F-actin Complex Research Articles

Fascin − F-actin interaction studied by molecular dynamics simulation and protein network analysis

Actin bundles are an important component of cellular cytoskeleton and participate in the movement of cells. The formation of actin bundles requires the participation of many actin binding proteins (ABPs). Fascin is a member of ABPs, which plays a key role in bundling filamentous actin (F-actin) to bundles. However, the detailed interactions between fascin and F-actin are unclear. In this study, we construct an atomic-level structure of fascin − F-actin complex based on a rather poor cryo-EM data with resolution of 20 nm. We first optimized the geometries of the complex by molecular dynamics (MD) simulation and analyzed the binding site and pose of fascin which bundles two F-actin chains. Next, binding free energy of fascin was calculated by MM/GBSA method. Finally, protein structure network analysis (PSNs) was performed to analyze the key residues for fascin binding. Our results show that residues of K22, E27, E29, K41, K43, R110, R149, K358, R408 and K471 on fascin are important for its bundling, which are in good agreement with the experimental data. On the other hand, the consistent results indicate that the atomic-level model of fascin − F-actin complex is reliable. In short, this model can be used to understand the detailed interactions between fascin and F-actin, and to develop novel potential drugs targeting fascin. Communicated by Ramaswamy H. Sarma

How SipA, a toxin from Salmonella, increases stability of F-actin.

Salmonella infection is a disease that is mainly limited to the intestinal tract, but can also be systemic and may be deadly for young children or people with a weakened immune system. Salmonella strains produce protein toxins that target cytoplasmic actin to promote bacterial uptake by a host cell, which is essential for pathogenicity. The SipA protein is a Salmonella effector delivered into the host cell by bacterial type III secretion system. SipA has been reported to stabilize F-actin upon binding, and to promote actin polymerization in vitro. Only very low resolution reconstructions from negative stain existed to show how SipA binds to actin. Here, we present a 3.5Å cryo-EM reconstruction of the SipA425-685/F-actin complex. Our reconstruction shows that SipA binds actin with nearest neighbor exclusion along actin's long-pitch strand. The globular SipA domain binds actin at subunit n through ionic and hydrophobic interactions with both actin strands with nanomolar affinity. The extended C-terminal tail is inserted in between the two actin strands at subunit n+2 preventing the binding of another SipA molecule at this site. Thus, SipA will be bound at n, n+1, n+4, n+5, etc. The C-terminal tail of SipA binds to the same site as two F-actin toxins, phalloidin (Pha) and jasplakinolide (Jasp). All three molecules interact with actin mainly through hydrophobic interactions, however SipA binds F-actin with higher affinity than Pha or Jasp, and outcompetes the latter two in competitive binding assay. The SipA binding involves conformational changes of actin's D-loop which provides additional intra-strand interactions. The observed stabilization of actin filaments upon SipA binding is therefore likely due to the introduction of additional inter- and intra-strand interactions within the actin filament.

CD44/ERM/F-actin complex mediates targeted nuclear degranulation and excessive neutrophil extracellular trap formation during sepsis.

Neutrophils release neutrophil extracellular traps (NETs) to capture and kill pathogens, but excessive NET release can damage the surrounding tissues. Myeloperoxidase (MPO) and neutrophil elastase (NE) are thought to be important in promoting histone depolymerization and DNA breakage in the nucleus. However, the detailed path by which MPO and NE enter the nucleus is unknown. In the present study, we observed that delayed fusion of azurophilic granules with the nuclear membrane 15–20 min after extracellular degranulation in activated neutrophils. In a subsequent experiment, we further demonstrated that this fusion leads to MPO entry into the nucleus and promotes nuclear histone depolymerization and DNA breakage, a process called ‘targeted nuclear degranulation’. This process can be effectively inhibited by dexamethasone and accompanied by the continuous low levels of MPO in the nucleus after PMA stimulation. Meanwhile, we found that ‘targeted nuclear degranulation’ is dependent on the CD44 translocation and subsequent redistribution of CD44 / ERM (Ezrin/Radixin/Moesin) / F‐actin complexes, which guides the movement of azurophilic granules towards the nucleus. Application of ERM phosphorylation inhibitors and importin activity inhibitors significantly reduced the complexes formation and redistribution. Taken together, these findings indicate for the first time that delayed ‘targeted nuclear degranulation’ after neutrophil activation is a key mechanism of NET formation. CD44/ERM/F‐actin complex mediates this process, which providing targets with promising prospects for the precise regulation of NET formation.

