scholarly journals Structure of the Lifeact–F-actin complex

2020 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Binding Pocket ◽  
Actin Binding ◽  
Binding Assays ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
Hydrophobic Binding ◽  
Binding Loop

AbstractLifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy. The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over two adjacent actin subunits, stabilizing the DNase I-binding loop of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as TccC3HVR from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

scholarly journals Structure of the Lifeact–F-actin complex

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000925 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Hypervariable Region ◽  
Binding Pocket ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
D Loop ◽  
Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

scholarly journals Characterization of calcium- and integrin-binding protein 1 (CIB1) knockout platelets: Potential compensation by CIB family members

2008 ◽  
Vol 100 (05) ◽  
pp. 847-856 ◽  
Author(s):  
Brenda R. Temple ◽  
Holly R. Gentry ◽  
Jan C. DeNofrio ◽  
Weiping Yuan ◽  
Leslie V. Parise
Keyword(s):  
Thrombus Formation ◽  
Mrna Level ◽  
Family Members ◽  
Cytoplasmic Tail ◽  
Binding Pocket ◽  
Vitro Binding ◽  
Cytoplasmic Tails ◽  
Hydrophobic Binding

SummaryPlatelet aggregation requires activation of the αIIbβ3 integrin,an event regulated by the integrin cytoplasmic tails. CIB1 binds to the cytoplasmic tail of the integrin αIIb subunit. Previous overexpression and knockdown studies in murine megakaryocytes demonstrated that CIB1 inhibits integrin αIIbβ3 activation.Here we analyzed Cib1-/- mice to determine the function of CIB1 in platelets in vitro and in vivo. We found that although these mice had no overt platelet phenotype, mRNA level of CIB1 homolog CIB3 was increased in Cib1-/- megakaryocytes. In vitro binding experiments showed that recombinant CIB1, -2 and -3 bound specifically to an αIIb cytoplasmic tail peptide. Subsequent protein modeling experiments indicated that CIBs 1–3 each have a highly conserved hydrophobic binding pocket. Therefore, the potential exists for compensation for the loss of CIB1 by these CIB family members, thereby preventing pathologic thrombus formation in Cib1-/- mice.

scholarly journals Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

2012 ◽  
Vol 444 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Keefe T. Chan ◽  
David W. Roadcap ◽  
Nicholas Holoweckyj ◽  
James E. Bear
Keyword(s):  
Binding Site ◽  
Leading Edge ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Unique Region ◽  
Equivalent Residue ◽  
Filament Networks ◽  
Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function

2007 ◽  
Vol 293 (3) ◽  
pp. C874-C884 ◽  
Author(s):  
Lixin Zhu ◽  
Rihong Zhou ◽  
Shelley Mettler ◽  
Tim Wu ◽  
Aennes Abbas ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Resonance Energy ◽  
Actin Binding ◽  
Live Cells ◽  
Filamentous Actin ◽  
High Turnover ◽  
Binding Conformation ◽  
Phosphorylated Form

In its dormant state, the membrane cytoskeletal linker protein ezrin takes on a NH2 terminal-to-COOH terminal (N-C) binding conformation. In vitro evidence suggests that eliminating the N-C binding conformation by Thr567 phosphorylation leads to ezrin activation. Here, we found for resting gastric parietal cells that the levels of ezrin phosphorylation on Thr567 are low and can be increased to a small extent (∼40%) by stimulating secretion via the cAMP pathway. Treatment of cells with protein phosphatase inhibitors led to a rapid, dramatic increase in Thr567 phosphorylation by 400% over resting levels, prompting the hypothesis that ezrin activity is regulated by turnover of phosphorylation on Thr567. In vitro and in vivo fluorescence resonance energy transfer analysis demonstrated that Thr567 phosphorylation opens the N-C interaction. However, even in the closed conformation, ezrin localizes to membranes by an exposed NH2 terminal binding site. Importantly, the opened phosphorylated form of ezrin more readily cosediments with F-actin and binds more tightly to membrane than the closed forms. Furthermore, fluorescence recovery after photobleaching analysis in live cells showed that the Thr567Asp mutant had longer recovery times than the wild type or the Thr567Ala mutant, indicating the Thr567-phosphorylated form of ezrin is tightly associated with F-actin and the membrane, restricting normal activity. These data demonstrate and emphasize the functional importance of reversible phosphorylation of ezrin on F-actin binding. A novel model is proposed whereby ezrin and closely associated kinase and phosphatase proteins represent a motor complex to maintain a dynamic relationship between the varying membrane surface area and filamentous actin length.

