Mutual effects of α-actinin,calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (α-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin α-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of α-actinin and calponin to actin bundles. Higher ability of calponin to depress α-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin–α-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with α-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that α-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Mutual effects of alpha-actinin, calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (alpha-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin alpha-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of alpha-actinin and calponin to actin bundles. Higher ability of calponin to depress alpha-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin-alpha-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with alpha-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that alpha-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Prediction and Dissection of Widely-Varying Association Rate Constants of Actin-Binding Proteins

Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as k _a = k _a0 , where k _a0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold k _a variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.     

Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin- binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin- binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.     

Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis

Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.     

Actin-binding proteins--a unifying hypothesis

Actin participates in more protein-protein interactions than any other known protein, including the interaction of actin with itself to form the helical polymer F-actin. The vast majority of actin-binding proteins (ABPs) can be grouped into conserved families. Only a handful of structures of complexes of actin with ABPs have been determined so far. These structures are starting to reveal how certain ABPs, including gelsolin, vitamin D-binding protein and Wiskott-Aldrich syndrome protein (WASP)-homology domain-2-related proteins, share a common actin-binding motif. It is proposed here that other ABPs, including actin itself, might share this motif, providing a mechanism whereby ABPs and actin compete for a common binding site. Of particular interest is a hydrophobic pocket that mediates important interactions in five of the existing structures of actin complexes. As the pocket remains accessible in F-actin, it is proposed that this pocket represents a primary target for F-actin-binding proteins, such as calponin-homology-related proteins and myosin.     

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid.

An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.     

Biochemical characterisation of the actin-binding properties of utrophin

Utrophin is a large ubiquitously expressed cytoskeletal protein that is important for maturation of vertebrate neuromuscular junctions. It is highly homologous to dystrophin, the protein defective in Duchenne and Becker muscular dystrophies. Utrophin binds to the actin cytoskeleton via an N-terminal actin-binding domain, which is related to the actin-binding domains of members of the spectrin superfamily of proteins. We have determined the actin-binding properties of this utrophin domain and investigated its binding site on F-actin. An F-actin cosedimentation assay confirmed that the domain binds more tightly to beta-F-actin than to alpha-F-actin and that the full-length utrophin domain binds more tightly to both actin isoforms than a truncated construct, lacking a characteristic utrophin N-terminal extension. Both domain constructs exist in solution as compact monomers and bind to actin as 1:1 complexes. Analysis of the products of partial proteolysis of the domain in the presence of F-actin showed that the N-terminal extension was protected by binding to actin. The actin isoform dependence of utrophin binding could reflect differences at the N-termini of the actin isoforms, thus localising the utrophin-binding site on actin. The involvement of the actin N-terminus in utrophin binding was also supported by competition binding assays using myosin subfragment S1, which also binds F-actin near its N-terminus. Cross-linking studies suggested that utrophin contacts two actin monomers in the actin filament as does myosin S1. These biochemical approaches complement our structural studies and facilitate characterisation of the actin-binding properties of the utrophin actin-binding domain.     

Actin-binding proteins: how to reveal the conformational changes

Actin is the most abundant protein in eukaryotes. Under physiological conditions, it can polymerize into polarized filaments. At the heart of these processes are actin-binding proteins that stimulate actin assembly. Most of them are composed of multiple domains that perform both regulatory and signaling functions. Many actin-binding proteins, including WASP and formin family proteins, are auto-inhibited through intramolecular interactions that mask the actin-regulating sites of these proteins. The large flexible molecules of formins have so far eluded crystallization, and have been crystallized only partially. The information from the available crystal structures is valuable, but somewhat difficult to interpret without a larger framework on which to pose the actin-binding mechanism. Single-particle electron microscopy and electron tomography could provide such a large framework with the full-length structures of protein complexes. The recent advances in determining the molecular interactions in protein complexes predict that the molecular modeling and molecular dynamics methods could be employed to study conformational changes in molecules.     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

Versatility of the small heat shock protein HSPB6 (Hsp20)