Acidosis modifies effects of phosphorylated tropomyosin on the actin-myosin interaction in the myocardium.

Phosphorylation of α-tropomyosin (Tpm1.1), a predominant Tpm isoform in the myocardium, is one of the regulatory mechanisms of the heart contractility. The Tpm 1.1 molecule has one site of phosphorylation, Ser283. The degree of the Tpm phosphorylation decreases with age and also changes in heart pathologies. Myocardial pathologies, in particular ischemia, are usually accompanied by pH lowering in the cardiomyocyte cytosol. We studied the effects of acidosis on the structural and functional properties of the pseudo-phosphorylated form of Tpm1.1 with the S283D substitution. We found that in acidosis, the interaction of the N- and C-ends of the S283D Tpm molecules decreases, whereas that of WT Tpm does not change. The pH lowering increased thermostability of the complex of F-actin with S283D Tpm to a greater extent than with WT Tpm. Using an in vitro motility assay with NEM- modified myosin as a load, we assessed the effect of the Tpm pseudo-phosphorylation on the force of the actin-myosin interaction. In acidosis, the force generated by myosin in the interaction with thin filaments containing S283D Tpm was higher than with those containing WT Tpm. Also, the pseudo-phosphorylation increased the myosin ability to resist a load. We conclude that ischemia changes the effect of the phosphorylated Tpm on the contractile function of the myocardium.

A crosslinked and ribosylated actin trimer does not interact productively with myosin.

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein-protein interactions to produce a physiologically relevant short F-actin complex.

Interaction of Phospho‐Moesin and CD44 in Pericytes Attenuated the Maturation of Neovessles in AGE‐induced Angiogenesis

