Journal: bioRxiv

Article Title: Ligand binding promiscuity of αVβ3 integrin is enlarged in response to mechanical force

doi: 10.1101/200493

Figure Lengend Snippet: αVβ3 integrin favors binding to vitronectin (Vn) compared to fibronectin (Fn). ( a ) Microcontact printing of 2×2 μm squares of Alexa Fluor 647 labeled Fn (blue) onto glass cover slips and backfilling the pattern with Alexa Fluor 568 labeled Vn (red) leads to differential Fn/Vn patterns as shown by the fluorescence intensity profile along the arrow. Geometrical coverage varies dependent on the pattern quality: 44-49% Fn, 56-51% Vn. ( b ) Height profiles of Fn patterns (left) and Fn/Vn patterns (right) measured with atomic force microscopy (AFM) in contact mode. Profiles along the white lines indicate a monolayer of Fn and a uniform topography of the binary choice substrates. ( c ) Super-resolution structured illumination microscopy (SR-SIM) image of a NIH 3T3 cell transfected with β3-wt GFP integrin (green), cultured on a Fn/Vn pattern (Fn in blue), and immunostained for paxillin (red). The cell contour is outlined with a dashed white line. ( d ) Quantification of colocalization of β3-wt GFP integrin with Fn or Vn for fixed cells (n = 66; N = 4). ( e ) Quantification of colocalization of β3-wt GFP integrin with Fn for all adhesions (“general”) or only for those bigger than the indicated threshold (re-analysis of the data from d). ( f ) Total number of αVβ3-mediated adhesions that initiated on Vn, Fn, or at the Fn/Vn-interface for cells imaged with live cell SR-SIM. Quantification is based on 246 initiated adhesions from six cells out of three independent experiments. ( g ) NIH 3T3 cell transfected with β3-wt GFP integrin (white) seeded on Fn/Vn patterns (Fn in blue) was monitored with live cell SR-SIM (also see Supplementary Video 1). Magnifications show initiation and maturation of αVβ3-mediated adhesions (time is given in h:min). Green arrows point to newly established adhesions. Yellow arrows follow adhesions that initiated on Vn while they translocate to Fn. The red arrow at 13 min indicates an adhesion that appeared at the Fn/Vn-interface. ( h ) Representative curves for Fn and Vn association (0 sec to 300 sec) and dissociation (300 sec to 550 sec) to αVβ3 integrin measured with biolayer interferometry. ( i ) K D values for the interaction of purified αVβ3 integrin to Vn or Fn measured with biolayer interferometry (values calculated from n = 9 measurements in N = 3 independent experiments). ( j ) Single-cell force spectroscopy of GD25 cells (expressing αVβ3, but no β1 integrin). Detachment forces on Fn and Vn measured after the indicated contact time points. Typically 10 force measurement repetitions were performed for each cell and time point, and a total of 8 cells were tested. ( a, c, g ) Scale bars: 10 μm in overviews, 2 μm in zoom-ins.

Article Snippet: Binary choice susbtrates were produced with human plasma fibronectin (Sigma-Aldrich, #F2006 or Millipore, #FC010), human plasma vitronectin (Sigma-Aldrich, #V8379), recombinant human vitronectin (Sigma-Aldrich, #SRP3186), native human fibrinogen (Bio-Rad, #4440-8604), recombinant human thrombospondin-1 (R&D systems, #3074-TH-050), or with osteopontin from bovine milk (Sigma-Aldrich, #O3514).

Techniques: Binding Assay, Labeling, Fluorescence, Microscopy, Transfection, Cell Culture, Purification, Spectroscopy, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

doi: 10.1074/jbc.RA117.001382

Figure Lengend Snippet: Oxidation of immobilized TG2 activity assay. Reduced, recombinant TG2 (green) was immobilized onto fibronectin-coated plates and incubated with oxidants (blue) for up to 30 min at 37 °C, producing inactivated TG2 (red). Oxidants were then washed away, and 200 μm 5BP and 5 mm CaCl2 were added to analyze TG2 activity. 5BP is a biotinylated primary amine that is a good substrate of TG2 and is therefore cross-linked to Gln residues on extracellular matrix proteins, such as fibronectin, in the presence of catalytically active extracellular TG2 (26). Streptavidin-HRP binds to cross-linked 5BP and turns over TMB, producing a blue color that can be spectrophotometrically monitored at 655 nm. The steady-state slope was extracted and was proportional to TG2 activity.

Article Snippet: Transamidation activity of immobilized TG2 10 μg/ml human plasma fibronectin (fc010, EMD Millipore) was diluted in PBS and coated onto an ELISA 96-well plate overnight at 4 °C.

