Altered Matrix-Cells and Intermolecular Interactions

描述（由申请人提供）：本提案将利用一个控制良好的实验系统，涉及重组胶原II和软骨细胞，研究纤维状胶原的突变如何影响细胞的行为，改变它们的空间排列，并影响受影响组织中细胞外基质的组装。突变对突变胶原与其他大分子相互作用的影响也将被分析。本申请是根据下列观察提出的：(i)软骨细胞的附着和运动取决于它们与胶原蛋白II的特定结构域的相互作用；（II）表达突变的人类COL2A1基因的小鼠中，胶原蛋白II原纤维的密度、肥大软骨细胞的组织和软骨细胞分泌胱氨酸的形态因arg - α -519取代的Cys而改变。（iii） II型胶原中arg - α 1 -519取代的Cys改变了其与正常的IX型胶原的相互作用。本研究的总体目标是验证纤维性胶原中的突变不仅影响细胞外，还影响突变蛋白与其他大分子的细胞内相互作用，改变细胞外基质的结构，改变细胞的空间排列的假设。为了验证这些假设，将采用利用软骨细胞和重组胶原II的细胞基质实验系统。为了消除正常内源性胶原II对细胞行为的影响，将利用现有的Col2al基因灭活的转基因小鼠获得不产生前胶原II但表达其他软骨大分子的软骨细胞。这些软骨细胞将被植入工程三维纳米纤维支架上，支架上涂有突变的重组胶原II。在另一系列实验中，将不表达Col2al但表达正常人类C0L2A1的小鼠软骨细胞转染突变DNA构建物，以表达除正常链外含有突变链的前胶原II。将分析表达突变蛋白的细胞在长期悬浮培养中组装软骨细胞外基质的能力。此外，将研究类似于细胞内非折叠胶原蛋白的单突变胶原a链与其他大分子的相互作用。具体目的是：(1)确定沉积在细胞外基质中的突变原纤维胶原的细胞外信号如何影响细胞的行为和空间组织。(2)分析细胞分泌加工的突变前胶原组装成的胶原原纤维的密度和分布。(3)研究突变原纤维胶原与其他分子的胞内胞外相互作用。

DESCRIPTION (provided by applicant): 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.