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1.
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.  相似文献   

2.
The purpose of this study was to address the paradox of calponin localization with alpha-actinin and filamin, two proteins with tandem calponin homology (CH) domains, by determining the effect of these proteins on the binding of calponin to actin. The results show that actin can accommodate near-saturating concentrations of either calponin and alpha-actinin or calponin and filamin with little change or no change in ligand affinity. Little direct interaction occurred between alpha-actinin and calponin in the absence of actin, so this effect is not likely to explain the co-distribution of these proteins. Calponin, like alpha-actinin, induced elastic gel formation when added to actin. When alpha-actinin was added to newly formed calponin/actin gels, no change was seen in the mechanical properties of the gel compared to calponin and actin alone. However, when calponin was added to newly formed alpha-actinin/actin gels, the resulting gel was much stronger than the gels formed by either ligand alone. Furthermore, gels formed by the addition of calponin to alpha-actinin/actin exhibited a phenomenon known as strain hardening, a characteristic of mechanically resilient gels. These results add weight to the concept that one of the functions of calponin is to stabilize the actin cytoskeleton.  相似文献   

3.
Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy.  相似文献   

4.
Syndecan-4, a cell surface heparan sulfate proteoglycan, is known to regulate the organization of the cytoskeleton, and oligomerization is crucial for syndecan-4 function. We therefore explored a possible regulatory effect of syndecan-4 oligomerization on the cytoskeleton. Glutathione-S-transferase-syndecan-4 proteins were used to show that syndecan-4 interacted specifically with alpha-actinin, but not paxillin, talin, and vinculin. Interestingly, only dimeric, and not monomeric, recombinant syndecan-4 interacted with alpha-actinin in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), and PIP2 potentiated the interaction of both the cytoplasmic domain syndecan-4 peptide and recombinant syndecan-4 proteins with alpha-actinin, implying that oligomerization of syndecan-4 was important for this interaction. Consistent with this notion, alpha-actinin interaction was largely absent in syndecan-4 mutants defective in transmembrane domain-induced oligomerization, and alpha-actinin-associated focal adhesions were decreased in rat embryo fibroblasts expressing mutant syndecan-4. Besides, this interaction was consistently lower with the phosphorylation-mimicking syndecan-4 mutant S183E which is known to destabilize the oligomerization of the syndecan-4 cytoplasmic domain. Taken together, the data suggest that the oligomeric status of syndecan-4 plays a crucial role in regulating the interaction of syndecan-4 with alpha-actinin.  相似文献   

5.
Individual cardiomyocytes are lengthened in dilated cardiomyopathy. However, it is not known how the new sarcomeres are added to preexisting myofibrils. Using a three-dimensional microtextured culturing system, a 10% mechanical static strain was applied to aligned, well-attached cardiomyocytes from neonatal rat. The morphology of the myofibrils and the ends of the myocytes were examined. Disruptions of the sarcomeric pattern for actin showed a progression from weak to intense staining over 4 hr. The lightly stained sarcomeres were common at 1 hr after being strained, peaked at 2 hr, and then subsided. In contrast, the numbers of intensely stained sarcomeres were initially low, peaked at 3 hr, and then began to decline when compared with control values. The myocyte ends showed elongations and convolutions after 3 hr and 4 hr of mechanical strain when observed with alpha-actinin and N-cadherin staining. We suggest that myocytes from neonatal rat hearts remodel by insertion of new sarcomeres throughout the cell length and also by enhancement at the intercalated discs.  相似文献   

6.
7.
Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.  相似文献   

8.
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.  相似文献   

9.
Dictyostelium discoideum alpha-actinin (D.d. alpha-actinin) is a calcium and pH-regulated actin-binding protein that can cross-link F-actin into a gel at a submicromolar free calcium concentration and a pH less than 7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. alpha-actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic-pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. alpha-actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to approximately 65% of the control value. If the D.d. alpha-actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. alpha-actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. alpha-actinin binding to the actin filaments was static over the time course of measurement (approximately 5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. alpha-actinin. These results demonstrate that 1) actin filaments need not be cross-linked into an immobile, static array in order to have macroscopic properties of a gel; 2) interpretation of the rheological properties of actin:alpha-actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and 3) a fraction of D.d. alpha-actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.  相似文献   

