Lipid Rafts and Signal Transduction by MHC Class II Molecules

本项目的目标是探索脂筏（细胞膜上具有独特脂质成分的离散小区域）与B淋巴细胞（一种白色血细胞）表面上称为主要组织相容性复合物（MHC）II类分子的一类重要蛋白质的相互作用。 脂筏被认为是膜蛋白跨细胞表面传递有关细胞外条件的信息以启动细胞内反应的位点。 MHC II类分子有助于调节B细胞对T细胞和外来抗原肽的应答。 具体目的是1）检查特定II类分子在何处和何时与脂筏缔合，以及这种缔合如何与细胞上II类分子的数量和类型相关，以及2）评估II类结构突变和用肽和/或交联剂处理如何影响脂筏缔合。这些研究将采用创新的光学测量，允许蛋白筏协会进行评估，对完整的细胞，包括荧光能量转移标记的蛋白质筏脂质和霍乱毒素扰动的蛋白质旋转，支持膜裂解物的密度梯度离心分析。 筏假说，即脂筏是信号转导事件的关键位点，正在成为细胞生物学的主要范式。然而，这一领域的大多数研究都是通过生物化学方法对破碎的细胞进行的。 该项目将采用光学方法来评估活细胞表面上的筏-蛋白质相互作用的性质。 因此，这些结果将对理解所有类型的细胞（而不仅仅是项目中要检查的细胞）如何对环境因素做出反应具有广泛的意义。 为了促进教学、培训和学习，两名研究生将是拟议项目的主要参与者，项目期间将改进的生物物理技术将有助于生物科学的研究基础设施。将与达特茅斯学院的研究人员合作开展工作，这将有助于在不同领域的研究人员之间传播现代科学。

The goal of this project is to explore interactions of lipid rafts, discrete small regions of the cell membrane having unique lipid compositions, with an important class of proteins, called Major Histocompatibility Complex (MHC) class II molecules, on the surfaces of B lymphocytes, a type of white blood cell. Lipid rafts are believed to be the sites where membrane proteins transmit information concerning extracellular conditions across the cell surface to initiate intracellular responses. MHC class II molecules help regulate B cell responses to T cells and to foreign antigenic peptides. The specific objectives are 1) to examine where and when specific class II molecules associate with lipid rafts and how this association relates to the number and type of class II molecules on the cell and 2) to assess how class II structural mutations and treatment with peptides and/or crosslinking reagents affect raft association. These studies will employ innovative optical measurements allowing protein-raft association to be evaluated on intact cells, including fluorescence energy transfer from labeled proteins to raft lipids and cholera toxin perturbation of protein rotation, as supported by density gradient centrifugation analysis of membrane lysates. The raft hypothesis, namely that lipid rafts are a key site for signal transduction events, is becoming a major paradigm in cell biology. However, most studies in this area have been performed by biochemical methods on disrupted cells. This project will employ optical approaches to evaluate the nature of raft-protein interactions on living cell surfaces. Thus the results will have wide implication for understanding how cells of all types, not just those to be examined in the project, respond to environmental factors. To promote teaching, training and learning, two graduate students will be key participants in the proposed project and the biophysical techniques to be refined during the project will contribute to research infrastructure of the biological sciences generally. Work will be conducted in collaboration with investigators at Dartmouth College which will facilitate disseminating modern science between researchers in diverse fields.