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Vol. 10, Issue 11, 3595-3605, November 1999

Characterization of an In Vitro Model of Elastic Fiber Assembly

Bruce W. Robb,* Hiroshi Wachi,* Theresa Schaub,dagger Robert P. Mecham,*Dagger and Elaine C. Davisdagger §

 *Department of Cell Biology and Physiology and  Dagger Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110; and  dagger Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039

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.

§   Corresponding author. E-mail address: davis16{at}utsw.swmed.edu.

Molecular Biology of the Cell
Vol. 10, 3595-3605, November 1999
Copyright © 1999 by The American Society for Cell Biology


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