Actin-binding Properties Research Articles

New anti-actin drugs in the study of the organization and function of the actin cytoskeleton.

The high degree of structural and molecular complexity of the actin-based cytoskeleton, combined with its ability to reorganize rapidly and locally in response to stimuli, and its force-generating properties, have made it difficult to assess how the different actin structures are assembled in cells, and how they regulate cell behavior. An obvious approach to study the relationships between actin organization, dynamics, and functions is the specific perturbation of actin structures using pharmacological means. Until recently there were only a few agents available that interfered with cellular activities by binding to actin and most of our knowledge concerning the involvement of actin in basic cellular processes was based on the extensive use of the cytochalasins. In recent years we have identified an increasing number of actin-targeted marine natural products, including the latrunculins, jasplakinolides (jaspamides), swinholide A, misakinolide A, halichondramides, and pectenotoxin II, which are discussed in this article. All these marine-sponge-derived compounds are unusual macrolides and can be classified into several major families, each with its own distinct chemical structures. We describe the current state of knowledge concerning the actin-binding properties of these compounds and show that each class of drugs alters the distribution patterns of actin in a unique way, and that even within a chemical class, structurally similar compounds can have different biochemical properties and cellular effects. We also discuss the effects of these new drugs on fenestrae formation in liver endothelial cells as an example of their usefulness as powerful tools to selectively unmask actin-mediated dynamic processes.

Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP

The vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.

Effects of single amino acid substitutions in the actin-binding site on the biological activity of bovine profilin I.

For a detailed analysis of the profilin-actin interaction, we designed several point mutations in bovine profilin I by computer modeling. The recombinant proteins were analyzed in vitro for their actin-binding properties. Mutant proteins with a putatively higher affinity for actin were produced by attempting to introduce an additional bond to actin. However, these mutants displayed a lower affinity for actin than wild-type profilin, suggesting that additional putative bonds created this way cannot increase profilin's affinity for actin. In contrast, mutants designed to have a reduced affinity for actin by eliminating profilin-actin bonds displayed the desired properties in viscosity assays, while their binding sites for poly(L)proline were still intact. The profilin mutant F59A, with an affinity for actin reduced by one order of magnitude as compared to wild-type profilin, was analyzed further in cells. When microinjected into fibroblasts, F59A colocalized with the endogenous profilin and actin in ruffling areas, suggesting that profilins are targeted to and tethered at these sites by ligands other than actin. Profilin null cells of Dictyostelium were transfected with bovine wild-type profilin I and F59A. Bovine profilin I, although expressed to only approximately 10% of the endogenous profilin level determined for wild-type Dictyostelium, caused a substantial rescue of the defects observed in profilin null amoebae, as seen by measuring the growth of colony surface areas and the percentage of polynucleated cells. The mutant protein was much less effective. These results emphasize the highly conserved biological function of profilins with low sequence homology, and correlate specifically their actin-binding capacity with cell motility and proliferation.

Human myosin-IXb is a mechanochemically active motor and a GAP for rho.

The heavy chains of the class IX myosins, rat myr5 and human myosin-IXb, contain within their tail domains a region with sequence homology to GTPase activating proteins for the rho family of G proteins. Because low levels of myosin-IXb expression preclude purification by conventional means, we have employed an immunoadsorption strategy to purify myosin-IXb, enabling us to characterize the mechanochemical and rho-GTPase activation properties of the native protein. In this report we have examined the light chain content, actin binding properties, in vitro motility and rho-GTPase activity of human myosin-IXb purified from leukocytes. The results presented here indicate that myosin-IXb contains calmodulin as a light chain and that it binds to actin with high affinity in both the absence and presence of ATP. Myosin-IXb is an active motor which, like other calmodulin-containing myosins, exhibits maximal velocity of actin filaments (15 nm/second) in the absence of Ca2+. Native myosin-IXb exhibits GAP activity on rho. Class IX myosins may be an important link between rho and rho-dependent remodeling of the actin cytoskeleton.

Conformational and functional studies of three gelsolin subdomain‐1 synthetic peptides and their implication in actin polymerization

Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin—actin interfaces were synthesized. The peptides (15–28, 42–55, and 96–114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42–55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin—actin interface is essential in the actin polymerization process. © 1997 John Wiley & Sons, Inc. Biopoly 41: 647–655, 1997

Conformational and functional studies of three gelsolin subdomain-1 synthetic peptides and their implication in actin polymerization

Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin-actin interfaces were synthesized. The peptides (15-28, 42-55, and 96-114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42-55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin-actin interface is essential in the actin polymerization process.

Complete cDNA sequence and tissue localization of N-RAP, a novel nebulin-related protein of striated muscle.

