Characterization of an in vitro model of elastic fiber assembly

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic...

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Bibliographic Details
Published in:Molecular biology of the cell 1999-11, Vol.10 (11), p.3595-3605
Main Authors: Robb, B W, Wachi, H, Schaub, T, Mecham, R P, Davis, E C
Format: Article
Language:English
Subjects:
Animals
Cattle
Cells, Cultured
Chondrocytes
Ciliary Body - metabolism
Contractile Proteins - metabolism
Desmosine - analysis
Elastic Tissue - metabolism
Elastin - genetics
Elastin - metabolism
Extracellular Matrix - ultrastructure
Extracellular Matrix Proteins
Fibrillins
Fluorescent Antibody Technique
Humans
In Vitro Techniques
Microfibrils - metabolism
Microfibrils - ultrastructure
Microfilament Proteins - chemistry
Microscopy, Electron
Transfection
Tropoelastin - genetics
Tropoelastin - metabolism
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Summary:Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.
ISSN:1059-1524
1939-4586
DOI:10.1091/mbc.10.11.3595