Techniques: Activity Assay, Recombinant, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

doi: 10.1074/jbc.RA117.001382

Figure Lengend Snippet: Pseudo-first order kinetic profile of TG2 oxidation by common biological small molecule oxidants at 37 °C (pH 7.5). All experiments were conducted with 10 nm reduced TG2 immobilized on fibronectin-coated plates (Fig. 1). Each oxidant was added at a concentration of 1 mm to the assay mixture. Experiments were performed in quadruplicate with three technical replicates of each time point, and data were fit to a decaying exponential in GraphPad Prism 7.0 to obtain an apparent rate constant, kapp. The bimolecular rate constant was derived by dividing the kapp by the concentration of oxidant (1 mm). Data are presented as average ± S.D.

Article Snippet: Transamidation activity of immobilized TG2 10 μg/ml human plasma fibronectin (fc010, EMD Millipore) was diluted in PBS and coated onto an ELISA 96-well plate overnight at 4 °C.

Techniques: Concentration Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

doi: 10.1074/jbc.RA117.001382

Figure Lengend Snippet: Colocalization of CXXC proteins with extracellular TG2. HUVEC cultures were fixed without permeabilization and stained with antibodies against TG2 (red) and individual CXXC proteins (green). Fibronectin, a known protein partner of extracellular TG2, and VE cadherin, an extracellular protein with no known affinity for TG2, were evaluated as controls. White arrows in the Overlay images denote areas of colocalization between TG2 and the partner protein. Images were taken in triplicate; a representative image is presented. Scale bar, 100 μm.

Article Snippet: Transamidation activity of immobilized TG2 10 μg/ml human plasma fibronectin (fc010, EMD Millipore) was diluted in PBS and coated onto an ELISA 96-well plate overnight at 4 °C.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

doi: 10.1074/jbc.RA117.001382

Figure Lengend Snippet: ERp57 specifically oxidizes TG2 in vitro. A, dose dependence of TG2 oxidation by CXXC proteins. 10 nm reduced TG2 was immobilized onto fibronectin and incubated with each oxidized CXXC protein for 30 min at 37 °C. After a series of washes with PBS, 200 μm 5BP and 5 mm CaCl2 were added to probe for TG2 activity (see Fig. 1). Experiments were performed in quadruplicate with three technical replicates per concentration. B, time dependence of TG2 oxidation by CXXC proteins. 10 nm reduced TG2 was immobilized onto fibronectin and oxidized with 1 μm PDI, ERp57, or ERp72 for up to 30 min at 37 °C. After a series of PBS washes, 200 μm 5BP and 5 mm CaCl2 was added to probe for TG2 activity. Experiments were performed in triplicate with three technical replicates per time point, and the first-order kinetic profile was fit to obtain an apparent rate constant, kapp. The bimolecular rate constant was derived by dividing the kapp by the initial oxidant concentration (1 μm). C, TG2 remains bound to fibronectin even after oxidation by PDI, ERp57, or ERp72. Immobilized TG2 was incubated with each oxidant for up to 30 min at 37 °C and probed with a rabbit anti-TG2 antibody. The amount of bound TG2 was determined by incorporation of an anti-rabbit HRP-conjugated secondary antibody and TMB liquid substrate system. TMB turnover was monitored continuously for 30 min, and the steady-state slope from 0 to 5 min is presented. D, either CXXC active site of ERp57 can oxidatively inactivate TG2. ERp57 mutants were generated to inactivate the redox-active Cys residues in domain a (SSCC), domain a′ (CCSS), or both domains (SSSS). 10 nm reduced TG2 was immobilized onto fibronectin, and incubated with oxidized proteins for 30 min at 37 °C. After a series of PBS washes, 200 μm 5BP, and 5 mm CaCl2 was added to probe for TG2 activity. Experiments were performed in duplicate with three technical replicates per condition. Data are presented as average ± S.D.

Article Snippet: Transamidation activity of immobilized TG2 10 μg/ml human plasma fibronectin (fc010, EMD Millipore) was diluted in PBS and coated onto an ELISA 96-well plate overnight at 4 °C.

Techniques: In Vitro, Incubation, Activity Assay, Concentration Assay, Derivative Assay, Generated

Journal: microPublication Biology

Article Title: Filamin A interacting protein 1-like (FILIP1L) has mitochondrial localization

doi: 10.17912/micropub.biology.001572

Figure Lengend Snippet:

Article Snippet: Fibronectin Human Purified , Krackeler Scientific, 45-FC010.

Techniques: Modification, Purification, Electron Microscopy