Transformation of actin-encapsulating liposomes induced by cytochalasin D.

Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction.     

Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.     

Membrane-associated actin from the microvillar membranes of ascites tumor cells

A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

激光原子力显微镜观察肌动蛋白体外自组织纤维结构多态性的初步研究

利用原子力显微镜(atomic force microscope,AFM)技术,研究了肌动蛋白体外通过自组织过程形成的纤维结构及其多态性。肌动蛋白在体外通过自组织过程能够聚合形成离散的树状分支的纤维丛和具有不同直径的长纤维等高级纤维结构,表现出明显的结构多态性;与微丝工具药物鬼笔环肽干预下自装配形成的主要由单根微丝和微丝束等纤维成份构成的连续网络结构明显不同。     

Preliminarily investigating the polymorphism of self-organized actin filament in vitro by atomic force microscope

Actin is a major structural component of eukaryoticcytoskeleton and exists in monomer G-actin and filamen-tous F-actin. G-actin consists of 375 amino acid residueswith molecular weight 43 kD and is a highly conservedprotein expressed in most living organi…     

利用激光AFM和TEM探索肌动蛋白体外自装配聚合结构

细胞内肌动蛋白(actin)通过与actin结合蛋白(actin binding proteins,ABPs)相互作用,形成以F-actin为基础多种ABPs参与装配的高度有序的超分子聚合结构,行使各种重要生理功能。在体外聚合条件下,不存在F-actin稳定剂时纯化的actin主要通过自装配形成大尺度的聚集堆积结构;这种表观无序的结构体系由于被认为不具备细胞功能活性而受到忽视。利用激光原子力显微镜(atomic force microscope,AFM)和透射电子显微镜(transmission electron microscope,TEM)技术,对actin体外通过自装配过程形成的大尺度聚集结构进行了细致的观察和分析。研究发现,actin在体外通过自装配过程除了形成无序的蛋白堆积物之外,还能够聚合形成复杂的离散结构,包括树状分支的纤维丛、无规卷曲的纤维簇以及具有不同直径的长纤维等;这些大尺度纤维复合物明显不同于在ABPs或过量F-actin稳定剂参与下形成的由单根微丝和微丝束构成的聚合结构。表明无ABPs或F-actin稳定剂存在的情况下,体外聚合的F-actin在一定条件下可进一步聚集缠绕形成复杂的纤维结构或无序的蛋白堆积物。事实上,actin自装配过程反映了其固有的聚合热力学特性,深入探索将有助于理解ABPs在体内actin超分子聚合结构体系装配中的调控作用及其分子机制。     

Two of the three actin-binding domains of gelsolin bind to the same subdomain of actin. Implications of capping and severing mechanisms.

Gelsolin binds two monomers in the nucleating complex with G-actin in calcium and caps actin filaments. However, 3 actin-binding domains have been identified within its 6 repeating sequence segments corresponding to S1 S2-3 and S4-6. S1 and S4-6 bind only G-actin whereas S2-3 binds specifically to F-actin. Two of the three domains (S2-3 and S4-6) are required for nucleation and a different pair (S1 and S2-3) for severing. Here we show for the first time that the domains unique to nucleation (S4-6) or severing (S1) compete for the same region on subdomain 1 of G-actin. We further show that S2-3 binds actin monomers weakly in G-buffer conditions and that this interaction persists when S1 or S4-6 are also bound. Thus gelsolin associates with two distinct regions on actin. Since S2-3 does not bind monomeric actin in F-buffer, we suggest that its high affinity 1:1 stoichiometry for filament subunits reflects interaction with two adjacent subunits.     

The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for actin assembly, activation of ATP hydrolysis, and delayed phosphate release

Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.     

Mechanism of regulation of actin polymerization by Physarum profilin

Physarum profilin reduces the rates of nucleation and elongation of F- actin and also reduces the extent of polymerization of actin at the steady state in a concentration-dependent fashion. The apparent critical concentration for polymerization of actin is increased by the addition of profilin. These results can be explained by the idea that Physarum profilin forms a 1:1 complex with G-actin and decreases the concentration of actin available for polymerization. The dissociation constant for binding of profilin to G-actin is estimated from the kinetics of polymerization of G-actin and elongation of F-actin nuclei and from the increase of apparent critical concentration in the presence of profilin. The dissociation constants for binding of Physarum profilin to Physarum and muscle actins under physiological ionic conditions are in the ranges of 1.4-3.7 microM and 11.3-28.5 microM, respectively. When profilin is added to an F-actin solution, profilin binds to G-actin which co-exists with F-actin, and then G- actin is dissociated from F-actin to compensate for the decrease of the concentration of free G-actin and to keep it constant at the critical concentration. At the steady state, free G-actin of the critical concentration is in equilibrium not only with F-actin but also with profilin-G-actin complex. The stoichiometry of 1:1 for the formation of complex between profilin and G-actin is directly shown by means of chemical cross-linking.     

Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids

We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

An assessment of the models of thin filament of the sarcomere using small-angle X-ray data on rabbit relaxed muscle

Models of a thin filament based on various G-actin atomic structures and modes of their packing into helical structures were considered. The calculated intensities of actin layer lines were compared with the X-ray data for thin fiber bundles of a relaxed rabbit skeletal muscle. The effect of the main components of thin filament on the intensity of actin layer lines was also analyzed. The F-actin PDB 3BYH model gave the best fit to the experimental data.     

Preparation and characterization of biopolymeric nanoparticles used in drug delivery

Nanotechnology plays an important role in advanced biology and medicine research particularly in the development of potential site-specific delivery systems with lower drug toxicity and greater efficiency. These include microcapsules, liposomes, polymeric microspheres, microemulsions, polymer micelles, hydrogels, solid nanoparticles etc. In the present study, preparation and characterization of biopolymeric gelatin nanoparticles for encapsulating the antimicrobial drug sulfadiazine and its in vivo drug release in phosphate buffer saline (PBS) have been investigated. The nanoparticles prepared by second desolvation process varied in a size range 200 nm and 600 nm with a drug entrapment efficiency of 50% characterized by atomic force microscopy and dynamic light scattering. The drug release from the nanoparticles occurred up to 30% in a controlled manner.     

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent.

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.     

Differences in internal dynamics of actin under different structural states detected by neutron scattering

F-actin, a helical polymer formed by polymerization of the monomers (G-actin), plays crucial roles in various aspects of cell motility. Flexibility of F-actin has been suggested to be important for such a variety of functions. Understanding the flexibility of F-actin requires characterization of a hierarchy of dynamical properties, from internal dynamics of the actin monomers through domain motions within the monomers and relative motions between the monomers within F-actin to large-scale motions of F-actin as a whole. As a first step toward this ultimate purpose, we carried out elastic incoherent neutron scattering experiments on powders of F-actin and G-actin hydrated with D₂O and characterized the internal dynamics of F-actin and G-actin. Well established techniques and analysis enabled the extraction of mean-square displacements and their temperature dependence in F-actin and in G-actin. An effective force constant analysis with a model consisting of three energy states showed that two dynamical transitions occur at ∼150 K and ∼245 K, the former of which corresponds to the onset of anharmonic motions and the latter of which couples with the transition of hydration water. It is shown that behavior of the mean-square displacements is different between G-actin and F-actin, such that G-actin is “softer” than F-actin. The differences in the internal dynamics are detected for the first time between the different structural states (the monomeric state and the polymerized state). The different behavior observed is ascribed to the differences in dynamical heterogeneity between F-actin and G-actin. Based on structural data, the assignment of the differences observed in the two samples to dynamics of specific loop regions involved in the polymerization of G-actin into F-actin is proposed.     

Interaction of actin with carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) receptor in liposomes is Ca2+- and phospholipid-dependent

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Computational prediction of actin–actin interaction

Actin is one of the most abundant proteins in eukaryotic cells, where it plays key roles in cell shape, motility, and regulation. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerization of monomeric G-actin molecules through sequential rowing, is called F-actin. Recently, the crystal structure of an actin dimer has been reported, which details molecular interface in F-actin. In this study, the computational prediction model of actin and actin complex has been constructed base on the atomic model structure of G-actin. To this end, a docking simulation was carried out using predictive docking tools to obtain modeled structures of the actin–actin complex. Following molecular dynamics refinement, hot spots interactions at the protein interface were identified, that were predicted to contribute substantially to the free energy of binding. These provided a detailed prediction of key amino acid interactions at the protein–protein interface. The obtained model can be used for future experimental and computational studies to draw biological and functional conclusions. Also, the identified interactions will be used for designing next studies to understand the occurrence of F-actin structure.