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Regulatory mechanisms and interactions of extracellular molecules

细胞外分子的调控机制和相互作用

基本信息

批准号：
14207076
负责人：
YANAGISHITA Masaki
金额：
$ 31.28万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2005
项目状态：
已结题

项目摘要

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.

细胞外基质除了为动物体内各种结构提供机械支撑外，还为细胞提供生存环境。因此，它参与许多重要的生物功能，包括细胞分化、增殖、代谢和对环境刺激的调节反应。特别是来自细胞外的生物信号对于调节基本细胞功能非常重要。我们特别关注细胞表面发生的细胞外基质分子与细胞分子之间的分子相互作用的分析，作为研究细胞外基质分子功能意义的相关实验系统。具体来说，假设特殊的质膜结构域参与与细胞外基质的分子相互作用。通过用 Triton X-100 溶解细胞培养物，然后在蔗糖梯度中超速离心，制备耐去污剂的质膜制剂。通过蛋白质（例如 CD 38 和 Lyn）的共定位来鉴定特化膜结构域的存在。通过使用针对硫酸乙酰肝素蛋白多糖的抗体探测的蛋白质印迹分析来检查从甲状旁腺细胞缝合线中分离出的膜结构域制剂，这表明syndecan-4硫酸乙酰肝素蛋白多糖显着富集并且排除了syndecan-1。使用 MALDI-TOF 质谱进行进一步确认正在进行中。实验还表明硫酸乙酰肝素蛋白聚糖参与膜域的结构稳定性。硫酸乙酰肝素降解酶对体外细胞培养物膜结构域形成的影响正在研究中。未来的实验将包括详细检查硫酸乙酰肝素蛋白聚糖如何参与信号转导以及它们如何促进质膜的机械完整性。