10.
Tseng Y  Wirtz D 《Biophysical journal》2001,81(3):1643-1656
Cell morphology is controlled by the actin cytoskeleton organization and mechanical properties, which are regulated by the available contents in actin and actin regulatory proteins. Using rheometry and the recently developed multiple-particle tracking method, we compare the mechanical properties and microheterogeneity of actin filament networks containing the F-actin cross-linking protein alpha-actinin. The elasticity of F-actin/alpha-actinin networks increases with actin concentration more rapidly for a fixed molar ratio of actin to alpha-actinin than in the absence of alpha-actinin, for networks of fixed alpha-actinin concentration and of fixed actin concentration, but more slowly than theoretically predicted for a homogeneous cross-linked semiflexible polymer network. These rheological measurements are complemented by multiple-particle tracking of fluorescent microspheres imbedded in the networks. The distribution of the mean squared displacements of these microspheres becomes progressively more asymmetric and wider for increasing concentration in alpha-actinin and, to a lesser extent, for increasing actin concentration, which suggests that F-actin networks become progressively heterogeneous for increasing protein content. This may explain the slower-than-predicted rise in elasticity of F-actin/alpha-actinin networks. Together these in vitro results suggest that actin and alpha-actinin provides the cell with an unsuspected range of regulatory pathways to modulate its cytoskeleton's micromechanics and local organization in vivo.  相似文献   

11.
The dense-bodies in the body wall muscle of the nematode Caenorhabditis elegans function to anchor the actin thin filaments to the adjacent sarcolemma. One of the major components of the dense-bodies is the actin-binding protein alpha-actinin. To facilitate a genetic analysis of alpha-actinin, we have cloned a cDNA encoding the nematode protein, identified its position on the nematode physical map, and developed a unique PCR based approach to test the position of the cloned gene relative to known genetic deletions. The peptide sequence deduced from the cDNA shows that, apart from a few exceptional regions, the nematode protein shows strong similarity to other known alpha-actinins. Its position on the genetic map shows that none of the known muscle affecting mutations identified in C. elegans are in this alpha-actinin gene. This gene has been given the name atn-1 (alpha-actinin-1).  相似文献   

12.
We previously discovered a large titin-like protein-c-titin-in chicken epithelial brush border and human blood platelet extracts that binds alpha-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with alpha-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures.  相似文献   

13.
CRP2 is an autonomous actin-binding protein   总被引:4,自引:0,他引:4  
Grubinger M  Gimona M 《FEBS letters》2004,557(1-3):88-92
  相似文献   

14.
The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.  相似文献   

15.
Panasenko OO  Gusev NB 《IUBMB life》2000,49(4):277-282
Interaction of calponin and alpha-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, alpha-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin filaments were separated, and the stoichiometry of ABPs and actin in both types of filaments was determined. Binding of calponin to the single or bundled actin filaments was not dependent on the presence of alpha-actinin and did not displace alpha-actinin from actin. In the presence of calponin, however, less alpha-actinin was bound to the bundled actin filaments, and the binding of alpha-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin filaments. Calponin had no influence on the binding of alpha-actinin to the single actin filaments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and alpha-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs.  相似文献   

