Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

Structural insight into the UNC‐45–myosin complex

The UNC‐45 chaperone protein interacts with and affects the folding, stability, and the ATPase activity of myosins. It plays a critical role in the cardiomyopathy development and in the breast cancer tumor growth. Here we propose the first structural model of the UNC‐45–myosin complex using various in silico methods. Initially, the human UNC‐45B binding epitope was identified and the protein was docked to the cardiac myosin (MYH7) motor domain. The final UNC45B–MYH7 structure was obtained by performing of total 630 ns molecular dynamics simulations. The results indicate a complex formation, which is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosin‐actin complex. A significant interspecies difference in the myosin binding epitope is observed. Our results reveal the structural basis of MYH7 exons 15–16 hypertrophic cardiomyopathy mutations and provide directions for drug targeting. Proteins 2013; 81:1212–1221. © 2013 Wiley Periodicals, Inc.     

Dynamic polymorphism of actin as activation mechanism for cell motility

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.     

Solution structure of the calmodulin‐like C‐terminal domain of Entamoeba α‐actinin2

Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross‐links, or caps the filament ends have been identified and the actin cross‐linker α‐actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α‐actinin is believed to be required for infection. To better understand the role of α‐actinin in the infectious process we have determined the solution structure of the C‐terminal calmodulin‐like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium‐binding EF‐hand motifs, connected with a mobile linker. Proteins 2016; 84:461–466. © 2016 Wiley Periodicals, Inc.     

Learning,AMPA receptor mobility and synaptic plasticity depend on n‐cofilin‐mediated actin dynamics

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin‐binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n‐cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long‐term potentiation and long‐term depression. Loss of n‐cofilin‐mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n‐cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.     

PTRN‐1/CAMSAP promotes CYK‐1/formin‐dependent actin polymerization during endocytic recycling

Cargo sorting and membrane carrier initiation in recycling endosomes require appropriately coordinated actin dynamics. However, the mechanism underlying the regulation of actin organization during recycling transport remains elusive. Here we report that the loss of PTRN‐1/CAMSAP stalled actin exchange and diminished the cytosolic actin structures. Furthermore, we found that PTRN‐1 is required for the recycling of clathrin‐independent cargo hTAC‐GFP. The N‐terminal calponin homology (CH) domain and central coiled‐coils (CC) region of PTRN‐1 can synergistically sustain the flow of hTAC‐GFP. We identified CYK‐1/formin as a binding partner of PTRN‐1. The N‐terminal GTPase‐binding domain (GBD) of CYK‐1 serves as the binding interface for the PTRN‐1 CH domain. The presence of the PTRN‐1 CH domain promoted CYK‐1‐mediated actin polymerization, which suggests that the PTRN‐1‐CH:CYK‐1‐GBD interaction efficiently relieves autoinhibitory interactions within CYK‐1. As expected, the overexpression of the CYK‐1 formin homology domain 2 (FH2) substantially restored actin structures and partially suppressed the hTAC‐GFP overaccumulation phenotype in ptrn‐1 mutants. We conclude that the PTRN‐1 CH domain is required to stimulate CYK‐1 to facilitate actin dynamics during endocytic recycling.     

Multiscale modeling of structural dynamics underlying force generation and product release in actomyosin complex