BackgroundWe have previously demonstrated that advanced glycation endproducts (AGEs) promoted angiogenesis by inducing ERM phosphorylation, especially, moesin phosphorylation in human umbilical vein endothelial cells (HUVECs). This study was to investigate the effect of moesin phosphorylation on angiogenesis and neovessel maturation in aortic ring and retina in AGE‐treated mice. This study also focus on the impact of interaction between phospho‐moesin and CD44 on AGE‐induced retinal microvascular pericyte (RMP) migration and neovessel maturation.MethodsAGE‐treated mouse model was developed in WT mice, in receptor for AGEs (RAGE)‐knockout (RAGE(−/−)) mice and in WT postnatal mice by intraperitoneal injection of AGEs. retinal vessels were observed by whole mount immunofluorescence staining for isolectin B4 (IB4). Mouse aorta rings were embedded in 3D collagen and the sprouted vascular endothelial cells was identified by immunofluorescent staining IB4. Pericytes were identified with NG2 or α‐SMA in retina and aortic ring. Primary RMPs were isolated, purified from weanling rats, and identified by cellular markers α‐SMA, PDGFR‐β, NG2 and desmin using immunofluorescence microscopy. Migration of RMPs was induced in different condition and detected with transwell. The phosphorylation of moesin was measured using western blotting. Colocalization and interaction between CD44, phospho‐moesin and F‐actin was detected by confocal laser scanning microscopy and coimmunoprecipitation.ResultsThe number of neovessels in retina and the phosphorylation of moesin were increased in WT mice after AGE treatment for 1, 2, and 6 months, and the increase of retinal neovessels was more obvious with time extension. There were no significant alteration in RAGE(−/−) mice after AGE treatment for six months. The phosphorylation of moesin and the number of neovessels in retina were increased in WT postnatal mice after AGE treatment for 7 d. Both in vivo and in vitro AGE treatment could enhanced the phosphorylation of moesin and promoted neovessel sprouting in mouse aortic ring, while there were not significant changes in RAGE(−/−) mouse aortic ring. The pericyte cover rate was reduced in neovessels in AGE‐treated mouse aortic ring, as well as in retinal microvessels in AGE‐treated mice. AGE‐BSA increased moesin phosphorylation in RMPs, which was in consistent with RMP migration in a dose‐ and time‐dependent manner. AGE‐BSA promoted CD44 and phospho‐moesin interaction, as well as the F‐actin stress fiber reassembly. The inhibition of ROCK by specific inhibitor Y27632 decreased AGE‐induced moesin phosphorylation and subsequently attenuated pericyte migration, suppressed the formation of CD44‐phospho‐ERM complex and redistribution of F‐actin. The inhibiting mutant of phosphorylation of Thr 558 in moesin suppressed AGE‐induced migration and F‐actin rearrangement, while the activating mutant of moesin phosphorylation of Thr 558 promoted cell migration and F‐actin alteration.ConclusionsAGEs promoted angiogenesis in mice by inducing moesin phosphorylation. The interaction of phospho‐moesin and CD44 participates in the pathogenesis of AGE‐induced RMP migration through ROCK activation and might hamper the maturation of neovessels.Support or Funding InformationSponsored by General Program from Natural Science Foundation of China (81370226, 81170297, and 80971201)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel nuclear complex of DRR1, F-actin and COMMD1 involved in NF-κB degradation and cell growth suppression in neuroblastoma.

Downregulated in renal cell carcinoma 1 (DRR1) has important roles in tumor cell growth, neuron survival and spine formation, and was recently shown to bind actin. However, the roles of nuclear DRR1 remain largely unexplored. Here, we identified an interaction between filamentous actin (F-actin) and DRR1 in the nucleus, and demonstrated that copper metabolism MURR1 domain-containing 1 (COMMD1) is another binding partner of DRR1. Accordingly, DRR1, F-actin and COMMD1 were shown to form a complex in the nucleus, and the stability of COMMD1 was enhanced in this complex. Increased nuclear COMMD1 in turn promoted the degradation of NF-κB. In addition, DRR1 and COMMD1 suppressed the cyclin D1 expression, G1/S transition and cell proliferation of neuroblastoma cells. The binding between DRR1 and F-actin in the nucleus was required for these events. Consistent with these facts, low expressions of DRR1 were associated with tumorigenesis of human neuroblastoma and its mouse model. This study has thus revealed a novel nuclear complex of F-actin, DRR1 and COMMD1 that is involved in NF-κB degradation and cell cycle suppression in neuroblastoma cells.

The interchain disulfide cross-linking of tropomyosin alters its regulatory properties and interaction with actin filament

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca2+-regulated contraction of striated muscles. Two chains of Tpm can be cross-linked by formation of a disulfide bond between Cys-190 residues. Normally, the SH-groups of these residues in cardiac muscle are in reduced state but in heart pathologies the interchain cross-linking of Tpm was shown to occur. Previous studies have shown that this cross-linking increases the thermal stability of the C-terminal part of the Tpm molecule. However it was unclear how this affects its functional properties. In the current work, we studied functional features of cross-linked Tpm at the level of isolated proteins. The results have shown that the cross-linking greatly decreases affinity of Tpm for F-actin and stability of the Tpm–F-actin complex. It also increases sliding velocity of regulated thin filaments in an in vitro motility assay. This last effect was mostly pronounced when cardiac isoforms of myosin and troponin were used instead of skeletal ones. The results indicate that cross-linking significantly affects properties of Tpm and actin-myosin interaction and can explain, at least partly, the role of the interchain disulfide cross-linking of cardiac Tpm in human heart diseases.

Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection.

Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin

CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable “tense” state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin.

The interactions between SATB1 and F-actin are important for mechanisms of active cell death.

The direct involvement of nuclear actin filaments in gene transcription and remodeling of chromatin is still debatable. However, nuclear localization of F-actin and its interactions with other nuclear matrix proteins have been reported. The aim of the study was to estimate the interactions between nuclear F-actin and one of the matrix proteins, special AT-rich sequence-binding protein 1 (SATB1), during active cell death induced in vitro by geldanamycin (GA). The expression of SATB1 was modified by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The amount and localization of F-actin were altered by changes of cofilin-1 (CFL1) expression in MCF-7 cells. The association between SATB1 and F-actin during GA-induced cell death was analyzed using confocal and transmission electron microscopy. Our studies revealed the colocalization between nuclear F-actin and SATB1 protein, during GA-induced death of breast cancer MCF-7 cells. The colocalization was enhanced in cells with overexpressed SATB1 and cofilin-1. At the ultrastructural level the SATB1 and F-actin complexes were seen at the border of condensed and decondensed chromatin. The presence of SATB1/F-actin molecular complexes was confirmed by magnetic separation of F-actin and interacting proteins. We suggest that the molecular interactions between SATB1 and F-actin are necessary for active cell death to occur.

Structure of the Complex of F-Actin and DNGR-1, a C-Type Lectin Receptor Involved in Dendritic Cell Cross-Presentation of Dead Cell-Associated Antigens

DNGR-1 is a C-type lectin receptor that binds F-actin exposed by dying cells and facilitates cross-presentation of dead cell-associated antigens by dendritic cells. Here we present the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Unusually for F-actin binding proteins, the DNGR-1 ligand binding domain contacts three actin subunits helically arranged in the actin filament, bridging over two protofilaments, as well as two neighboring actin subunits along one protofilament. Mutation of residues predicted to mediate ligand binding led to loss of DNGR-1-dependent cross-presentation of dead cell-associated antigens, formally demonstrating that the latter depends on F-actin recognition. Notably, DNGR-1 has relatively modest affinity for F-actin but multivalent interactions allow a marked increase in binding strength. Our findings shed light on modes of actin binding by cellular proteins and reveal how extracellular detection of cytoskeletal components by dedicated receptors allows immune monitoring of loss of cellular integrity.

Mathematical modeling of sustainable synaptogenesis by repetitive stimuli suggests signaling mechanisms in vivo.

The mechanisms of long-term synaptic maintenance are a key component to understanding the mechanism of long-term memory. From biological experiments, a hypothesis arose that repetitive stimuli with appropriate intervals are essential to maintain new synapses for periods of longer than a few days. We successfully reproduce the time-course of relative numbers of synapses with our mathematical model in the same conditions as biological experiments, which used Adenosine-3′, 5′-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS) as external stimuli. We also reproduce synaptic maintenance responsiveness to intervals of Sp-cAMPS treatment accompanied by PKA activation. The model suggests a possible mechanism of sustainable synaptogenesis which consists of two steps. First, the signal transduction from an external stimulus triggers the synthesis of a new signaling protein. Second, the new signaling protein is required for the next signal transduction with the same stimuli. As a result, the network component is modified from the first network, and a different signal is transferred which triggers the synthesis of another new signaling molecule. We refer to this hypothetical mechanism as network succession. We build our model on the basis of two hypotheses: (1) a multi-step network succession induces downregulation of SSH and COFILIN gene expression, which triggers the production of stable F-actin; (2) the formation of a complex of stable F-actin with Drebrin at PSD is the critical mechanism to achieve long-term synaptic maintenance. Our simulation shows that a three-step network succession is sufficient to reproduce sustainable synapses for a period longer than 14 days. When we change the network structure to a single step network, the model fails to follow the exact condition of repetitive signals to reproduce a sufficient number of synapses. Another advantage of the three-step network succession is that this system indicates a greater tolerance of parameter changes than the single step network.

