Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis (labeling fibronectin fibrillogenesis).

Department of Cytology, Histology and Embryology, University of Sofia, 1164, Sofia, Bulgaria.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 522:261-74. DOI: 10.1007/978-1-59745-413-1_18
Source: PubMed

ABSTRACT Fibronectin fibrillogenesis is a cell-mediated, step-wise process that converts soluble fibronectin into insoluble fibronectin matrix. The deposition of fibronectin fibrils occurs at specific sites on the cell surface and depends on the unfolding of the fibronectin dimer. Fibronectin matrix provides positional information for cell migration during early embryogenesis and plays an important role in cell growth, differentiation, survival, and oncogenic transformation. Here we present simple techniques, based on the use of fluorescently labeled fibronectin and species-specific antifibronectin antibodies that allow determination of the fibronectin fibril growth in conventional in vitro cell cultures and in three-dimensional matrix environment.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Circulating plasma fibronectin (pFN), produced by hepatocytes, is a major component of the noncollagenous bone matrix where it was recently shown in vivo in mice to control the biomechanical quality as well as the mineral-to-matrix ratio in bone. FN fibrillogenesis is a process generally requiring FN binding to cellular integrins, and cellular tension to elongate and assemble the molecule. Whether soluble pFN (not synthesized by osteoblasts) undergoes cell-mediated assembly in bone is not fully established. FN is a well-known substrate for transglutaminases (TGs), which are protein-crosslinking enzymes capable of stabilizing macromolecular structures. The role of this modification regarding the function of FN in bone matrix has remained unknown. Osteoblasts express two TGs-transglutaminase 2 and Factor XIIIA-and we have shown that Factor XIIIA is the main TG active during osteoblast differentiation. In the present study, conducted using MC3T3-E1 osteoblast cultures and bone marrow stromal cells, pFN required a TG-mediated crosslinking step to form osteoblast matrix in vitro. This modification step was specific for pFN; cellular FN (EDA-FN) did not serve as a TG substrate. Inhibition of pFN assembly using a TG inhibitor, or depletion of pFN from cell culture serum, dramatically decreased total FN matrix assembly in the osteoblast cultures and affected both the quantity and quality of the type I collagen matrix, and decreased lysyl oxidase and alkaline phosphatase levels, resulting in decreased mineralization. Experiments with isozyme-specific substrate peptides showed that FXIIIA is responsible for the crosslinking of pFN. Addition of exogenous preactivated FXIIIA to osteoblast cultures promoted pFN assembly from the media into matrix. Exogenous TG2 had no effect. Analysis of pFN and EDa-FN networks by immunofluorescence microscopy demonstrated that they form distinct matrix network, albeit with minor overlap, suggesting different functions for the two FN forms. Further analysis using EDA-FN blocking antibody showed that it regulated preosteoblast proliferation whereas pFN depletion from the serum had no effect on this process. In conclusion, our study shows that pFN assembly into bone matrix in vitro requires FXIIIA transglutaminase activity making pFN assembly an active, osteoblast-mediated process.
    Bone 11/2013; 59. DOI:10.1016/j.bone.2013.11.006 · 4.46 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.
    Nature 07/2014; 511(7509):319-25. DOI:10.1038/nature13535 · 42.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects.
    Soft Matter 07/2014; 10(35). DOI:10.1039/c4sm00819g · 4.15 Impact Factor