This proposal will exploit a well-controlled experimental system that involves recombinant Collagen II and chondrocytes to study how mutations in fibrillar collagens influence the behavior of cells, change their spatial arrangement, and influence the assembly of the extracellular matrix in affected tissues. The effect of mutations on the interaction of mutant collagens with other macromolecules will also be analyzed. The present application is based on the following observations: (i) attachment and motility of chondrocytes depends on their interaction with specific domains of Collagen II, (ii) density of Collagen II fibrils, organization of hypertrophic chondrocytes, and morphology of chondrocytes secretory cysternae in mice expressing the mutated human COL2A1 gene are altered because of the Cys for Arg-alpha -519 substitution, and (iii) the Cys for Arg-alpha l -519 substitution in Collagen II alters its interaction with normal Collagen IX The overall goal of this proposal is to test hypotheses that mutations in fibrillar collagens affect not only extracellular but also affect the intracellular interaction of mutant proteins with other macromolecules, alter the structure of the extracellular matrix, and change the spatial arrangement of cells. To test these hypotheses, a cell-matrix experimental system, which exploits chondrocytes and recombinant Collagen II will be employed. To eliminate the influence of normal endogenous Collagen II on the behavior of cells, the existing transgenic mice with an inactivated Col2al gene will be exploited to obtain chondrocytes that do not produce procollagen II but express other cartilaginous macromolecules. These chondrocytes will be seeded onto engineered three-dimensional nanofibrillar scaffolds coated with mutated recombinant Collagen II. In another series of experiments, the mouse chondrocytes that do not express Col2al but express the normal human C0L2A1 will be transfected with mutant DNA constructs to express procollagen II that contains mutant chains in addition to normal ones. The cells that express the mutant protein will be analyzed for their ability to assemble a cartilaginous extracellular matrix in a long-term suspension culture. Moreover, the interaction of single mutant Collagen a-chains that resemble intracellular, non-folded Collagen, with other macromolecules will be studied.
The Specific Aims are: (1) To determine how extracellular signals from mutant fibrillar collagens deposited in extracellular matrix influence the behavior and spatial organization of cells. (2) To analyze the density and distribution of Collagen fibrils assembled from mutant procollagen secreted and processed by cells. (3) To study intra- and extracellular interactions of mutant fibrillar collaqen with other molecules.
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