Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural insights into actin filament recognition by commonly used cellular actin markers

ABSTRACT: Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

SUBMITTER: Dr. Archana Kumari

PROVIDER: S-SCDT-EMBOJ-2019-104006 | biostudies-other |

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

Structural insights into actin filament recognition by commonly used cellular actin markers.

Project description:Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

| S-EPMC7360965 | biostudies-literature

Structural basis of actin filament assembly and aging.

Project description:The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state1-3. It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+ at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca2+-actin shows slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.

| S-EPMC9646518 | biostudies-literature

Evaluation of commonly used ectoderm markers in iPSC trilineage differentiation.

Project description:Patient-derived induced pluripotent stem cells (iPSCs) have become a promising resource for exploring genetics of complex diseases, discovering new drugs, and advancing regenerative medicine. Increasingly, laboratories are creating their own banks of iPSCs derived from diverse donors. However, there are not yet standardized guidelines for qualifying these cell lines, i.e., distinguishing between bona fide human iPSCs, somatic cells, and imperfectly reprogrammed cells. Here, we report the establishment of a panel of 30 iPSCs from CD34+ peripheral blood mononuclear cells, of which 10 were further differentiated in vitro into all three germ layers. We characterized these different cell types with commonly used pluripotent and lineage specific markers, and showed that NES, TUBB3, and OTX2 cannot be reliably used as ectoderm differentiation markers. Our work highlights the importance of marker selection in iPSC authentication, and the need for the field to establish definitive standard assays.

| S-EPMC6570500 | biostudies-literature

Structural basis for cofilin binding and actin filament disassembly.

Project description:Actin depolymerizing factor (ADF) and cofilin accelerate actin dynamics by severing and disassembling actin filaments. Here, we present the 3.8 Å resolution cryo-EM structure of cofilactin (cofilin-decorated actin filament). The actin subunit structure of cofilactin (C-form) is distinct from those of F-actin (F-form) and monomeric actin (G-form). During the transition between these three conformations, the inner domain of actin (subdomains 3 and 4) and the majority of subdomain 1 move as two separate rigid bodies. The cofilin-actin interface consists of three distinct parts. Based on the rigid body movements of actin and the three cofilin-actin interfaces, we propose models for the cooperative binding of cofilin to actin, preferential binding of cofilin to ADP-bound actin filaments and cofilin-mediated severing of actin filaments.

| S-EPMC5945598 | biostudies-literature

Structural basis of actin filament nucleation by tandem W domains.

Project description:Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.

| S-EPMC3780403 | biostudies-literature

Structural insights into actin isoforms.

Project description:Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle differences in their design principles determine the interaction with myosin motors and actin-binding proteins. Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal muscle α-actin (3.37 Å), cardiac muscle α-actin (3.07 Å), ß-actin (2.99 Å), and γ-actin (3.38 Å) in the Mg2+·ADP state with their native post-translational modifications. The structures revealed isoform-specific conformations of the N-terminus that shift closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Collectively, the structures of single-isotype, post-translationally modified bare skeletal muscle α-actin, cardiac muscle α-actin, ß-actin, and γ-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

| S-EPMC10072879 | biostudies-literature

Structural basis and evolutionary origin of actin filament capping by twinfilin.

Project description:Dynamic reorganization of the actin cytoskeleton is essential for motile and morphological processes in all eukaryotic cells. One highly conserved protein that regulates actin dynamics is twinfilin, which both sequesters actin monomers and caps actin filament barbed ends. Twinfilin is composed of two ADF/cofilin-like domains, Twf-N and Twf-C. Here, we reveal by systematic domain-swapping/inactivation analysis that the two functional ADF-H domains of twinfilin are required for barbed-end capping and that Twf-C plays a critical role in this process. However, these domains are not functionally equivalent. NMR-structure and mutagenesis analyses, together with biochemical and motility assays showed that Twf-C, in addition to its binding to G-actin, interacts with the sides of actin filaments like ADF/cofilins, whereas Twf-N binds only G-actin. Our results indicate that during filament barbed-end capping, Twf-N interacts with the terminal actin subunit, whereas Twf-C binds between two adjacent subunits at the side of the filament. Thus, the domain requirement for actin filament capping by twinfilin is remarkably similar to that of gelsolin family proteins, suggesting the existence of a general barbed-end capping mechanism. Furthermore, we demonstrate that a synthetic protein consisting of duplicated ADF/cofilin domains caps actin filament barbed ends, providing evidence that the barbed-end capping activity of twinfilin arose through a duplication of an ancient ADF/cofilin-like domain.

| S-EPMC1805582 | biostudies-literature

Molecular and structural basis of actin filament severing by ADF/cofilin.

Project description:ADF/cofilin's cooperative binding to actin filament modifies the conformation and alignment of G-actin subunits locally, causing the filament to sever at "boundaries" formed among bare and ADF/cofilin-occupied regions. Analysis of the impact of the ADF/cofilin cluster boundary on the deformation behavior of actin filaments in a mechanically strained environment is critical for understanding the biophysics of their severing. The present investigation uses molecular dynamics simulations to generate atomic resolution models of bare, partially, and fully cofilin decorated actin filaments. Steered molecular dynamics simulations are utilized to determine the mechanical properties of three filament models when subjected to axial stretching, axial compression, and bending forces. We highlight differences in strain distribution, failure mechanisms in the three filament models, and biomechanical effects of cofilin cluster boundaries in overall filament rupture. Based on the influence of ADF/cofilin binding on intrastrand and interstrand G-actin interfaces, the cofilin-mediated actin filament severing model proposed here can help understand cofilin mediated actin dynamics.

| S-EPMC9379983 | biostudies-literature

Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying <i>Phytophthora</i> Species.

Project description:The genus Phytophthora is agriculturally and ecologically important. As the number of Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (β-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and β-tub. Resolution of these four markers also varied with (sub)clade. β-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99% PCR success rate) and β-tub (96%) were easier to amplify than cox1 (75%) and tigA (71%). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade.

| S-EPMC6178919 | biostudies-literature

Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization.

Project description:Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.

| S-EPMC4217450 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data