16.
We have applied correspondence analysis to electron micrographs of 2-D rafts of F-actin cross-linked with alpha-actinin on a lipid monolayer to investigate alpha-actinin:F-actin binding and cross-linking. More than 8000 actin crossover repeats, each with one to five alpha-actinin molecules bound, were selected, aligned, and grouped to produce class averages of alpha-actinin cross-links with approximately 9-fold improvement in the stochastic signal-to-noise ratio. Measurements and comparative molecular models show variation in the distance separating actin-binding domains and the angle of the alpha-actinin cross-links. Rafts of F-actin and alpha-actinin formed predominantly polar 2-D arrays of actin filaments, with occasional insertion of filaments of opposite polarity. Unique to this study are the numbers of alpha-actinin molecules bound to successive crossovers on the same actin filament. These "monofilament"-bound alpha-actinin molecules may reflect a new mode of interaction for alpha-actinin, particularly in protein-dense actin-membrane attachments in focal adhesions. These results suggest that alpha-actinin is not simply a rigid spacer between actin filaments, but rather a flexible cross-linking, scaffolding, and anchoring protein. We suggest these properties of alpha-actinin may contribute to tension sensing in actin bundles.  相似文献   

17.
Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.  相似文献   

18.
A single-site mutation of the flight-muscle-specific actin gene of Drosophila melanogaster causes a substitution of glutamic acid 93 by lysine in all the actin encoded in the indirect flight muscle (IFM). In these Act88FE93K mutants, myofibrillar bundles of thick and thin filaments are present but lack Z-discs and all sarcomeric repeats. Dense filament bundles, which are probably aberrant Z-discs, are seen in myofibrils of pupal flies, but early in adult life these move to the periphery of the fibrils and are not seen in skinned adult fibres. Consistent with this observation, alpha-actinin and other high molecular weight proteins, possibly associated with Z-discs, are not detected on SDS/polyacrylamide gels or Western blots of skinned adult IFM. The mutation lies at the beginning of a loop in the small domain of actin, near the myosin binding region. However, that the mutant actin binds myosin heads is shown by (1) rigor crossbridges in electron micrographs, (2) the appropriate rise in stiffness when ATP is withdrawn in mechanical experiments, and (3) equal protection against tryptic digestion provided by rigor binding between actin and myosin in both wild-type and mutant fibres. Reversal of rigor chevron angle along some thin filaments reflects reversal of thin-filament polarity due to lattice disorder. The absence of Z-discs, alpha-actinin and two high molecular weight proteins, and binding studies by others, suggest that the substitution at residue 93 affects the binding of the mutant actin to a protein, possibly alpha-actinin, which is necessary for Z-disc assembly or maintenance.  相似文献   

19.
ErbB2 is essential in the prevention of dilated cardiomyopathy   总被引:22,自引:0,他引:22  
Amplification of the gene encoding the ErbB2 (Her2/neu) receptor tyrosine kinase is critical for the progression of several forms of breast cancer. In a large-scale clinical trial, treatment with Herceptin (trastuzumab), a humanized blocking antibody against ErbB2, led to marked improvement in survival. However, cardiomyopathy was uncovered as a mitigating side effect, thereby suggesting an important role for ErbB2 signaling as a modifier of human heart failure. To investigate the physiological role of ErbB2 signaling in the adult heart, we generated mice with a ventricular-restricted deletion of Erbb2. These ErbB2-deficient conditional mutant mice were viable and displayed no overt phenotype. However, physiological analysis revealed the onset of multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall thinning and decreased contractility. Additionally, cardiomyocytes isolated from these conditional mutants were more susceptible to anthracycline toxicity. ErbB2 signaling in cardiomyocytes is therefore essential for the prevention of dilated cardiomyopathy.  相似文献   

20.
alpha-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between alpha-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific alpha-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, alpha-actinin is ubiquitously expressed. The non-muscle alpha-actinin mutant Actn(Delta233), which is viable and fertile, lacks alpha-actinin expression in ovarian germline cells, while somatic follicle cells express alpha-actinin at late oogenesis. Here we show that this latter population of alpha-actinin, termed FC-alpha-actinin, is absent from the dorsoanterior follicle cells, and we present evidence that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increased the F-actin bundling activity of ectopically expressed muscle-specific alpha-actinin. We also describe a novel morphogenetic event in the follicle cells that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior beta-integrin-dependent adhesion site accumulating alpha-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although alpha-actinin was not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling was perturbed, and Enabled did not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that alpha-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila.  相似文献   

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