Enolase exists in the fluid phase of cytoplasm in 3T3 cells

1989 ◽  
Vol 94 (2) ◽  
pp. 333-342
Author(s):  
L. Pagliaro ◽  
K. Kerr ◽  
D.L. Taylor
Keyword(s):  
Diffusion Coefficient ◽  
Fluid Phase ◽  
Glycolytic Enzyme ◽  
Binding Activity ◽  
Actin Binding ◽  
Filamentous Actin ◽  
3T3 Cells ◽  
Ratio Imaging

We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (F1-enolase) and carboxytetramethylrhodamine (Rh-enolase) In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack of interaction between enolase and F-actin. Fluorescence redistribution after photo-bleaching (FRAP) measurements of the diffusion coefficient (D) of F1-enolase in aqueous solutions gave a value of D37,aq = 6.08 × 10(−7) cm2s-1, and no immobile fraction, consistent with a native molecular weight of 90,000. These values were not significantly different with Rh-enolase, or in the presence of F-actin, 2-phosphoglycerate or F-actin-aldolase gels, demonstrating that neither F1-enolase nor Rh-enolase binds to F-actin or aldolase in vitro. FRAP measurements of F1- and Rh-enolase microinjected into living Swiss 3T3 cells revealed spatial differences in the diffusion coefficient, but not the mobile fraction. In the perinuclear cytoplasm, we measured an apparent diffusion coefficient of 1.1 × 10(−7) cm2s-1, compared to 7.1 × 10(−8) cm2s-1 in the peripheral cytoplasm, with approximately 100% mobility of F1- or Rh-enolase in both regions. Imaging of cells co-injected with Rh-enolase and size-fractionated FITC-dextran (FD-90) revealed that Rh-enolase entered the nucleus, while FD-90 was excluded. Ratio imaging showed a relatively high nuclear ratio of Rh-enolase/FD-90, and a uniform cytoplasmic ratio, with no indication of increased concentration of enolase around stress fibers. These data demonstrate that Rh- and F1-enolase do not bind to F-actin in vitro, and are 100% mobile in vivo. Together with our recent finding that a significant fraction of aldolase binds to F-actin in vitro and is immobile in vivo, these data suggest a correlation between actin-binding activity and cytoplasmic mobility of glycolytic enzymes.

scholarly journals Actin activatesPseudomonas aeruginosaExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

2015 ◽  
Author(s):  
Dorothee Raoux-Barbot ◽  
Cosmin Saveanu ◽  
Abdelkader Namane ◽  
Vasily Ogryzko ◽  
Lina Worpenberg ◽  
...  
Keyword(s):  
Actin Binding ◽  
Target Cells ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Severe Damage ◽  
Chronic Infections ◽  
Effector Domains ◽  
Host Target

Pseudomonas aeruginosa is a major cause of chronic infections in cystic fibrosis patients. The nucleotidyl cyclase toxin ExoY is a virulence factors injected by the pathogen and associated with severe damage to lung tissue. ExoY-like cyclases are also found in other Gram-negative pathogens and shown to contribute to virulence, although they remained poorly characterized. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown co-factor that activates P. aeruginosa ExoY within host target cells. Highly purified actin, when polymerized into filaments, potently stimulates (>10,000 fold) ExoY activity. ExoY co-localizes in vivo with actin filaments in transfected cells and, in vitro, it interferes with the regulation of actin assembly/disassembly-dynamics mediated by important F-actin-binding proteins. We further show that actin also activates an ExoY-like adenylate cyclase from a Vibrio species. Our results thus highlight a new sub-class within the class II adenylyl cyclase family, defined as actin-activated nucleotidyl cyclase (AA-NC) toxins.

scholarly journals Molecular mechanism for the recognition of sequence-divergent CIF peptides by the plant receptor kinases GSO1/SGN3 and GSO2