Remodeling of actin filaments by ADF/cofilin proteins

Cofilin/ADF proteins play key roles in the dynamics of actin, one of the most abundant and highly conserved eukaryotic proteins. We used cryoelectron microscopy to generate a 9-Å resolution three-dimensional reconstruction of cofilin-decorated actin filaments, the highest resolution achieved for a complex of F-actin with an actin-binding protein. We show that the cofilin-induced change in the filament twist is due to a unique conformation of the actin molecule unrelated to any previously observed state. The changes between the actin protomer in naked F-actin and in the actin-cofilin filament are greater than the conformational changes between G- and F-actin. Our results show the structural plasticity of actin, suggest that other actin-binding proteins may also induce large but different conformational changes, and show that F-actin cannot be described by a single molecular model.

Fast Magic Angle Sample Spinning NMR Yields a View of the F-actin - Cofilin Complex with Atomic Resolution

Proteins of the ADF/cofilin family are vital regulators of the actin cytoskeleton in eukaryotes. Binding of cofilin results in dramatic reorganization of the F-actin structure and potentiates filament severing, and its accelerated depolymerization from the pointed end. Although the X-ray structure of monomeric actin with cofilin-homology domain of twinfilin has been recently solved, F-actin evades crystallization and therefore the analysis of cofilin interaction with F-actin at the atomic level calls for alternative approaches. While considerable insight into the cofilin F-actin complex has been gained through chemical cross-linking and radiolytic footprinting, these approaches were unable to generate high resolution information on this interaction While considerable insight into the cofilin F-actin complex has been gained through chemical cross-linking and radiolytic footprinting, novel approaches are still desirable. Here we demonstrate that Fast-MAS Solid State NMR is a high sensitivity approach to studying this system. Isotopically labeled S. cerevisieae yeast cofilin, in complex with polymerized yeast actin, allows for an atomic resolution view of cofilin within the complex. Intramolecular conformational changes occurring in cofilin upon binding to actin can be deduced from dipolar and scalar coupling based spectra. Additional studies of the free cofilin have been performed in the solution and solid states for comprehensive comparisons. Therefore, our data demonstrates the general feasibility of the SS-NMR approach for studying the interaction between F-actin and actin binding proteins.

3E1346 三次元FRET解析によるFアクチン-トロポニンコアドメイン複合体のアトミックモデル構築(3E 筋肉2,日本生物物理学会第49回年会)

3E1346 三次元FRET解析によるFアクチン-トロポニンコアドメイン複合体のアトミックモデル構築(3E 筋肉2,日本生物物理学会第49回年会)

ADP-Ribosylation of Cross-Linked Actin Generates Barbed-End Polymerization-Deficient F-Actin Oligomers

Actin filament subunit interfaces are required for the proper interaction between filamentous actin (F-actin) and actin binding proteins (ABPs). The production of small F-actin complexes mimicking such interfaces would be a significant advance toward understanding the atomic interactions between F-actin and its many binding partners. We produced actin lateral dimers and trimers derived from F-actin and rendered polymerization-deficient by ADP-ribosylation of Arg-177. The degree of modification resulted in a moderate reduction in thermal stability. Calculated hydrodynamic radii were comparable to theoretical values derived from recent models of F-actin. Filament capping capabilities were retained and yielded pointed-end dissociation constants similar those of wild-type actin, suggesting native or near-native interfaces on the oligomers. Changes in DNase I binding affinity under low and high ionic strength suggested a high degree of conformational flexibility in the dimer and trimer. Polymer nucleation activity was lost upon ADP-ribosylation and rescued upon enzyme-mediated deADP-ribosylation, or upon binding to gelsolin, suggesting that interactions with actin binding proteins can overcome the inhibiting activities of ADP-ribosylation. The combined strategy of chemical cross-linking and ADP-ribosylation provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers that are able to act as F-actin binding protein scaffolds.

