Domain organization of alpha-actinin from the rabbit skeletal muscle

Limited trypsinolysis of native alpha-actinin by trypsin was carried out. This procedure resulted in the formation of two large fragments with Mr of 30 and 70 kD which cover almost the whole subunit of alpha-actinin. Using the sedimentation equilibrium method, it was demonstrated that the bisubunit structure of alpha-actinin is provided for by C-terminal fragments of the subunits. Data from circular dichroism analysis suggest that the fragments formed are independent structural units, i.e., domains.     

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.     

Substructure and higher structure of chicken smooth muscle alpha-actinin molecule

Substructure of chicken gizzard smooth muscle alpha-actinin molecule was deduced by domainal mapping of the proteolytic fragments with alpha-chymotrypsin. There were three chymotryptic cleavage sites (Sites I, II, and III, from the amino terminus). Cleavage at Site I generated two fragments, i.e. an NH2-terminal 36-kDa fragment and a COOH-terminal 70-kDa fragment. The 70-kDa fragment generated either a 55-kDa fragment by cleavage at Site II or a 65-kDa fragment by cleavage at Site III. Purified NH2-terminal 36-kDa fragment bound to F-actin, whereas the 55-kDa fragment formed a dimeric molecule. Circular dichroism and electron microscopic experiments demonstrated that the alpha-helical content of the 55-kDa fragment was 14% higher than that of native gizzard alpha-actinin, coinciding with the apparently rod-shaped configuration of this fragment. A 110-kDa product was generated from two 55-kDa fragments in a cross-linking study with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Two cross-linkable sites in the 55-kDa, A- and B-site, were shown to be involved in this reaction. Further, it was demonstrated by using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide labeling and immunoblotting analyses that the A-site on one 55-kDa fragment was cross-linked to the B-site on the other. These results suggest that smooth muscle alpha-actinin formed an antiparallel dimeric molecule in which the 55-kDa fragments connected the two actin-binding domains composed of the 36-kDa fragments.     

Analysis of the alpha-actinin/zyxin interaction.

The yeast two-hybrid system was used to search for interaction partners of human zyxin. Screening of two different cDNA libraries, one prepared from human placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, nebulette, and alpha-actinin. The zyxin/alpha-actinin interaction was analyzed in detail. By site-directed mutagenesis, a linear motif of 6 amino acids (Phe-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role. Replacement of a single amino acid within this motif abolished binding to alpha-actinin in blot overlays as well as in living cells. On the other hand, the interaction site in alpha-actinin was mapped to a conformational determinant present in the center of the protein as demonstrated by a fragment deletion analysis. This binding site involved a tandem array of two complete spectrin-like domains. Only fragments that were able to dimerize in yeast also bound to zyxin, suggesting that dimerization of alpha-actinin is essential for zyxin binding.     

Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations

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.     

Isolation and characterization of a cDNA encoding a chick alpha-actinin

We have isolated and sequenced a 2.1-kilobase cDNA encoding 86% of the sequence of alpha-actinin. The cDNA clone was isolated from a chick embryo fibroblast cDNA library constructed in the expression vector lambda gt11. Identification of this sequence as alpha-actinin was confirmed by immunological methods and by comparing the deduced protein sequence with the sequence of several CNBr fragments obtained from adult chicken smooth muscle (gizzard) alpha-actinin. The deduced protein sequence shows two distinct domains, one of which consists of four repeats of approximately 120 amino acids. This region corresponds to a previously identified 50-kDa tryptic peptide involved in formation of the alpha-actinin dimer. The last 19 residues of C-terminal sequence display an homology with the so-called E-F hand of Ca2+-binding proteins. Hybridization analysis reveals only one size of mRNA (approximately 3.5 kilobases) in fibroblasts, but multiple bands in genomic cDNA.     

Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin

Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.     

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.     

Supercontracted state of vertebrate smooth muscle cell fragments reveals myofilament lengths

Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, alpha-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and alpha-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged integral to 4.5 microns. Myosin filaments in the supercontracted dimers averaged 1.6 microns in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.     

The behavior and cytoskeletal system of chick gastrula mesodermal cells on substrata coated with lines of fibronectin.

