Regulatory mechanisms and interactions of extracellular molecules

细胞外基质除了为动物的各种身体结构提供机械支撑外，还为细胞提供生存环境。因此，它参与了许多重要的生物学功能，包括细胞分化、增殖、代谢和对环境刺激的调节反应。尤其是来自细胞外的生物信号对调节细胞的基本功能是非常重要的。我们特别关注细胞外基质分子之间的分子相互作用的分析，细胞分子发生在细胞表面，作为研究细胞外基质分子功能意义的相关实验系统。具体地说，假设特殊的质膜结构域参与了与细胞外基质的分子相互作用。用Triton X-100溶解细胞培养物，然后用蔗糖梯度超速离心法制备了一种耐洗涤剂的质膜。通过CD38和Lyn等蛋白质的共定位，鉴定了特定膜结构域的存在。从甲状旁腺细胞缝合中分离出的膜结构域制剂，通过与硫酸乙酰肝素蛋白多糖抗体的免疫印迹分析，表明Syndecan-4硫酸乙酰肝素蛋白多糖显著浓缩，而Syndecan-1被排除。使用MALDI-TOF质谱学的进一步确认正在进行中。实验还表明，硫酸乙酰肝素蛋白多糖参与了膜结构域的结构稳定性。正在研究硫酸乙酰肝素降解酶对体外细胞培养中膜结构域形成的影响。未来的实验将包括详细检查硫酸乙酰肝素蛋白多糖如何参与信号转导，以及它们如何有助于质膜的机械完整性。

In additionto providing mechanical supports for various body structures in animals,extracellular matrix also provides living environments for cells. Thus, it is involved in many crucial biological functions, including cell differentiation, proliferation, metabolism and regulated responses to environmental stimuli. Especially biological signals coming from outside cells are very important regulating fundamental cell functions. We have especially focused our attention on the analysis of molecular interactions between extracellular matrix molecules cellular molecules occurring at the cell surface, as a pertinent experimental system studying functional significance of extracellular matrix molecules. Specifically, a hypothesis that specialized plasma membrane domains are involved in the molecular interactions with extracellular matrix. A detergent-resistant plasma membrane preparation was made by solubilizing cell cultures with Triton X-100 followed by ultracentrifugation in a sucrose gradient. The presence of a specialized membrane domain was identified by colocalization of proteins, such as CD 38 and Lyn. A membrane domain preparation isolated from parathyroid cell sutures was examined by Western blot analyses probed with antibodies against heparan sulfate proteoglycans, which indicated significant enrichment syndecan-4 heparan sulfate proteoglycan and exclusion of syndecan-1. Further confirmation using MALDI-TOF mass spectroscopy has been on the way. Experiments also suggested that heparan sulfate proteoglycans participate in the structural stability of membrane domains. Effects of heparan sulfate degrading enzymes on the formation of membrane domains in the cell cultures in vitro are being studied. Future experiments will include detailed examination how heparan sulfate proteoglycans may be involved in the signal transduction and how they are contributing to the mechanical integrity of plasma membranes.