title: Early fibrillin-1 assembly monitored through a modifiable recombinant cell approach.
abstract: Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length fibrillin-1 assembly system that allows for rapid analysis of critical roles in fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.
authors.affiliation: Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University , Montreal, Quebec H3A 0C7, Canada.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hubmacher
authors.forename: Eric
authors.initials: E
authors.lastname: Bergeron
authors.forename: Christine
authors.initials: C
authors.lastname: Fagotto-Kaufmann
authors.forename: Lynn Y
authors.initials: LY
authors.lastname: Sakai
authors.forename: Dieter P
authors.initials: DP
authors.lastname: Reinhardt
mesh_terms: D000818:Animals; D002452:Cell Count; D018920:Coculture Techniques; D000071838:Fibrillin-1; D000071837:Fibrillins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D057809:HEK293 Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D020894:Microfibrils / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D009154:Mutation; D041681:NIH 3T3 Cells / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054392:Adipokines
chemical_list: D000071838:Fibrillin-1; D000071837:Fibrillins; D005353:Fibronectins; D006493:Heparin; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C000605722:FBN1 protein, human; D054392:Adipokines; C000605724:Fbn1 protein, mouse