Hierarchical structures of the actin/polycation complexes, investigated by ultra-small-angle neutron scattering and fluorescence microscopy

By employing a combined method of ultra-small-angle & small-angle neutron scattering (USANS&SANS) and fluorescence microscopy (FM), we investigated the solution mixtures of filamentous actin (F-actin) and the synthesized cationic polymer poly-N-[3-(dimethylamino)propyl]acrylamide methylchloride quaternary (PDMAPAA-Q). The combined USANS&SANS method, covering a wide range of length scales from 10 micrometres to nanometres, plays a crucial role in elucidating the hierarchical structure of the complex structure, as it is in an aqueous solution. FM determined that the complex of F-actin and PDMAPAA-Q appears with a finite size (>10μm), referred to as a superbundle, and its morphology changes from a globular one to a stretched one by increasing the salt (KCl) concentration Cs from 0.01 to 0.3 M. USANS&SANS revealed that the superbundle is composed of a structural unit of a protobundle, in which PDMAPAA-Q binds F-actins in order to form a hexagonal lattice. The diameter of the protobundle (DII), determined by USANS&SANS, increases from 40 to 290 nm as Cs increases from 0.01 to 0.3 M. In order to explain the finite-size & hierarchical condensation observed in the actin/PDMAPAA-Q solution, we employ a scenario of imperfect charge neutralization between F-actins and PDMAPAA-Q. Due to the chain connectivity of PDMAPAA-Q, a spatial distribution of positive charges around the F-actins becomes inhomogeneous, so that the repulsive electrostatic interaction appearing in the protobundle limits bundle formation with an infinite-size. The morphology of the superbundles is controlled by the bending rigidity due to individual protobundles, which significantly increases as DII increases.

Binding of N-Terminus Fragments of Cardiac Myosin-Binding C-protein to Actin

Myosin-binding protein C (C-protein) is a sarcomeric protein regularly associated with thick filaments of vertebrate skeletal and heart muscle and is involved in the regulating of muscle contraction. C-protein belongs to the immunoglobulin- fibronectin superfamily of proteins and it consists of 11 tandemly arranged immunoglobulin-like domains. Four Ig-domains at the N-terminus of the cardiac isoform (C0-C1-m-C2 or C0C2) influence the actin -myosin S1 interaction whereas the C-terminal domains (C7-C10) play a structural role in the sarcomere, binding to myosin thick filaments. It has been shown that C0C2 fragment stabilizes F-actin, decorating the filaments in a highly regular arrangement.We used electron microscopy and single particle image analysis to reconstruct complexes of F-actin with cardiac C-protein fragments containing 4 Ig-domains (C0C2) and 2 Ig-domains (C0C1). Both fragments regularly decorated actin filaments and image analysis has revealed substantial rod-shaped density connecting adjacent monomers along the actin filament. The fragments induced dendritic growth of actin filaments in the first several minutes of co-polymerization followed by formation of regular sheets of filaments and ordered bundles. The results will be discussed in the terms of suggested involvement of C-protein in the modulation of contractile cycles of heart muscles.

A novel approach to the structural analysis of partially decorated actin based filaments

We describe a novel set of single particle based procedures for the structural analysis of electron microscope images of muscle thin filaments and other partially decorated actin based filaments. The thin filament comprises actin and the regulatory proteins tropomyosin and troponin in a 7:1:1 M ratio. Prior to our work, structure analysis from electron microscope images of the thin filament has largely involved either helical averaging defined by the underlying actin helix or the use of single particle analysis but using a starting model as a reference structure. Our single particle based approach yields an accurate structure for the complete thin filament by avoiding the loss of information from troponin and tropomyosin associated with helical averaging and also removing the potential reference bias associated with the use of a starting model. The approach is more widely applicable to sub-stoichiometric complexes of F-actin and actin-binding proteins.