Membrane Organization in Cell Signaling and Adhesion

描述（由申请人提供）：拟议研究的总体目标是了解静息细胞和受体激活细胞质膜中蛋白质和脂质组织和分布的过程和机制。作为模型系统，我们将重点关注g蛋白偶联受体（gpcr）和整合素。趋化因子/趋化因子gpcr，如n -甲酰基肽受体（FPR），调节白细胞功能的许多方面，包括在生理损伤的情况下控制造血、趋化和激活。GPCR激活被认为导致受体重新分布到质膜中的筏中。这种激活也会导致整合素功能的改变，导致聚集并转化为高亲和力状态。整合素聚类已被认为是调节其结合相互作用的快速性及其生理功能的关键决定因素。因此，膜内蛋白质的组织（聚类）和蛋白质脂质分布（筏）可能是膜蛋白正常功能的关键决定因素。尽管人们普遍认为木筏在调节生物功能方面起着至关重要的作用，但事实证明，从大小、组成和动力学方面对木筏进行可靠的表征是极其困难的。值得注意的是，由于囊泡的运输，脂质代谢和脂质成分的插入和去除，膜的化学成分是动态的。我们建议结合光谱技术，通过测量荧光标记的脂质探针和与荧光标记的小分子配体结合的受体之间的能量转移，来实时成像活细胞中脂质膜横向组织的变化。数据将根据供体（在脂质探针上或与蛋白质结合）和受体（在脂质探针或蛋白质上）之间最接近的距离进行分析。膜中最接近的距离反映了蛋白质或构象（整合素）的大小，排除受体的边界脂质的存在，或供体在膜上的距离。目标是：描述与整合素激活和连接膜组织相关的结构功能关系。目标2。实时评估GPCR信号与膜重组的关系。目标3。生成整合素GFP嵌合体和细胞质尾部α /L和α 4β 1突变体，并评估K562细胞的横向组织。拟议的工作提供了一个独特的培训机会，使用生物物理工具和概念结合分子和细胞生物学方法来解决细胞生物学的巨大兴趣问题。完成所提出的工作将更好地掌握膜组织作为信号传导和细胞粘附因素的动力学机制。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to understand processes and mechanisms involved in the organization and distribution of proteins and lipids in the plasma membrane of both resting and receptor activated cells. As model systems, we will focus on G-protein coupled receptors (GPCRs) and integrins. Chemokine/chemoattractant GPCRs, such as the N-Formyl peptide receptor (FPR), regulate numerous aspects of leukocyte function, including controlling hematopoiesis, chemotaxis and activation as in the event of physiological insult. GPCR activation has been suggested to lead to a redistribution of receptor into rafts in the plasma membrane. This activation also leads to alterations in the functions of integrins, resulting in clustering and conversion to a high affinity state. Integrin clustering has been suggested to be a critical determinant in regulating the avidity of its binding interactions and hence its physiological function. Thus, both the organization of the protein within the membrane (clustering) and the distribution of lipids about the protein (rafts) are likely to represent critical determinants in the proper functioning of membrane proteins. Although rafts are widely believed to play a critical role in mediating biological functions, it has proven exceedingly difficult to provide a robust characterization of rafts in terms of size, composition, and dynamics. It is noted that chemical composition of membranes is dynamic due to trafficking of vesicles, metabolism of lipids and insertion and removal of lipid components. We propose to use a combination of spectroscopic techniques to image real time changes in the lateral organization of lipid membranes in live cells by measuring energy transfer between fluorescently-labeled lipid probes and receptors bound to fluorescently labeled small molecule ligands. The data will be analyzed in terms of distances of closest approach between donors (on lipid probes or bound to proteins) and acceptors (on lipid probes or proteins). Distance of closest approach in membranes reflect the size of a protein, or conformation (integrins), the presence of boundary lipid which excludes the acceptor, or the distance of the donor above the membrane. The aims are: Aim 1. To characterize the structure function relationship associated with integrin activation and connection to membrane organization. Aim 2. To assess in real time the relationship between GPCR signaling and membrane reorganization. Aim 3. To generate integrin GFP chimerae and cytoplasmic tail alpha/L and beta2 mutants of alpha4beta1 and assess lateral organization in K562 cells. The proposed work provides a unique training opportunity to use biophysical tools and concepts combined with molecular and cell biological approaches to address problems of immense interest to cell biology. Completion of the proposed work will yield a better mechanistic grasp of the dynamics of membrane organization as a factor in signaling and cell adhesion.