Biophysical Mechanisms of Protein Recruitment to Raft Domains

目前在细胞生物学和膜生物物理学中讨论的最吸引人的研究问题之一是与理解纳米尺度膜异质性(结构域形成)和膜功能之间的关系的问题。特别是，富含鞘脂(SL)-胆固醇(Chol)的结构域，也被称为脂筏，已被确定在生物膜的功能中发挥关键作用。虽然已经确定了RAFT结构域的重要功能作用，但对RAFT组装的生物物理仍知之甚少。这项研究项目试图解决与这个仍然悬而未决的话题相关的一个重要问题：膜蛋白招募到RAFT结构域的生物物理机制是什么？我们的目标是探索三种最可能的募集机制：(1)通过结合配体；(2)通过膜蛋白周围富含SL-Chol的脂膜；(3)通过其他RAFT相关蛋白和脂类。为了解决蛋白质募集到RAFT结构域的问题，实验策略将是利用荧光显微镜、广场单分子荧光显微镜和荧光相关光谱(FCS)来研究平面模型膜的募集过程。模型膜方法使膜蛋白募集研究能够在受控的膜条件下进行，而不需要在细胞中发现的巨大复杂性。成像实验将在膜蛋白上进行，膜蛋白被认为是永久排除RAFT的(转铁蛋白受体)，永久与RAFT相关的[尿激酶型纤溶酶原激活剂受体(UPAR)]，以及暂时与RAFT相关的(avb3和a5b1整合素)。这种互补成像方法将使PI能够研究蛋白质-RAFT共定位(荧光显微镜)、依赖于膜成分的蛋白质横向迁移率(广场单分子荧光显微镜)以及小的蛋白质-脂和蛋白质-蛋白质聚集体(FCS)的形成。该项目将是PI在古典科学和工程学科之间发展创新的、跨学科的研究和教学计划目标的重要组成部分。为了达到这一目标，研究成果将被落实到国际和平研究所教授的课程中。此外，该项目还将为一名本科生和两名研究生提供科学培训。由于跨学科团队合作和沟通是培训计划的关键要素，PI研究小组的学生将有机会与来自德国优秀研究小组的学生参与合作研究。最后，作为新成立的IUPUI纳米级成像中心的联席主任，PI还寻求通过为生物分子成像提供尖端设施来扩展到当地社区，以促进跨学科研究。

One of the most fascinating research problems currently discussed in cell biology and membrane biophysics is that which relates to understanding the relationship between nanoscale membrane heterogeneity (domain formation) and membrane function. In particular, sphingolipid (SL)-cholesterol (CHOL)-rich domains, also known as lipid rafts, have been identified to play a key role in the functionality of biomembranes. Though the important functional role of raft domains has been established, the biophysics of raft assembly remains poorly understood. This research project seeks to address an important question related to this still open topic: What are the biophysical mechanisms that are involved in the recruitment of membrane proteins to raft domains? The goal will be to explore the three most likely mechanisms of recruitment: (1) via binding ligands; (2) via SL-CHOL-rich lipid shells around membrane proteins; and (3) via other raft-associated proteins and lipids. To address the question of protein recruitment to raft domains, the experimental strategy will be to study the recruitment processes in planar model membranes using epifluorescence microscopy, wide-field single molecule fluorescence microscopy, and fluorescence correlation spectroscopy (FCS). The model membrane approach enables membrane protein recruitment studies to be conducted under controlled membrane conditions without the great complexity found in cells. Imaging experiments will be performed on membrane proteins, which are considered to be permanently raft-excluded (transferrin receptor), permanently raft-associated [urokinase plasminogen activator receptor (uPAR)], and temporarily raft-associated (avb3 and a5b1 integrins). The complementary imaging approach will allow the PI to study the protein-raft co-localization (epifluorescence microscopy), the membrane composition-dependent protein lateral mobility (wide-field single molecule fluorescence microscopy), and the formation of small protein-lipid and protein-protein aggregates (FCS). This project will be an important part of the PI's goal to develop innovative, interdisciplinary research and teaching programs at the interface between classical sciences and engineering disciplines. To reach this goal, research results will be implemented into courses taught by the PI. Furthermore, this project will provide scientific training for one undergraduate and two graduate students. Because interdisciplinary teamwork and communication are key elements of the training program, students of the PI's research group will have the opportunity to participate in collaborative research with students from excellent research groups in Germany. Finally, as co-director of the newly established IUPUI Nanoscale Imaging Center, the PI seeks also to outreach into the local community to promote interdisciplinary research by providing cutting-edge facilities for biomolecular imaging.