The recently published review by Dreiza et al. (Cell Stress and Chaperones DOI ) dealing with the functional role of HSPB6 in muscle regulation is critically analyzed. Published data indicate that the chaperone-like activity of HSPB6 is comparable with that of HSPB5 and that phosphorylation of HSPB6 does not affect its oligomeric structure. Different hypotheses concerning the molecular mechanisms of HSPB6 action on smooth muscle contraction and on the reorganization of the cytoskeleton are compared, and it is concluded that although HSPB6 is not a genuine actin-binding protein, it can affect the actin cytoskeleton indirectly. Phosphorylated HSPB6 interacts with 14-3-3 and thereby displaces other binding partners of 14-3-3; among them, certain phosphatases, protein kinases, and various actin-binding proteins, which can participate in the reorganization of the actin cytoskeleton. In addition, HSPB6 seems to regulate the activity of certain protein kinases. All of these processes are dependent on HSPB6 phosphorylation which in turn might be regulated by the formation of heterooligomeric complexes of HSPB6 with other small heat shock proteins.     

Focal adhesions - the cytoskeletal connection

Cellular contacts with the extracellular matrix are regulated by the Rho family of GTPases through their effects on both the actin and the microtubule cytoarchitecture. Recent genetic, biochemical and structural data have highlighted the role played by a subset of actin-binding proteins in coupling integrins to cytoskeletal actin and in assembling signalling complexes that are important for cell motility and cell proliferation.     

A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin

Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (K(d)=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.     

Caldesmon phosphorylation in actin cytoskeletal remodeling

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.     

Polarity and division site specification in yeast

A significant component of polarization in budding yeast involves the regulated restructuring of the actin cytoskeleton in response to defined cellular signals. Recent evidence suggests that such cytoskeletal organization arises through the action of large protein complexes that form in response to signals from small GTP-binding proteins, such as Cdc42, Rho, and Ras. These actin-organizing complexes may be fairly diverse, but generally consist of one or more central scaffold proteins, such as those of the formin class, that bind to signaling molecules and recruit actin-binding proteins to bring about desired polarizing events.     

Actin and pollen tube growth

Summary Actin microfilaments (MFs) are essential for the growth of the pollen tube. Although it is well known that MFs, together with myosin, deliver the vesicles required for cell elongation, it is becoming evident that the polymerization of new actin MFs, in a process that is independent of actomyosin-dependent vesicle translocation, is also necessary for cell elongation. Herein we review the recent literature that focuses on this subject, including brief discussions of the actin-binding proteins in pollen, and their possible role in regulating actin MF activity. We promote the view that polymerization of new actin MFs polarizes the cytoplasm at the apex of the tube. This process is regulated in part by the apical calcium gradient and by different actin-binding proteins. For example, profilin binds actin monomers and gives the cell control over the initiation of polymerization. A more recently discovered actin-binding protein, villin, stimulates the formation of unipolar bundles of MFs. Villin may also respond to the apical calcium gradient, fragmenting MFs, and thus locally facilitating actin remodeling. While much remains to be discovered, it is nevertheless apparent that actin MFs play a fundamental role in controlling apical cell growth in pollen tubes.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.     

A generalized Flory-Stockmayer kinetic theory of connectivity percolation and rigidity percolation of cytoskeletal networks

Actin networks are essential for living cells to move, reproduce, and sense their environments. The dynamic and rheological behavior of actin networks is modulated by actin-binding proteins such as α-actinin, Arp2/3, and myosin. There is experimental evidence that actin-binding proteins modulate the cooperation of myosin motors by connecting the actin network. In this work, we present an analytical mean field model, using the Flory-Stockmayer theory of gelation, to understand how different actin-binding proteins change the connectivity of the actin filaments as the networks are formed. We follow the kinetics of the networks and estimate the concentrations of actin-binding proteins that are needed to reach connectivity percolation as well as to reach rigidity percolation. We find that Arp2/3 increases the actomyosin connectivity in the network in a non-monotonic way. We also describe how changing the connectivity of actomyosin networks modulates the ability of motors to exert forces, leading to three possible phases of the networks with distinctive dynamical characteristics: a sol phase, a gel phase, and an active phase. Thus, changes in the concentration and activity of actin-binding proteins in cells lead to a phase transition of the actin network, allowing the cells to perform active contraction and change their rheological properties.