To decrypt the mechanistic basis of myosin motor function, it is essential to probe the conformational changes in actomyosin with high spatial and temporal resolutions. In a computational effort to meet this challenge, we have performed a multiscale modeling of the allosteric couplings and transition pathway of actomyosin complex by combining coarse‐grained modeling of the entire complex with all‐atom molecular dynamics simulations of the active site. Our modeling of allosteric couplings at the pre‐powerstroke state has pinpointed key actin‐activated couplings to distant myosin parts which are critical to force generation and the sequential release of phosphate and ADP. At the post‐powerstroke state, we have identified isoform‐dependent couplings which underlie the reciprocal coupling between actin binding and nucleotide binding in fast Myosin II, and load‐dependent ADP release in Myosin V. Our modeling of transition pathway during powerstroke has outlined a clear sequence of structural events triggered by actin binding, which lead to subsequent force generation, twisting of central β‐sheet, and the sequential release of phosphate and ADP. Finally we have performed atomistic simulations of active‐site dynamics based on an on‐path “transition‐state” myosin conformation, which has revealed significantly weakened coordination of phosphate by Switch II, and a disrupted key salt bridge between Switch I and II. Meanwhile, the coordination of MgADP by Switch I and P loop is less perturbed. As a result, the phosphate can be released prior to MgADP. This study has shed new lights on the controversy over the structural mechanism of actin‐activated phosphate release and force generation in myosin motor. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Dynamic polymorphism of single actin molecules in the actin filament

Actin filament dynamics are critical in cell motility. The structure of actin filament changes spontaneously and can also be regulated by actin-binding proteins, allowing actin to readily function in response to external stimuli. The interaction with the motor protein myosin changes the dynamic nature of actin filaments. However, the molecular bases for the dynamic processes of actin filaments are not well understood. Here, we observed the dynamics of rabbit skeletal-muscle actin conformation by monitoring individual molecules in the actin filaments using single-molecule fluorescence resonance energy transfer (FRET) imaging with total internal reflection fluorescence microscopy (TIRFM). The time trajectories of FRET show that actin switches between low- and high-FRET efficiency states on a timescale of seconds. If actin filaments are chemically cross-linked, a state that inhibits myosin motility, the equilibrium shifts to the low-FRET conformation, whereas when the actin filament is interacting with myosin, the high-FRET conformation is favored. This dynamic equilibrium suggests that actin can switch between active and inactive conformations in response to external signals.     

An ionic–chemical–mechanical model for muscle contraction

The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar‐like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical‐chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress‐generating conformational changes in the myosin cross bridge, and relief of built‐up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin–myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin–myosin engagement during the weak‐to‐strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin–myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre‐power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle.     

Aberrant post‐translational modifications compromise human myosin motor function in old age

Novel experimental methods, including a modified single fiber in vitro motility assay, X‐ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging‐related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force‐generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X‐ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age‐specific myosin post‐translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.     

Apple S‐RNase interacts with an actin‐binding protein,MdMVG, to reduce pollen tube growth by inhibiting its actin‐severing activity at the early stage of self‐pollination induction

In S‐RNase‐mediated self‐incompatibility, S‐RNase secreted from the style destroys the actin cytoskeleton of the self‐pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S‐RNase does not bind or sever filamentous actin (F‐actin). In apple (Malus domestica), we identified an actin‐binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S‐RNase and directly binds and severs F‐actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S‐RNase inhibits the F‐actin‐severing activity of MdMVG in vitro. In vivo, the addition of S‐RNase to self‐pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self‐pollination induction in the presence of MdMVG overexpression. By generating 25 single‐, double‐ and triple‐point mutations in the amino acid motif E‐E‐K‐E‐K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple‐point mutant, MdMVG^{(E167A/E171A/K185A)}, that no longer has F‐actin‐severing activity or interacts with any of the four S‐haplotype S‐RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S‐RNases. We conclude that apple S‐RNase interacts with MdMVG to reduce self‐pollen tube growth by inhibiting its F‐actin‐severing activity.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

During clathrin‐mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G‐actin) and a central‐acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3‐dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G‐actin‐binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G‐actin‐binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two‐hybrid system, GST‐pulldown, fluorescence polarization and pyrene‐actin polymerization assays, we show that LGM binds G‐actin and is necessary for normal Arp2/3‐mediated actin polymerization in vitro. Live‐cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G‐actin‐binding motif, WH2. These results establish a second G‐actin‐binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.      

