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 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 Isolation and characterization of a regulated form of actin depolymerizing factor

1993 ◽  
Vol 122 (3) ◽  
pp. 623-633 ◽  
Author(s):  
TE Morgan ◽  
RO Lockerbie ◽  
LS Minamide ◽  
MD Browning ◽  
JR Bamburg
Keyword(s):  
Muscle Development ◽  
Peptide Mapping ◽  
Actin Binding ◽  
Actin Assembly ◽  
Actin Depolymerizing Factor ◽  
Isolation And Characterization ◽  
Relative Molecular Weight

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

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 Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals Cofilin is an essential component of the yeast cortical cytoskeleton.

1993 ◽  
Vol 120 (2) ◽  
pp. 421-435 ◽  
Author(s):  
A L Moon ◽  
P A Janmey ◽  
K A Louie ◽  
D G Drubin
Keyword(s):  
Sequence Similarity ◽  
Actin Binding ◽  
Actin Assembly ◽  
The Family ◽  
Multiple Copies ◽  
Complete Disruption ◽  
Diploid Cells ◽  
Related Proteins

We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin-like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.

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 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.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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