scholarly journals Mapping the binding site of thymosin β4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin

1997 ◽  
Vol 327 (3) ◽  
pp. 787-793 ◽  
Author(s):  
Edda BALLWEBER ◽  
Ewald HANNAPPEL ◽  
Thomas HUFF ◽  
Hans Georg MANNHERZ
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Ternary Complex ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Critical Concentration ◽  
Dnase I ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Actin Binding Proteins

The β-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin β4 was determined by analysing the binding of thymosin β4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin β4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin β4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin β4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin β4 was obtained only when using the chemically cross-linked actin-thymosin β4 complex, indicating that thymosin β4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin β4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

scholarly journals Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Minkyo Jung ◽  
Doory Kim ◽  
Ji Young Mun
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Actin Binding ◽  
Direct Visualization ◽  
Actin Binding Proteins ◽  
Synapse Plasticity

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP–actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP–actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.

scholarly journals Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins

1999 ◽  
Vol 344 (1) ◽  
pp. 117-123 ◽  
Author(s):  
Barbara D. LEINWEBER ◽  
Paul C. LEAVIS ◽  
Zenon GRABAREK ◽  
C.-L. Albert WANG ◽  
Kathleen G. MORGAN
Keyword(s):  
Binding Site ◽  
Potential Function ◽  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Calponin Homology ◽  
Signalling Molecule ◽  
Fluorescence Change ◽  
Extracellular Regulated Kinase ◽  
Overlay Assay

An interaction between extracellular regulated kinase 1 (ERK1) and calponin has previously been reported (Menice, Hulvershorn, Adam, Wang and Morgan (1997) J. Biol. Chem.272(40), 25157-25161) and has been suggested to reflect a function of calponin as a signalling molecule. We report in this study that calponin binds to both ERK1 and ERK2 under native conditions as well as in an overlay assay. Using chymotryptic fragments of calponin, the binding site of ERK on calponin was identified as the calponin homology (CH) domain, an N-terminal region of calponin found in other actin-binding proteins. ERK also bound, in a gel overlay assay, α-actinin, a protein with two tandem CH domains, as well as a 27 kDa thermolysin product of α-actinin containing the CH domains of α-actinin. The CH domain of calponin could compete with intact calponin or α-actinin for ERK binding. Titration of acrylodan-labelled calponin with ERK gave a Ka of 6×106 M-1 and titration of acrylodan-labelled calponin with a peptide from the αL16 helix of ERK gave a Ka of 1×106 M-1. Recombinant ERK was found to co-sediment with purified actin and induced a fluorescence change in pyrene-labelled F-actin (Ka = 5×106 M-1). The interaction of ERK with CH domains points to a new potential function for CH domains. The interaction of ERK with actin raises the possibility that actin may provide a scaffold for ERK signalling complexes in both muscle and non-muscle cells.

scholarly journals Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells

1997 ◽  
Vol 327 (3) ◽  
pp. 909-915 ◽  
Author(s):  
C. Bryan GIBBON ◽  
Haiyun REN ◽  
J. Christopher STAIGER
Keyword(s):  
Binding Proteins ◽  
Dissociation Constants ◽  
Critical Concentration ◽  
Tryptophan Fluorescence ◽  
Actin Binding ◽  
Live Cells ◽  
Post Translational Modification ◽  
Actin Binding Proteins ◽  
Maize Pollen

Profilin is a small, 12-15 kDa, actin-binding protein that interacts with at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actin-binding proteins have been shown to behave differently with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin-profilin complexes (Kd = 1.0-1.5 μM). The ability of maize profilin isoforms to bind poly-L-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actin-binding proteins in living cells. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.

scholarly journals The utrophin actin-binding domain binds F-actin in two different modes

2002 ◽  
Vol 157 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Vitold E. Galkin ◽  
Albina Orlova ◽  
Margaret S. VanLoock ◽  
Inna N. Rybakova ◽  
James M. Ervasti ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
New Approach ◽  
Calponin Homology ◽  
Multiple Binding Sites ◽  
Actin Binding Domain ◽  
Multiple Binding

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

scholarly journals Artemether Targets Raptor-Induced Actin Polymerization and Suppresses Migration of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

2021 ◽  
Author(s):  
Jian-Da Ma ◽  
Jun Jing ◽  
Jun-Wei Wang ◽  
Xue-Pei Zhang ◽  
Qian-Hua Li ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Rho Gtpase ◽  
Actin Binding ◽  
Migration And Invasion ◽  
Actin Binding Proteins ◽  
Activation Assay ◽  
Fibroblast Like Synoviocytes

Abstract Background: Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) may cause articular damage as a result of its aggressive features including direct adhesion and invasion of surface cartilage in joints. Artemether (ART), one of the artemisinin derivatives with antimalarial properties, showed inhibitory effect on inflammation and destruction of joints in collagen-induced arthritis rats, which might be applied in RA treatment. However, whether ART has effects on the aggressive properties of human RA-FLS remains unexplored. Methods: Synovium was obtained from patients with active RA (n=18) and FLS were isolated in vitro. RA-FLS were subjected to cell migration, invasion assays, live-cell imaging analysis and Rho GTPase activation assay after ART treatment. To identify the therapeutic target of ART, key signaling molecules of PI3K/Akt, AMPK, MAPK, NF-κB and mTOR pathways from RA-FLS were examined by Western Blot after ART treatment. Raptor was knockdown or overexpressed by siRNA or lentivirus transfection to reveal its role on regulating the aggressive properties of RA-FLS.Results: ART treatment significantly suppressed the transwell migration and invasion of synovial FLS from RA patients. Time-lapse microscopy revealed that ART treatment reduced random migration velocity of RA-FLS, as well as the directional persistence. ART also impaired the formation of filopodia and lamellipodia in RA-FLS. Further mechanism investigation showed that ART reduced the protein level of Raptor, a critical component of the mTOR pathway, and its downstream target 4E-BP1. It also inhibited the activation of Rho GTPases and the expression of actin binding proteins, including Profilin 1 and p-Cofilin. Raptor overexpression could reverse the anti-migration and anti-invasion effects of ART on RA-FLS as well as the suppression of Rho GTPases activation and the expression of actin binding proteins. Conclusion: ART can inhibit migration of RA-FLS by blocking Raptor-induced actin polymerization. ART might be a potential agent targeting FLS in RA treatment.

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