraction fibers and in less adherent pseudopods.

Alpha-actinin and calmodulin interact with distinct sites on the arms of the clathrin trimer.

In the present study, the interaction of alpha-actinin and calmodulin with clathrin heavy chain are demonstrated using Western blot analysis and rotary shadowing electron microscopy. The results show that alpha-actinin and calmodulin bind the clathrin heavy chain. The interaction is specific and affected by calcium. However, the interaction of both proteins with the clathrin heavy chain is distinct; the proteins do not block each other's ability to bind, and they interact with different protein fragments of the clathrin heavy chain. Furthermore, using rotary shadowing the results show that alpha-actinin differentially affected the terminal region of the clathrin trimer. Whereas, the effects of calmodulin were most noticeably detected along the length of trimer arms. The possible existence of distinct binding sites on the arms of the clathrin trimer for these cytosolic proteins supports the contention that these cytosolic proteins play an important role in cellular trafficking.

Receptor and non receptor-mediated activation of blood platelets. Effect on membrane-cytoskeleton interaction

Platelets were activated by receptor and non receptor-mediated reagents. The effect of these reagents on aggregation, secretion, cytoskeleton formation, interaction of alpha-actinin and other membrane proteins with the cytoskeleton was studied. Results show that receptor-mediated activation (e.g. ADP or thrombin activation) leads to a high extent of association of alpha-actinin with the cytoskeleton while non receptor-mediated activation (e.g. ionophore A-23187, arachidonic acid) leads to a low association between the two species. The degrees of aggregation, secretion and total amount of protein in the two modes of activation were the same.

Interaction of alpha-actinin and vinculin with actin: opposite effects on filament network formation.

The interaction of actin filaments with two actin-associated proteins, alpha-actinin and vinculin (Mr 130,000 protein), was studied in vitro with viscometry and light and electron microscopy. Vinculin, like alpha-actinin, binds to F-actin, and the two proteins were found to have different effects on the formation of filament networks: alpha-actinin crosslinks individual filaments in a manner strongly dependent on temperature and acts as a spacer, whereas vinculin forms actin bundles that display a paracrystalline substructure. In viscometric assays, alpha-actinin mimics the effect of actin gelation factors, whereas vinculin acts as a gelation inhibitor. These findings imply complementary functions of these proteins in the regulation of cellular mobility.

Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells.

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.

The Interaction of α‐Actinin with F‐Actin and its Abolition by Tropomyosin

The interaction of α‐actinin with F‐actin was examined with the use of the viscometric technique under various conditions. α‐Actinin causes the gelation of F‐actin when added both before and after polymerization. The interaction of α‐actinin with F‐actin seems to be much stronger at 0° than at 21°. In the latter case it takes place only when actin is polymerized with 0.1 M KCl, but not with 1 mM MgCl2 or CaCl2. Tropomyosin, when added before α‐actinin, prevents the formation of F‐actin gel, and when added to already formed gel, causes its disappearance.This abolishing effect of tropomyosin is much stronger at 21° than at 0°. The results are discussed in view of the possible competition between tropomyosin and α‐actinin for the binding to F‐actin.