We have cloned and sequenced the full-length cDNA of N-RAP, a novel nebulin-related protein, from mouse skeletal muscle. The N-RAP message is specifically expressed in skeletal and cardiac muscle, but is not detected by Northern blot in non-muscle tissues. The full-length N-RAP cDNA contains an open reading frame of 3,525 base pairs which is predicted to encode a protein of 133 kDa. A 587 amino acid region near the C-terminus is 45% identical to the actin binding region of human nebulin, containing more than 2 complete 245 residue nebulin super repeats. The N-terminus contains the consensus sequence of a cysteine-rich LIM domain, which may function in mediating protein-protein interactions. These data suggest that the encoded protein may link actin filaments to some other proteins or structure. We expressed full-length N-RAP in Escherichia coli, as well as the nebulin-like super repeat region of N-RAP (N-RAP-SR) and the region between the LIM domain and N-RAP-SR (N-RAP-IB). An anti-N-RAP antibody raised against a 30 amino acid peptide corresponding to sequence from N-RAP-IB detected recombinant N-RAP and N-RAP-IB, but failed to detect N-RAP-SR. This antibody specifically identified a 185 kDa band as N-RAP on immunoblots of mouse skeletal and cardiac muscle proteins. In an assay of actin binding to electrophoresed and blotted proteins, we detected significant actin binding to expressed nebulin super repeats and N-RAP-SR, but only a trace amount of binding to N-RAP-IB. In immunofluorescence experiments, N-RAP was found to be localized at the myotendinous junction in mouse skeletal muscle and at the intercalated disc in cardiac muscle. Based on its domain organization, actin binding properties, and tissue localization, we propose that N-RAP plays a role in anchoring the terminal actin filaments in the myofibril to the membrane and may be important in transmitting tension from the myofibrils to the extracellular matrix.

Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin.

Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, epsilon ADP and TNP-ADP. The complexes of epsilon ADP and BeFx or AIF4- were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.AIF4- and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca(2+)-ATPase (Ki = 1.6 microM). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP approximately ADP.AIF4- approximately ADP.BeFx > ATP gamma S > ADP > PPi. In the case of S1dC, the effects of different nucleotides were smaller and indistinguishable from each other. S1dC bound actin tighter than S1 (Kd = 7 nM and 60 nM, respectively). The actin activated MgATPase activity of S1dC varied between preparations, and the Vmax and K(m) values ranged between 3 and 7 s-1 and 60 and 190 microM, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inactivations at 35 degrees C. These results show that the nucleotide and actin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and the communication between the nucleotide and actin binding sites in these proteins.

Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long α-helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197–226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F-actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.

Enzymatic Characterization and Functional Domain Mapping of Brain Myosin-V

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.

Effects of calponin on force generation by single smooth muscle cells.

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.

Elastic Properties of Single Titin Molecules Made Visible through Fluorescent F-Actin Binding

Titin (also known as connection) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere [1-4]. Several indirect observations have implicated titin as playing a fundamental role in the generation of passive force of muscle [5,6], driven by titin's elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstrated. Here we have used the recently shown strong actin-binding property of titin [7-9] to indirectly visualize and manipulate single molecules of titin. Titin molecules were immobilized on a microscope coverslip by attaching them to anti-titin antibody. The titin molecules were detected by attaching fluorescent actin filaments to them. The titin molecules were subsequently stretched by manipulating the free end of the attached actin filaments with a glass microneedle. Titin is shown here to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followed by recoil. Titin's unloaded elastic recoil proceeded in two stages: an initial rapid process (15 ms time constant) was followed by a slower one (400 ms time constant). The force necessary to fully extend titin—estimated by observing the breakage of the titin-bound actin filaments—may reach above ∼100 pN (longitudinal tensile strength of actin [10]). Attachment of fluorescent actin filaments to titin provides a useful tool to further probe titin's molecular properties.

Actin and tubulin binding properties of immobilized ezrin

Actin and tubulin binding properties of immobilized ezrin

Fragmin, a microfilament regulatory protein from Physarum polycephalum, is phosphorylated by casein kinase II-type enzymes.

