Actin-binding Site Research Articles

Molecular Mechanism of Fascin Function in Filopodial Formation

Filopodia are cell surface protrusions that are essential for cell migration. This finger-like structure is supported by rigid tightly bundled actin filaments. The protein responsible for actin bundling in filopodia is fascin. However, the mechanism by which fascin functions in filopodial formation is not clear. Here we provide biochemical, cryo-electron tomographic, and x-ray crystal structural data demonstrating the unique structural characteristics of fascin. Systematic mutagenesis studies on 100 mutants of fascin indicate that there are two major actin-binding sites on fascin. Crystal structures of four fascin mutants reveal concerted conformational changes in fascin from inactive to active states in the process of actin bundling. Mutations in any one of the actin-binding sites impair the cellular function of fascin in filopodial formation. Altogether, our data reveal the molecular mechanism of fascin function in filopodial formation.

Actin Filament Nucleation by Smooth Muscle Leiomodin-1

The formation of new filaments is an essential, but kinetically unfavorable, step in actin cytoskeleton remodeling. In cells nucleation has to be catalyzed by proteins called nucleators that bring together at least two actin monomers and stabilize a polymerization nucleus. Among known filament nucleators Leiomodin (Lmod) is the only one specifically expressed in muscle cells. Lmod is related to the F-actin pointed-end capping protein tropomodulin (Tmod), with which it shares two actin-binding sites: a flexible N-terminal region and a leucine-rich repeat (LRR) domain. In addition, Lmod contains a unique C-terminal extension that features a WH2 domain, another actin-binding site.There are three muscle-type specific isoforms of Lmod: smooth muscle Lmod1, cardiac and striated muscle Lmod2, and the fetal isoform Lmod3. To date, only Lmod2 has been shown to nucleate actin filaments. Lmod1 is quite different in sequence from Lmod2 (37% identity, most of which is concentrated in the LRR). Here we demonstrate that despite these differences smooth muscle Lmod1 is also an actin filament nucleator. However its activity and mechanism of nucleation vary significantly from that of Lmod2.

More than Just a Cargo Adapter: Melanophilin Prolongs Slow Processive Runs of Myosin Va

Melanophilin (Mlph) is a cargo adapter protein that links melanosomes via Rab27a to myosin Va (myoVa) for transport along actin. In the absence of cargo, full length myoVa is in equilibrium between a folded, inactive and an extended, active conformation. This equilibrium causes a deviation from a hand-over-hand stepping pattern due to altered gating between the heads (Armstrong et al. 2012). Cargo binding has been suggested to activate myoVa for transport. Here we used single molecule TIRF assays at near physiological ionic strength (150mM KCl) to determine the effect of Mlph on myoVa processivity. In the absence of Mlph, Qdot labeled full-length myoVa moved at a median velocity of 443nm/s, and showed the altered stepping pattern previously seen at lower ionic strength. Addition of Mlph recruited 14-times more motors to move processively, consistent with a simple model of cargo activation. The myoVa-Mlph complex also showed increased run lengths, with many motors traveling to the ends of the actin filament. In the presence of Mlph, myoVa moved much more slowly (median velocity=75nm/s). The speed distribution was asymmetrical and similar to speeds of melanosome movement observed in vivo. In the presence of Mlph, myoVa showed normal gating between the heads, and hand-over-hand steps on actin typical of a fully-active motor. Based on mutagenesis of Mlph the enhanced processivity depended on a positively charged cluster of amino acids in the actin binding site of Mlph. This suggests that Mlph acts as an electrostatic tether to limit myoVa dissociation from actin, a property likely to favor the transfer of melanosomes to adjacent keratinocytes in vivo. More generally, our results suggest that adapter proteins which link motors to cargo can affect motor properties in ways favorable for their biological role.

Hydrogen-Deuterium Exchange Mass Spectrometry of Phosphorylation Induced Changes in Smooth Muscle Myosin

Inter-molecular interactions between two smooth muscle myosin heads have been observed using chemical cross linking and visualized by electron microscopy. In order to probe the nature of the phosphorylation-dependent head-head interactions, we performed Hydrogen-Deuterium exchange electrospray ionization mass spectrometry (HDX-MS) on phosphorylated and unphosphorylated smooth muscle heavy mero myosin (HMM) from chicken gizzard. HDX rates are determined by solvent accesibility and the strength of hydrogen bonding and thus are a good fingerprint of intra- and intermolecular structural changes. The observed decrease in deuterium uptake upon phosphorylation in the coiled-coil and the hinge region of the heavy chain (residues 864-877), is consistent with the unraveling of the myosin dimer at the hinge. Unexpectedly, we have observed phosphorylation dependent changes in the regulatory light chain (RLC 58-69), which are not implicated in the RLC-RLC or RLC-heavy chain interface. No changes were observed for N-terminal region of RLC that was postulated to undergo major secondary structure changes, or in the heavy chain actin binding site (residues 397-437), or any of the residues that form the head-head interface in the EM model of Wendt & Taylor. This latter finding indicates that the interaction between the heads is probably transient in solution, or in the unlikely case, interacting heads constitute a very small fraction of total protein.

