Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.

Maurer, Lisa M.; Tomasini-Johansson, Bianca R.; Ma, Wenjiang; Annis, Douglas S.; Eickstaedt, Nathan L.; Ensenberger, Martin G.; Satyshur, Kenneth A.; Mosher, Deane F.

doi:10.1074/jbc.a110.153692

Cited by 8 publications

(19 citation statements)

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“…The N-terminus of Fn1 is essential for Fn1 fibrillogenesis (Schwarzbauer, 1991). FUD binds tightly to the N-terminal domain of Fn1 (K D <2.6 nM), with a fast k on and a slow k off , and acts as a competitive inhibitor of Fn1-Fn1 interactions (Ma et al, 2015; Maurer et al, 2010; Tomasini-Johansson et al, 2001). Although in the absence of FUD, individual fibrils in an established matrix are stable and can be tracked for over 16 hours (S.A., unpublished observations), when FUD is added to cells, it localizes with Fn1 fibrils and dismantles the mature Fn1 ECM (Filla et al, 2017; Tomasini-Johansson et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

Tomer

Arriagada

Munshi

et al. 2020

Preprint

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Fibronectin (Fn1) is an essential ECM glycoprotein important for embryonic development and homeostasis. The functions of Fn1 in regulating cell fate decisions, morphogenesis and cellular responses to injury are intimately linked to the process of Fn1 fibrillogenesis. Therefore, understanding the mechanisms by which Fn1 proteins assemble into fibrils is necessary to gain insights into diverse functions of Fn1. Using CRISPR/Cas9 mutagenesis, we generated mice and cell lines wherein a sequence encoding a fluorescent protein (FP) was knocked into the Fn1 locus replacing the termination codon, resulting in the expression of Fn1-FP proteins subject to endogenous regulation. Live imaging and super-resolution microscopy revealed that Fn1 fibrils are not continuous fibers as was thought before, instead, they are comprised of a discontinuous array of small nanodomains. Live imaging showed that Fn1 nanodomains are mobile and that they become arranged into progressively longer linear arrays as they move toward the nucleus in parallel with the rearward actin flow. The organization of Fn1 nanodomains into linear fibrillar arrays but not the formation of Fn1 nanodomains is regulated by the interactions mediated by the Fn1 N-terminal assembly domain. The nanodomain architecture of Fn1 fibrils is observed in multiple contexts: in three-dimensional ECM in vivo, on substrata of different composition and stiffness, and is retained when the linkage of Fn1 fibrils to cells is disrupted. The modular assembly and structure of Fn1 fibrils bears important implications for mechanisms of ECM remodeling and signal transduction.

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Section: Discussionmentioning

confidence: 99%

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

Tomer

Arriagada

Munshi

et al. 2020

Preprint

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“…Primers were designed to obtain PCR amplicons with 5′ Kpn I and 3′ Nhe I recognition sites. These sites were utilized for insert ligation into the pET‐ELMER vector, which contains sequences for a 6xHis tag and thrombin cleavage site 5′ of the Kpn I restriction site . After sequence verification, plasmids were transformed into BL21(DE3)‐competent Escherichia coli .…”

Section: Methodsmentioning

confidence: 99%

“…After sequence verification, plasmids were transformed into BL21(DE3)‐competent Escherichia coli . The 6xHis‐tagged recombinant proteins were expressed and purified as described, except that proteins were eluted from NiNTA agarose (Qiagen, Valencia, CA, USA) using pH 7.0 elution buffer containing 300 mM imidazole, which was removed through dialysis to obtain purified samples. PN0 was dialysed into phosphate‐buffered saline (PBS), pH 7.4; periostin FAS1 2 was dialysed into 3‐(N‐morpholino) propanesulfonic acid (MOPS), pH 7.5; and the rest of the protein samples were dialysed into 1 mM acetic acid, pH 4.5.…”

Section: Methodsmentioning

confidence: 99%

IL‐5‐stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8‐dependent migration

Johansson

Khanna

Bortnov

et al. 2017

Clin Experimental Allergy

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Summary Background IL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the “nucleopod”, which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators. Objective Determine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin. Methods Blood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed. Results After 10 min in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30–60 min, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target.

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“…Review articles and methods papers Protein interactions with other proteins Protein interactions with small ligands (including co‐factors and drugs) …”

Section: Introductionmentioning

confidence: 99%

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Ghai

Falconer

Collins

2011

J of Molecular Recognition

163

108

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Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.

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Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.

Cited by 8 publications

References 0 publications

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

IL‐5‐stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8‐dependent migration

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

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