In order to investigate the mechanism of the formation of the mesodermal layer during chick gastrulation, we observed the behavior of fragments of mesodermal cells explanted and cultured on substrata coated with parallel lines of fibronectin (FN). We also examined the distribution of F-actin, alpha-actinin, and vinculin in explanted fragments by immunocytochemical methods noting particularly their distribution with respect to FN lines. Explants of mesodermal cells flattened on FN-coated substrata and then became elliptical with the major axis of the ellipse oriented along the FN lines and migrated along them. The peripheral cells of explants extended filopodia and lamellipodia which attached preferentially to FN lines and then contracted, pulling other mesodermal cells in explants along passively. Vinculin and alpha-actinin in peripheral anchoring filopodia and lamellipodia co-localized with the terminations of F-actin bundles and with FN lines, suggesting that the peripheral cells were the moving force for explant translocation. We propose based on these results that in vivo, peripheral cells of invaginated cell mass are guided by the known FN-rich fibrous extracellular matrix on the basal surface of epiblast to move outwards; the rest linked to the peripheral cells are pulled away from the primitive streak to spread in tandem to form the mesodermal layer.     

Targeting of nebulin fragments to the cardiac sarcomere

Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.     

Phosphoinositide binding regulates alpha-actinin CH2 domain structure: analysis by hydrogen/deuterium exchange mass spectrometry

alpha-Actinin is an actin bundling protein that regulates cell adhesion by directly linking actin filaments to integrin adhesion receptors. Phosphatidylinositol (4,5)-diphosphate (PtdIns (4,5)-P(2)) and phosphatidylinositol (3,4,5)-triphosphate (PtdIns (3,4,5)-P(3)) bind to the calponin homology 2 domain of alpha-actinin, regulating its interactions with actin filaments and integrin receptors. In this study, we examine the mechanism by which phosphoinositide binding regulates alpha-actinin function using mass spectrometry to monitor hydrogen-deuterium (H/D) exchange within the calponin homology 2 domain. The overall level of H/D exchange for the entire protein showed that PtdIns (3,4,5)-P(3) binding alters the structure of the calponin homology 2 domain increasing deuterium incorporation, whereas PtdIns (4,5)-P(2) induces changes in the structure decreasing deuterium incorporation. Analysis of peptic fragments from the calponin homology 2 domain showed decreased local H/D exchange within the loop region preceding helix F with both phosphoinositides. However, the binding of PtdIns (3,4,5)-P(3) also induced increased exchange within helix E. This suggests that the phosphate groups on the fourth and fifth position of the inositol head group of the phosphoinositides constrict the calponin homology 2 domain, thereby altering the orientation of actin binding sequence 3 and decreasing the affinity of alpha-actinin for filamentous actin. In contrast, the phosphate group on the third position of the inositol head group of PtdIns (3,4,5)-P(3) perturbs the calponin homology 2 domain, altering the interaction between the N and C terminus of the full-length alpha-actinin antiparallel homodimer, thereby disrupting bundling activity and interaction with integrin receptors.     

Purification of human smooth muscle filamin and characterization of structural domains and functional sites

A method was developed to purify human smooth muscle filamin in high yield and structural domains were defined by using mild proteolysis to dissect the molecule into intermediate-sized peptides. Unique domains were defined and aligned by using high-resolution peptide mapping of iodinated peptides on cellulose plates. The amino- and carboxyl-terminal orientation of these domains within the molecule was determined by amino acid sequence analysis of several aligned peptides. In addition to the three unique domains which were identified, a number of smaller and larger fragments were also characterized and aligned within the intact molecule. These structural domains and related peptides provide a useful set of defined fragments for further elucidation of structure-function relationships. The two known functionally important binding sites of filamin, the self-association site and the actin-binding site, have been localized. Self-association of two monomers in a tail-to-tail orientation involves a small protease-sensitive region near the carboxyl terminal of the intact polypeptide chain. Sedimentation assays indicate that an actin-binding site is located near the blocked amino terminal of the filamin molecule. Sequences derived from large peptides mapping near the amino terminal show homology to the amino-terminal actin-binding site of alpha-actinin (chicken fibroblast and Dictyostelium), Dictyostelium 120-kDa actin gelation factor, beta-spectrin (human red cell and Drosophila), and human dystrophin. This homology is particularly interesting for two reasons. The functional form of filamin is single stranded, in contrast to alpha-actinin and spectrin which are antiparallel double-stranded actin cross-linkers. Also, no homology to the spectrin-like segments which comprise most of the mass of spectrin, alpha-actinin, and dystrophin was found. Instead, the sequence of a domain located near the center of the filamin molecule (tryptic 100-kDa peptide, T100) shows homology to the published internal repeats of the Dictyostelium 120-kDa actin gelation factor. On the basis of these results, a model of human smooth muscle filamin substructure is presented. Also, comparisons of human smooth muscle filamin, avian smooth muscle filamin, and human platelet filamin are reported.     

Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Microinjection of fluorophore-tagged cytoskeletal proteins has been a useful tool in studies of formation of focal adhesions (FA). We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (TJ) of epithelial Madin-Darby bovine kidney cells. We chose alpha-actinin and vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterized. Isolated FITC-labelled chicken alpha-actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade within an hour after injection and the cells were joined to polykaryons having 5 to 13 nuclei. Short fragments of cell membranes containing injected proteins, actin, beta-catenin, cadherin, claudin, occludin and ZO-1 were visible inside the polykaryons indicating that both AJs and TJs were disintegrated as a single complex. Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidly induced the detachment of the cells from the substratum probably due to high affinity of vinculin head to talin. Vinculin tail domain had no apparent effect on the cell morphology. Since small GTPases are involved in the building up of AJs, we injected active and inactive forms of cdc42 and rac proteins together with vinculin to see their effect. Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect. We suggest that excess alpha-actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskeleton which leads to fragmentation of junctional complexes and subsequent cell fusion. The results show that cell-cell adhesion sites are more dynamic and more sensitive than FAs to an imbalance in the amount of free alpha-actinin and intact vinculin.     

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.     

Chemotactically directed redistribution of alpha-actinin precedes morphological polarization and reversal of polarity in human polymorphonuclear leucocytes (PMNs)

We have determined the temporal and spatial relationship between cell polarization and alpha-actinin localization by analysing the redistribution of alpha-actinin and F-actin in spherical PMNs developing polarity and in polarized cells reversing polarity following localized stimulation with chemotactic peptide using micropipettes. Initially spherical PMNs develop a one-sided accumulation of alpha-actinin before lamellipodia enriched in alpha-actinin are formed. In polarized cells, alpha-actinin is concentrated at the leading front. When polarity is reversed, alpha-actinin redistribution to the uropod precedes reversal of morphological polarity and formation of new lamellipodia at the uropod. Later, lamellipodia enriched in F-actin and alpha-actinin develop at the former uropod to form a new front. The data document that redistribution of alpha-actinin is a very early event in the development of polarity, which precedes formation of lamellipodia.     

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.     

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.     

Differential binding of cross-reactive anti-DNA antibodies to mesangial cells: the role of alpha-actinin

Target Ag display is a necessary requirement for the expression of certain immune-mediated kidney diseases. We previously had shown that anti-DNA Abs that cross-react with alpha-actinin may be important in the pathogenesis of murine and human lupus nephritis; in murine models, we had found that a significant proportion of pathogenic serum and kidney-deposited Igs are alpha-actinin reactive. Furthermore, a pathogenic anti-DNA/alpha-actinin Ab showed enhanced binding to immortalized mesangial cells (MCs) derived from a lupus prone MRL-lpr/lpr mouse as compared with MCs from BALB/c mice which are not susceptible to spontaneous lupus, suggesting that kidney alpha-actinin expression may be contributing to nephritis. In the current study, we established that two isoforms of alpha-actinin that are present in the kidney, alpha-actinin 1 and alpha-actinin 4, can both be targeted by anti-alpha-actinin Abs. We found novel sequence polymorphisms between MRL-lpr/lpr and BALB/c in the gene for alpha-actinin 4. Moreover, alpha-actinin 4 and a splice variant of alpha-actinin 1 were both expressed at significantly higher levels (mRNA and protein) in MCs from the lupus prone MRL-lpr/lpr strain. Significantly, we were able to confirm these differences in intact kidney by examining glomerular Ig deposition of anti-alpha-actinin Abs. We conclude that enhanced alpha-actinin expression may determine the extent of Ig deposition in the Ab-mediated kidney disease in lupus. Modulation of Ag expression may be a promising approach to down-regulate immune complex formation in the target organ in individuals with circulating pathogenic Abs.     

Alpha-actinin is required for tightly regulated remodeling of the actin cortical network during cytokinesis

Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.