Functional synergy of actin filament cross-linking proteins

The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.     

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.     

Relative distribution of myosin, actin, and alpha-actinin in adherent monocytes.

The characteristic amoeboid movement of human leucocytes uses mechanical energy derived from the hydrolysis of adenosine triphosphate through a mechanochemical system of the contractile proteins myosin, actin, and the actin-associated protein alpha-actinin. We observed the relative distribution of myosin, actin, and alpha-actinin in adherent monocytes during movement by a double-fluorescence staining procedure. The results indicate that myosin and alpha-actinin are closely associated with the actin cable network, and that alpha-actinin is in close association with the plasma membrane and anchors filamentous actin (F-actin) beneath the plasma membrane; F-actin and alpha-actinin play an important role at the leading edge during the formation of lamellipodia. These findings should be helpful in clarifying the mechanism of leucocyte movement from a morphologic standpoint.     

The filamentous actin cross-linking/bundling activity of mammalian formins

Formins are multidomain proteins that regulate actin filament dynamics and are defined by the formin homology 2 domain. Biochemical assays suggest that mammalian formins display actin-filament nucleation, severing, and bundling activities. Whether formins can cross-link actin filaments into viscoelastic arrays and the effectiveness of formins' bundling activity compared with that of important filamentous actin (F-actin) cross-linking/bundling proteins are unknown. Here, we used rigorous in vitro rheologic assays to deconvolve the dynamic cross-linking activity from the bundling activity of formin FRL1 and the closely related mDia1 and mDia2. In addition, we compared these formins with the canonical F-actin bundling protein fascin and cross-linking/bundling proteins alpha-actinin and filamin. We found that FRL1 and mDia2, but not mDia1, can help F-actin form highly elastic networks. FRL1 and mDia2 mediate the formation of highly elastic F-actin networks as effectively and rapidly as alpha-actinin and filamin but only past a relatively high actin-to-formin molar ratio of 50:1. Past that threshold molar ratio, the mechanical properties of F-actin/formin networks are independent of formin concentration, similar to fascin. Moreover, unlike those for alpha-actinin and filamin but similar to those for fascin, F-actin/formin networks show no strain-induced hardening. mDia1 cannot bundle F-actin but can weakly cross-link filaments at high concentrations. Point mutagenesis reveals that reducing the barbed-end binding activity of FRL1 and mDia2 greatly enhances the rate of formation of F-actin gels but does not significantly affect the mechanical properties of the resulting networks at steady state. Together, these results suggest that the mechanical behaviors of FRL1 and mDia2 are fundamentally different from those of cross-linking/bundling proteins alpha-actinin and filamin but qualitatively similar to the mechanical behavior of the bundling protein fascin, albeit with a dramatically increased (>10-fold) threshold concentration for transition to bundling, which nevertheless leads to much stiffer F-actin networks than fascin.     

Mechanics and multiple-particle tracking microheterogeneity of alpha-actinin-cross-linked actin filament networks

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.     

Adhesion of L1210 cells to sulfonated styrene copolymer surfaces: imaging of F-actin AND alpha-actinin

The static adhesion of living L1210 cells to sulfonated copolymer surfaces of different sulfonic group content and the actin cytoskeleton organization in the adhering cells were studied. The strength of the cell-substratum interaction was estimated by determining the relative number of cells remaining adherent despite experiencing a shearing force equal to 1.25 x 10(-11) N caused by the laminar flow of the medium. The cell-substratum interaction took place in a medium with or without serum. The distribution of F-actin and alpha-actinin in the adhering cells was determined in sequences of fluorescent images of cell optical slices with the use of a computer method of cell image analysis. It was shown that the surface sulfonic groups affect not only the rate and strength of cell-substratum adhesion but also the F-actin and alpha-actinin distribution (in the cell regions near the substratum surface) in cells adhering in the medium containing serum. These proteins, concentrated in the tips of microvilli, were observed as dots. The distinctness (discernibleness) and sizes of these dots depend on the surface content of sulfonic groups. F-actin is located at the periphery of the cells in cells adhering in the medium without serum and alpha-actinin is concentrated in small dots at the periphery and in the central part of the cells.     