2020 ◽  
Vol 117 (5) ◽  
pp. 2693-2703 ◽  
Author(s):  
Satohiro Okuda ◽  
Satoshi Fujita ◽  
Andrea Moretti ◽  
Ulrich Hohmann ◽  
Verónica G. Doblas ◽  
...  
Keyword(s):  
Higher Plants ◽  
Binding Pocket ◽  
Peptide Hormones ◽  
Receptor Activation ◽  
Missense Mutations ◽  
Binding Assays ◽  
Biochemical Interaction ◽  
Receptor Kinases

Plants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0-Å crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3–CIF2 represents one of the strongest receptor–ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover previously uncharacterized CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK proteins as essential coreceptor kinases required for GSO1/SGN3 and GSO2 receptor activation. Our work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.

scholarly journals Viscoelasticity of F-actin measured with magnetic microparticles.

1989 ◽  
Vol 109 (5) ◽  
pp. 2233-2243 ◽  
Author(s):  
K S Zaner ◽  
P A Valberg
Keyword(s):  
Mechanical Properties ◽  
Magnetic Particles ◽  
Magnetic Particle ◽  
Particle Method ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Particle Measurements ◽  
Probe System

Dispersed submicroscopic magnetic particles were used to probe viscoelasticity for cytoplasm and purified components of cytoplasm. An externally applied magnetic field exerted force on particles in cells, in filamentous actin (F-actin) solutions, or in F-actin gels formed by the addition of the actin gelation factor, actin-binding protein (ABP). The particle response to magnetic torque can be related to the viscoelastic properties of the fluids. We compared data obtained on F-actin by the magnetic particle method with data obtained on F-actin by means of a sliding plane viscoelastometer. F-actin solutions had a significant elasticity, which increased by 20-fold when gels were formed by ABP addition. Both methods gave consistent results, but the dispersed magnetic particles indicated quantitatively greater rigidity than the viscoelastometer (two and six times greater for F-actin solutions and for F-actin plus ABP gels, respectively). These differences may be due to the fact that, compared with traditional microrheometers, dispersed particle measurements are less affected by long-range heterogeneity or domain-like structure. The magnetometric method was used to examine the mechanical properties of cytoplasm within intact macrophages; the application of the same magnetometric technique to both cells and well-defined, purified protein systems is a first step toward interpreting the results obtained for living cells in molecular terms. The magnetic particle probe system is an effective nonoptical technique for determining the motile and mechanical properties of cells in vitro and in vivo.

scholarly journals Molecular mechanism for the recognition of sequence-divergent CIF peptides by the plant receptor kinases GSO1/SGN3 and GSO2

2019 ◽  
Author(s):  
Satohiro Okuda ◽  
Satoshi Fujita ◽  
Andrea Moretti ◽  
Ulrich Hohmann ◽  
Verónica G. Doblas ◽  
...  
Keyword(s):  
Higher Plants ◽  
Plant Root ◽  
Binding Pocket ◽  
Peptide Hormones ◽  
Receptor Activation ◽  
Missense Mutations ◽  
Binding Assays ◽  
Receptor Kinases

AbstractPlants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0 Å crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino-acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3 – CIF2 represents one of the strongest receptor-ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro, and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover novel CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK LRR-RKs as essential co-receptor kinases required for GSO1/SGN3 and GSO2 receptor activation. 0ur work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.Significance StatementTwo sequence-related plant membrane receptor kinases and their shape-complementary co-receptors are shown to selectively sense members of a small family of secreted peptide hormones to control formation of an important diffusion barrier in the plant root.

scholarly journals An epilepsy-causing mutation leads to co-translational misfolding of the Kv7.2 channel

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Janire Urrutia ◽  
Alejandra Aguado ◽  
Carolina Gomis-Perez ◽  
Arantza Muguruza-Montero ◽  
Oscar R. Ballesteros ◽  
...  
Keyword(s):  
Structural Instability ◽  
Binding Assays ◽  
Iq Motif ◽  
Channel Function ◽  
Human Epilepsy ◽  
Pathogenic Variants ◽  
In Silico Studies ◽  
Nascent Chain

Abstract Background The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. Results We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. Conclusions Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitro.

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