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

Cloning and characterization of a fish specific gelsolin family gene, ScinL, in olive flounder (Paralichthys olivaceus)

Scinderin like (ScinL) gene is a unique gelsolin family gene found only in fish. In this study ScinL gene was cloned in olive flounder for the first time and characterized its expression and function. Flounder ScinL cDNA consists of 2911 nucleotides encoding a putative protein of 720 amino acids (79.4 kDa). In phylogenetic analysis, flounder ScinL is closely related to ScinL of zebra fish, anableps, and fugu with the similarity of 51-72%. Fish ScinLs are positioned between gelsolin and scinderin of other species. Flounder ScinL protein has the highly conserved actin and PIP2 binding sites, Ca(2+) coordination site, and a C-terminal latch helix preventing the activation of ScinL protein in the absence of Ca(2+). Putative binding sites for NFAT and AP-1 were found in 5' flanking region. Constitutive ScinL expression was found in most organs and the expression level was higher in gill, head kidney, trunk kidney, spleen and skin than muscle, stomach, intestine and brain. In Q-PCR analysis ScinL and CYP1A1 gene expression were significantly upregulated by BaP in head kidney in vivo and in vitro, and in macrophage cells. Upregulated ScinL expression by BaP was blocked by EGTA, indicating a calcium dependent regulation of ScinL expression.

Nuclear actin and myosins at a glance

Actin and myosin II form the archetypical molecular motor complex. Myosin II, like all members of the myosin superfamily, is an actin-activated ATPase that uses the energy released when ATP is hydrolyzed to do work. Although we typically associate work with muscle contraction, cell motility and cell

Determination of the critical residues responsible for cardiac myosin binding protein C's interactions

Despite early demonstrations of myosin binding protein C's (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure–function relationships is needed to fully understand cMyBP-C's ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C's N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C's C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.

The myosin interacting-heads motif is present in the relaxed thick filament of the striated muscle of scorpion

Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures.

Processive Steps in the Reverse Direction Require Uncoupling of the Lead Head Lever Arm of Myosin VI

Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

Identification of the Actin and Plasminogen Binding Regions of Group B Streptococcal Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in significantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.

Arabidopsis Vacuolar H+-ATPase (V-ATPase) B Subunits Are Involved in Actin Cytoskeleton Remodeling via Binding to, Bundling, and Stabilizing F-actin

Vacuolar H(+)-ATPase (V-ATPase) is a membrane-bound multisubunit enzyme complex composed of at least 14 different subunits. The complex regulates the physiological processes of a cell by controlling the acidic environment, which is necessary for certain activities and the interaction with the actin cytoskeleton through its B and C subunits in both humans and yeast. Arabidopsis V-ATPase has three B subunits (AtVAB1, AtVAB2, and AtVAB3), which share 97.27% sequence identity and have two potential actin-binding sites, indicating that these AtVABs may have crucial functions in actin cytoskeleton remodeling and plant cell development. However, their biochemical functions are poorly understood. In this study, we demonstrated that AtVABs bind to and co-localize with F-actin, bundle F-actin to form higher order structures, and stabilize actin filaments in vitro. In addition, the AtVABs also show different degrees of activities in capping the barbed ends but no nucleating activities, and these activities were not regulated by calcium. The functional similarity and differences of the AtVABs implied that they may play cooperative and distinct roles in Arabidopsis cells.

The C-terminal tail domain of metavinculin, vinculin’s splice variant, severs actin filaments

Vinculin and its splice variant, metavinculin (MV), are key elements of multiple protein assemblies linking the extracellular matrix to the actin cytoskeleton. Vinculin is expressed ubiquitously, whereas MV is mainly expressed in smooth and cardiac muscle tissue. The only difference in amino acid sequence between the isoforms is a 68-residue insert in the C-terminal tail domain of MV (MVt). Although the functional role of this insert remains elusive, its importance is exemplified by point mutations that are associated with dilated and hypertrophic cardiomyopathy. In vinculin, the actin binding site resides in the tail domain. In this paper, we show that MVt binds actin filaments similarly to the vinculin tail domain. Unlike its splice variant, MVt did not bundle actin filaments. Instead, MVt promoted severing of actin filaments, most efficiently at substoichiometric concentrations. This surprising and seemingly contradictory alteration of vinculin function by the 68-residue insert may be essential for modulating compliance of vinculin-induced actin bundles when exposed to rapidly increasing external forces.

Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

Molecular Basis of Dynamic Relocalization of Dictyostelium Myosin IB

Class I myosins have a single heavy chain comprising an N-terminal motor domain with actin-activated ATPase activity and a C-terminal globular tail with a basic region that binds to acidic phospholipids. These myosins contribute to the formation of actin-rich protrusions such as pseudopodia, but regulation of the dynamic localization to these structures is not understood. Previously, we found that Acanthamoeba myosin IC binds to acidic phospholipids in vitro through a short sequence of basic and hydrophobic amino acids, BH site, based on the charge density of the phospholipids. The tail of Dictyostelium myosin IB (DMIB) also contains a BH site. We now report that the BH site is essential for DMIB binding to the plasma membrane and describe the molecular basis of the dynamic relocalization of DMIB in live cells. Endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The BH site is required for association of DMIB with the plasma membrane at all stages where it colocalizes with phosphoinositide bisphosphate/phosphoinositide trisphosphate (PIP(2)/PIP(3)). The charge-based specificity of the BH site allows for in vivo specificity of DMIB for PIP(2)/PIP(3) similar to the PH domain-based specificity of other class I myosins. However, DMIB-head is required for relocalization of DMIB to the front of migrating cells. Motor activity is not essential, but the actin binding site in the head is important. Thus, dynamic relocalization of DMIB is determined principally by the local PIP(2)/PIP(3) concentration in the plasma membrane and cytoplasmic F-actin.

Highlights from the Literature

Highlights from the Literature

The role of the ERM protein family in maintaining cellular polarity, adhesion and regulation of cell motility

Ezrin, radixin and moesin, forming the ERM protein family, act as molecular crosslinkers between actin filaments and proteins anchored in the cell membrane. By participating in a complex intracellular network of signal transduction pathways, ERM proteins play a key role in the regulation of adhesion and polarity of normal cells through interactions with membrane molecules, e.g. E-cadherin. Dynamic cytoskeletal transformations, in which the ERM and Rho GTPases are involved, lead to the formation of membrane-cytoplasmic structures, such as filopodia and lamellipodia, which are responsible for cellular motility. The interactions of ERM proteins with active Akt kinase cause the acquisition of antiapoptotic cellular features by downregulation of the proapoptotic protein Bad. ERM protein activity is regulated by phosphorylation/dephosphorylation reactions and linking phosphatidylinositols. The model of activation based on the molecular conformation changes by breaking the intramolecular bonds and exposing actin binding sites is essential for the proper functioning of the ERM proteins. Additionally, the connection types between the ERM and membrane proteins (direct or indirect by EBP50 and E3KARP) play an important role in transduction of signals from the extracellular matrix. Due to the wide range of ezrin, radixin and moesin cytophysiological features, detailed exploration of the ERM biochemistry will provide a series of answers to questions about ambiguous functions in many intracellular signal transduction pathways.

A novel actin binding site of myosin required for effective muscle contraction

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Myosin IIIB Uses an Actin-Binding Motif in Its Espin-1 Cargo to Reach the Tips of Actin Protrusions

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

Mechanical Stability of the Cardiac Myosin Binding Protein-C (CMybP-C) Motif is Phosphorylation-Dependent

Enhanced cardiac contractility results from phosphorylation within the cMyBP-C motif domain by affecting the interaction of cMyBP-C's N-terminus with actin and/or myosin. We investigated whether motif (i.e. C1C2 linker) phosphorylation alters its structural conformation by characterizing the force-extension behavior of expressed N-terminal cMyBP-C fragments (i.e.C0C3) that contain the motif using atomic force spectroscopy. Proteins were adsorbed onto glass slides, bound non-specifically to SiNi AFM probes, and stretched at 700nm/s. Force-extension traces showed the characteristic sawtooth shape of unfolding immunoglobulin domains, and were well fitted by the wormlike chain model. The model fits produced a median persistence length of 0.36nm and an average C0C3 contour length of 71.5nm, suggesting that the motif is disordered and freely extensible when unphosphorylated. PKA phosphorylation did not change immunoglobulin unfolding force or persistence length but decreased the contour length by 27nm, suggesting that the motif assumed a folded configuration that is stable under load. This was investigated further by varying the extent of phosphorylation using phosphomimetic mutants, in which serines 273, 282, and 302 were replaced with different combinations of alanines or aspartic acids in order to mimic the unphosphorylated and phosphorylated states respectively. While substitution with all alanines or all aspartic acids produced similar results to the control and PKA treated fragments respectively, phosphorylation at only one or two sites did not reduce contour length significantly. This implies that phosphorylation at all three sites is required for motif folding to be stable under load, while partial phosphorylation may result in a fold which is readily extended at low force. In conclusion, cMyBP-C phosphorylation could modulate the cMyBP-C's N-terminal domain interactions with actin and/or myosin by folding the motif in such a way that binding sites for actin and/or myosin are hidden.