The effect of alpha-actinin on the length distribution of F-actin

Actin filament length distribution in cells is often regulated to fit specific tasks. In comparison to the well-studied regulation of the average filament length (e.g., using capping proteins), controlling the width of the distribution is less well understood. We utilize two complementary methods to measure the effect of alpha-actinin on the width of the distribution of lengths of F-actin in vitro. Analyzing transmission electron micrographs shows that crosslinking by alpha-actinin reduces the width of the length distribution of F-actin, decreasing the coefficient of variation by two- to threefold. Analysis of fluorescence data from depolymerization assays confirms this observation. We suggest a mechanistic molecular model in which a local (weak) stabilization of crosslinked monomers in the filament is the physical origin of the decrease in the variance of lengths. Although alpha-actinin is known to bind reversibly to F-actin, our model shows that even weak binding can produce this effect, and that in fact it persists throughout a wide range of binding strengths.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.     

Enaptin, a giant actin-binding protein, is an element of the nuclear membrane and the actin cytoskeleton

Enaptin belongs to a family of recently identified giant proteins that associate with the F-actin cytoskeleton as well as the nuclear membrane. It is composed of an N-terminal alpha-actinin type actin-binding domain (ABD) followed by a long coiled coil rod and a transmembrane domain at the C-terminus. The ABD binds to F-actin in vivo and in vitro and leads to bundle formation. The human Enaptin gene spreads over 515 kb and gives rise to several splicing isoforms (Nesprin-1, Myne-1, Syne-1, CPG2). The longest assembled cDNA encompasses 27,669 bp and predicts a 1014 kDa protein. Antibodies against the ABD of Enaptin localise the protein at F-actin-rich structures throughout the cell and in focal contacts as well as at the nuclear envelope. In COS7 cells, the protein is also present within the nuclear compartment. With the discovery of the actin-binding properties of Enaptin and the highly homologous Nuance, we define a family of proteins that integrate the cytoskeleton with the nucleoskeleton.     

A novel nonmuscle alpha-actinin. Purification and characterization of chicken lung alpha-actinin.

Two distinct alpha-actinin-like proteins were detected in chicken lung extract by immunoblot analysis with monoclonal antibodies against alpha-actinin. The mobilities of these proteins on SDS-polyacrylamide gel electrophoresis are very close (approximately 100 kDa). On SDS-polyacrylamide gel electrophoresis in the presence of 6 M urea, however, one of the proteins migrates at 115 kDa and is clearly separated from the other protein (105 kDa). The 115-kDa protein was purified and shown to have at least three unique amino acid sequences which were not detected in other kinds of alpha-actinins: one locates at the extreme NH2-terminal region, and the others locate at the COOH-terminal half region. Immunoblot and proteolytic cleavage analyses revealed that the 115-kDa protein has structural divergence at the COOH-terminal region that includes Ca(2+)-binding EF-hand motifs. Falling-ball viscometric studies showed that although the 115-kDa protein-induced gelation of F-actin is sensitive to Ca2+, the gelation activity of the 115-kDa protein is much higher than that of Ca(2+)-insensitive gizzard alpha-actinin regardless of Ca2+. This indicates that the 115-kDa protein is distinct from other nonmuscle alpha-actinins by its Ca2+ sensitivity.     

Microfilament gel rigidity cooperates negatively with the binding of actin gelling proteins

At 37 degrees C, in the presence of 0.1 M KC1 and 2 mM MgCl2, the binding of alpha-actinin to F-actin increases with the concentration of alpha-actinin but not with the concentration of F-actin. This implies that binding is determined by additional factors, beside the alpha-actinin - F-actin association constant. We propose that one of these factors is the rigidity of the gel, which cooperates negatively to the binding by increasing the work needed to bring two actin filaments at the reaction distance with alpha-actinin.     

Human lymphocyte-specific protein 1, the protein overexpressed in neutrophil actin dysfunction with 47-kDa and 89-kDa protein abnormalities (NAD 47/89), has multiple F-actin binding domains

Human lymphocyte-specific protein 1 (LSP1) is an F-actin binding protein, which has an acidic N-terminal half and a basic C-terminal half. In the basic C-terminal half, there are amino acid sequences highly homologous to the actin-binding domains of two known F-actin binding proteins: caldesmon and the villin headpieces (CI, CII, VI, VII). However, the exact numbers and locations of the F-actin binding domains within LSP1 are not clearly defined. In this report, we utilized 125I-labeled F-actin ligand blotting and high-speed F-actin cosedimentation assays to analyze the F-actin binding properties of truncated LSP1 peptides and to define the F-actin binding domains. Results show that LSP1 has at least three and potentially a fourth F-actin binding domain. All F-actin binding domains are located in the basic C-terminal half and correspond to the caldesmon and villin headpiece homologous regions. LSP1 181-245 and LSP1 246-295, containing sequences homologous to caldesmon F-actin binding site I and II, respectively (CI, CII), binds F-actin; similarly, LSP1 306-339 can bind F-actin and contains two inseparable villin headpiece-like F-actin binding domains (VI, VII). Although LSP1 1-305, which does not contain VI and VII regions, retains F-actin binding activity, its binding affinity for F-actin is much weaker than that of full-length LSP1. Site-directed mutagenesis of the basic amino acids in the KRYK (VI) or KYEK (VII) sequences to acidic amino acids create mutants that bind F-actin with lower affinity than full-length wild-type LSP1. High KCl concentrations decrease full-length LSP1 binding to F-actin, suggesting the affinity between LSP1 and F-actin is mainly through electrostatic interaction.     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with alpha-actinin.

Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.     

Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism

Utrophin is a large ubiquitously expressed cytoskeletal protein, homologous to dystrophin, the protein disrupted in Duchenne muscular dystrophy. The association of both proteins with the actin cytoskeleton is functionally important and is mediated by a domain at their N termini, conserved in members of the spectrin superfamily, including alpha-actinin, beta-spectrin and fimbrin. We present the structure of the actin-binding domain of utrophin in complex with F-actin, determined by cryo-electron microscopy and helical reconstruction, and a pseudo-atomic model of the complex, generated by docking the crystal structures of the utrophin domain and F-actin into the reconstruction. In contrast to the model of actin binding proposed for fimbrin, the utrophin actin-binding domain appears to associate with actin in an extended conformation. This conformation places residues that are highly conserved in utrophin and other members of the spectrin superfamily at the utrophin interface with actin, confirming the likelihood of this binding orientation. This model emphasises the importance of protein flexibility in modeling interactions and presents the fascinating possibility of a diversity of actin-binding mechanisms among related proteins.     

Amorphin is phosphorylase; phosphorylase is an alpha-actinin-binding protein

In a study of myofibrillar proteins, Chowrashi and Pepe [1982: J. Cell Biol. 94:565-573] reported the isolation of a new, 85-kD Z-band protein that they named amorphin. We report that partial sequences of purified amorphin protein indicate that amorphin is identical to phosphorylase, an enzyme important in the metabolism of glycogen. Anti-amorphin antibodies also reacted with purified chicken and rabbit phosphorylase. To explore the basis for phosphorylase's (amorphin's) localization in the Z-bands of skeletal muscles, we reacted biotinylated alpha-actinin with purified amorphin and with purified phosphorylase and found that alpha-actinin bound to each. Radioimmune assays also indicated that phosphorylase (amorphin) bound to alpha-actinin, and, with lower affinity, to F-actin. Negative staining of actin filaments demonstrated that alpha-actinin mediates the binding of phosphorylase to actin filaments. There are several glycolytic enzymes that bind actin (e.g., aldolase, phosphofructokinase, and pyruvate kinase), but phosphorylase is the first one demonstrated to bind alpha-actinin. Localization of phosphorylase in live cells was assessed by transfecting cultures of quail embryonic myotubes with plasmids expressing phosphorylase fused to Green Fluorescent Protein (GFP). This resulted in targeting of the fusion protein to Z-bands accompanied by a diffuse pattern in the cytoplasm.     

Strain hardening of actin filament networks. Regulation by the dynamic cross-linking protein alpha-actinin

Mechanical stresses applied to the plasma membrane of an adherent cell induces strain hardening of the cytoskeleton, i.e. the elasticity of the cytoskeleton increases with its deformation. Strain hardening is thought to mediate the transduction of mechanical signals across the plasma membrane through the cytoskeleton. Here, we describe the strain dependence of a model system consisting of actin filaments (F-actin), a major component of the cytoskeleton, and the F-actin cross-linking protein alpha-actinin, which localizes along contractile stress fibers and at focal adhesions. We show that the amplitude and rate of shear deformations regulate the resilience of F-actin networks. At low temperatures, for which the lifetime of binding of alpha-actinin to F-actin is long, F-actin/alpha-actinin networks exhibit strong strain hardening at short time scales and soften at long time scales. For F-actin networks in the absence of alpha-actinin or for F-actin/alpha-actinin networks at high temperatures, strain hardening appears only at very short time scales. We propose a model of strain hardening for F-actin networks, based on both the intrinsic rigidity of F-actin and dynamic topological constraints formed by the cross-linkers located at filaments entanglements. This model offers an explanation for the origin of strain hardening observed when shear stresses are applied against the cellular membrane.     

Purification and characterization of an alpha-actinin-like protein from porcine kidney

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.     

Novel structures for alpha-actinin:F-actin interactions and their implications for actin-membrane attachment and tension sensing in the cytoskeleton

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.