Fragmin is a 42 kDa regulatory protein involved in actin microfilament organization in Physarum polycephalum. We show that fragmin is a target of casein kinase II (CK II) enzymes isolated from evolutionarily divergent species. In Physarum microplasmodia, two such kinases were identified. A serine residue located in the sequence Gly-Gly-Ser-Asp-Leu-Glu constitutes the phosphorylation site and was identified by phosphopeptide sequencing, mass spectometry analysis, and inhibition studies with a synthetic peptide corresponding to this site. Interestingly, the actin-fragmin dimer (A--F) as well as the actin2-fragmin trimer (A2--F) are equally efficient targets, and phosphorylation had no effect on the actin-binding properties of fragmin. Actin-fragmin isolated from microplasmodia revealed a minor acidic fragmin isoform, suggesting that fragmin is phosphorylated in vivo. The actin-fragmin complex is also phosphorylated on the actin subunit by an endogenous actin-fragmin kinase [Gettemans, J., De Ville, Y., Vandekerckhove, J., & Waelkens, E. (1992) EMBO J. 11, 3185-3191]. We show that the two phosphorylation events act independently of each other.

Distinct Biochemical Characteristics of the Two Human Profilin Isoforms

The biochemical characteristics of a new human profilin isoform are described. We refer to this recently described isoform as profilin II (isoelectric point 5.9) in comparison to profilin I (pI 8.4). We expressed both isoforms in bacteria and compared their actin-binding properties, binding to poly(L-proline), affinities for phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and their effects on nucleotide exchange on actin. Profilin I and profilin II have similar affinities for PtdIns(4,5)P2 and poly(L-proline), and both accelerate nucleotide exchange on monomeric actin to the same extent. However, the affinity of profilin I for monomeric actin is about five times higher than the affinity of profilin II for actin. Potential structural differences of profilin I and profilin II that might explain the difference in actin binding are discussed.

The Actin-binding Properties of the Physarum Actin-Fragmin Complex: REGULATION BY CALCIUM, PHOSPHOLIPIDS, AND PHOSPHORYLATION

The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with respect to regulation by Ca2+, phospholipids, and phosphorylation of the actin subunit by the endogenous actin-fragmin kinase. Fragmin possesses two high affinity actin-binding sites and probably also a third, low affinity site. Its nucleating and F-actin severing activities are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin-fragmin specifically binds PIP2 which competes with actin for the Ca(2+)-sensitive site. However, PIP2 cannot dissociate the actin-fragmin complex nor the actin2-fragmin trimer. Efficient F-actin nucleating activity by actin-fragmin is only observed with unphosphorylated actin-fragmin, in the absence of PIP2 and at high Ca2+ (> microM) concentrations. In the presence of PIP2, actin-fragmin only caps actin filaments when unphosphorylated. The results suggest that in the cell, hydrolysis of PIP2, concomitant with the increase of cytosolic Ca2+, could promote subcortical actin polymerization.

Coagulation factor Va is an actin filament binding and cross-linking protein.

Bovine coagulation cofactor factor Va is shown to bind to filament of skeletal muscle actin with a dissociation constant of 40-50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-linkis actin filaments. Factor Va bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va-factor Xa-phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.

A gelsolin-related actin-severing protein with fully reversible actin-binding properties from the tail muscle of crayfish, Astacus leptodactylus.

A Ca(2+)-dependent actin-severing protein was purified from the tail muscle of the crayfish Astacus leptodactylus. The isolation procedure involved extraction at low ionic strength in the presence of EGTA, followed by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The purified crayfish actin modulator appeared as a single band with a molecular mass of 105 kDa on SDS/PAGE. The crustacean actin modulator revealed basic functional properties in common with vertebrate gelsolin, like the Ca(2+)-activated severing of F-actin and the nucleation of actin polymerization. However, both proteins differed in major aspects: Ca2+ activation of crayfish actin modulator started at lower threshold concentrations (0.1 microM). The effect of the modulator on shortening the nucleation phase of actin polymerization was significantly weaker at lower modulator/actin ratios. The modulator formed three distinct stoichiometric complexes with G-actin, identified as binary, ternary and quaternary. Binding of G-actin occurred in a low cooperative manner and was completely reversible by EGTA. Despite some properties being similar to those of villin, crayfish actin modulator did not cross-link actin filaments. It is regarded in principle as a gelsolin-type protein, but with characteristic functional deviations from vertebrate gelsolin.

Interaction of G-actin with thymosin ?4 and its variants thymosin ?9 and thymosin ? 9 met

Thymosin beta 4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin beta 9 and thymosin beta met9 are minor variants of thymosin beta 4. The possible function of these peptides has been investigated by comparing the actin binding properties of these beta-thymosins. Thymosin beta 9 and thymosin beta met9 were found to inhibit polymerization of ATP-actin with identical KDs of 0.7-0.8 microM (as compared to 2 +/- 0.3 microM for thymosin beta 4); like thymosin beta 4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin beta 4 and thymosin beta met9 with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin beta 4 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin beta 4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of approximately 1.5 microM-1 s-1 and 2 s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin beta 4 function in vivo is discussed.