Differentiation of human adipose‐derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca²⁺ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study

Detailed residue-wise interactions involved in the binding of myosin to actin in the rigor conformation without nucleotides have been examined using molecular dynamics simulations of the chicken skeletal myosin head complexed with two actin monomers, based on the cryo-microscopic model of Holmes et al. (Nature 2003;425:423-427). The overall interaction is largely electrostatic in nature, because of the charged residues in the four loops surrounding the central primary binding site. The 50k/20k loop, disordered in crystal structures and in simulations of free myosin in solution, was found to be in a conformation stabilized with 1 - 2 internal salt bridges. The cardiomyopathy loop forms 2 - 3 interprotein salt bridges with actin monomers upon binding, whereas its Arg405 residue, the mutation site associated with the hypertrophic cardiomyopathy, forms a strong salt bridge with Glu605 in the neighboring helix away from actin in the actin-bound myosin. The myopathy loop of the R405Q mutant maintains a high degree of two-strand beta-sheet character when bound to actin with the corresponding salt bridges broken.     

Oryza sativa actin‐interacting protein 1 is required for rice growth by promoting actin turnover

Rapid actin turnover is essential for numerous actin‐based processes. However, how it is precisely regulated remains poorly understood. Actin‐interacting protein 1 (AIP1) has been shown to be an important factor by acting coordinately with actin‐depolymerizing factor (ADF)/cofilin in promoting actin depolymerization, the rate‐limiting factor in actin turnover. However, the molecular mechanism by which AIP1 promotes actin turnover remains largely unknown in plants. Here, we provide a demonstration that AIP1 promotes actin turnover, which is required for optimal growth of rice plants. Specific down‐regulation of OsAIP1 increased the level of filamentous actin and reduced actin turnover, whereas over‐expression of OsAIP1 induced fragmentation and depolymerization of actin filaments and enhanced actin turnover. In vitro biochemical characterization showed that, although OsAIP1 alone does not affect actin dynamics, it enhances ADF‐mediated actin depolymerization. It also caps the filament barbed end in the presence of ADF, but the capping activity is not required for their coordinated action. Real‐time visualization of single filament dynamics showed that OsAIP1 enhanced ADF‐mediated severing and dissociation of pointed end subunits. Consistent with this, the filament severing frequency and subunit off‐rate were enhanced in OsAIP1 over‐expressors but decreased in RNAi protoplasts. Importantly, OsAIP1 acts coordinately with ADF and profilin to induce massive net actin depolymerization, indicating that AIP1 plays a major role in the turnover of actin, which is required to optimize F‐actin levels in plants.     

Coarse‐grained simulations of proton‐dependent conformational changes in lactose permease

During lactose/H⁺ symport, the Escherichia coli lactose permease (LacY) undergoes a series of global conformational transitions between inward‐facing (open to cytoplasmic side) and outward‐facing (open to periplasmic side) states. However, the exact local interactions and molecular mechanisms dictating those large‐scale structural changes are not well understood. All‐atom molecular dynamics simulations have been performed to investigate the molecular interactions involved in conformational transitions of LacY, but the simulations can only explore early or partial global structural changes because of the computational limits (< 100 ns). In this work, we implement a hybrid force field that couples the united‐atom protein models with the coarse‐grained MARTINI water/lipid, to investigate the proton‐dependent dynamics and conformational changes of LacY. The effects of the protonation states on two key glutamate residues (Glu325 and Glu269) have been studied. Our results on the salt‐bridge dynamics agreed with all‐atom simulations at early short time period, validating our simulations. From our microsecond simulations, we were able to observe the complete transition from inward‐facing to outward‐facing conformations of LacY. Our results showed that all helices have participated during the global conformational transitions and helical movements of LacY. The inter‐helical distances measured in our simulations were consistent with the double electron‐electron resonance experiments at both cytoplasmic and periplasmic sides. Our simulations indicated that the deprotonation of Glu325 induced the opening of the periplasmics side and partial closure of the cytoplasmic side of LacY, while protonation of the Glu269 caused a stable cross‐domain salt‐bridge (Glu130‐Arg344) and completely closed the cytoplasmic side. Proteins 2016; 84:1067–1074. © 2016 Wiley Periodicals, Inc.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.