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  • Stimulated single-cell force spectroscopy to quantify cell adhesion receptor crosstalk. 20127696

    To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single-cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen-binding integrin alpha(1)beta(1) and fibronectin-binding integrin alpha(5)beta(1) in HeLa cells. This crosstalk was unidirectional, from integrin alpha(1)beta(1) to integrin alpha(5)beta(1), and functioned by regulating the endocytosis of integrin alpha(5)beta(1). The single-cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1973
    Product Catalog Name:
    Anti-Integrin α1 Antibody, clone FB12

  • Structural modeling and electron paramagnetic resonance spectroscopy of the human Na+/H+ exchanger isoform 1, NHE1. 20974853

    We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3140
    Product Catalog Name:
    Anti-Na+/H+ Exchanger-1 Antibody, CT, clone 4E9

  • Protein secondary structure from Fourier transform infrared spectroscopy: a data base analysis. 1867384

    An infrared (ir) method to determine the secondary structure of proteins in solution using the amide I region of the spectrum has been devised. The method is based on the circular dichroism (CD) matrix method for secondary structure analysis given by Compton and Johnson (L. A. Compton and W. C. Johnson, 1986, Anal. Biochem. 155, 155-167). The infrared data matrix was constructed from the normalized Fourier transform infrared spectra from 1700 to 1600 cm-1 of 17 commercially available proteins. The secondary structure matrix was constructed from the X-ray data of the seventeen proteins with secondary structure elements of helix, beta-sheet, beta-turn, and other (random). The CD and ir methods were compared by analyzing the proteins of the CD and ir databases as unknowns. Both methods produce similar results compared to structures obtained by X-ray crystallographic means with the CD slightly better for helix conformation, and the ir slightly better for beta-sheet. The relatively good ir analysis for concanavalin A and alpha-chymotrypsin indicate that the ir method is less affected by the presence of aromatic groups. The concentration of the protein and the cell path length need not be known for the ir analysis since the spectra can be normalized to the total ir intensity in the amide I region. The ir spectra for helix, beta-sheet, beta-turn, and other, as extracted from the data-base, agree with the literature band assignments. The ir data matrix and the inverse matrix necessary to analyze unknown proteins are presented.
    Document Type:
    Reference
    Product Catalog Number:
    15-101
    Product Catalog Name:
    Akt phosphorylation Pathway Explorer MiniPack

  • β-Connectin studies by small-angle x-ray scattering and single-molecule force spectroscopy by atomic force microscopy. 21728583

    The three-dimensional structure and the mechanical properties of a β-connectin fragment from human cardiac muscle, belonging to the I band, from I(27) to I(34), were investigated by small-angle x-ray scattering (SAXS) and single-molecule force spectroscopy (SMFS). This molecule presents an entropic elasticity behavior, associated to globular domain unfolding, that has been widely studied in the last 10 years. In addition, atomic force microscopy based SMFS experiments suggest that this molecule has an additional elastic regime, for low forces, probably associated to tertiary structure remodeling. From a structural point of view, this behavior is a mark of the fact that the eight domains in the I(27)-I(34) fragment are not independent and they organize in solution, assuming a well-defined three-dimensional structure. This hypothesis has been confirmed by SAXS scattering, both on a diluted and a concentrated sample. Two different models were used to fit the SAXS curves: one assuming a globular shape and one corresponding to an elongated conformation, both coupled with a Coulomb repulsion potential to take into account the protein-protein interaction. Due to the predominance of the structure factor, the effective shape of the protein in solution could not be clearly disclosed. By performing SMFS by atomic force microscopy, mechanical unfolding properties were investigated. Typical sawtooth profiles were obtained and the rupture force of each unfolding domain was estimated. By fitting a wormlike chain model to each peak of the sawtooth profile, the entropic elasticity of octamer was described.
    Document Type:
    Reference
    Product Catalog Number:
    5125

  • Metabolic distinction between vesicular and cytosolic GABA in cultured GABAergic neurons using 13C magnetic resonance spectroscopy. 11170185

    GABA exists in at least two different intracellular pools, i.e., a cytoplasmic or metabolic pool and a vesicular pool. This study was performed to gain information about the quantitative role of the tricarboxylic acid (TCA) cycle in biosynthesis of GABA from glutamine when GABA was selectively released from either one of these two pools. Cultured cerebral cortical neurons (GABAergic) were incubated in a medium containing 0.5 mM [U-13C]glutamine and subsequently depolarized for release of GABA from either the vesicular or the cytoplasmic pool. The vesicular release was induced by 55 mM K+ in the presence of tiagabine, a nontransportable inhibitor of the plasma membrane GABA carriers, whereas the cytoplasmic release via a reversal of the GABA carrier was induced by exposure to N-methyl-D-aspartate (NMDA; 50 microM) in the presence of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA; 50 microM). Cell extracts were analyzed by 13C magnetic resonance spectroscopy subsequent to the incubation or depolarization. The percentage of GABA generated from glutamine via the TCA cycle decreased from 60% to 46% during depolarization, inducing GABA release from the cytoplasmic pool, whereas a significant change in this parameter was not observed after release from the vesicular pool. These observations indicate that, during release from the cytoplasmic pool, the fraction of GABA synthesized directly from glutamine without involvement of the TCA cycle is more pronounced than that occurring during resting conditions and when release occurs from the vesicular pool. This might be explained by differences in the regulation of the two isoforms of glutamate decarboxylase (GAD(65) and GAD(67)), which presumably play different roles in the maintenance of GABA in the two pools. Both isoforms were found in the cultured cerebral cortical neurons, as shown by Western blotting employing an antibody recognizing GAD(65) as well as GAD(67).
    Document Type:
    Reference
    Product Catalog Number:
    AB1511