Biophysical Mechanisms of Protein Recruitment to Raft Domains

细胞生物学和膜生物物理学中最引人注目的研究问题之一是如何理解纳米尺度膜非均质性（结构域形成）与膜功能之间的关系。特别是鞘脂(SL)-胆固醇(CHOL)-富结构域，也被称为脂筏，已被确定在生物膜的功能中起关键作用。虽然筏结构域的重要功能作用已经确立，但筏组装的生物物理学仍然知之甚少。这个研究项目旨在解决一个与这个仍然开放的话题相关的重要问题：什么是参与膜蛋白募集到筏结构域的生物物理机制？目标将是探索三种最可能的招募机制：(1)通过结合配体；(2)通过膜蛋白周围富含sl - choll的脂质壳；(3)通过其他筏子相关的蛋白质和脂质。为了解决蛋白质募集到筏结构域的问题，实验策略将是使用表观荧光显微镜、宽视场单分子荧光显微镜和荧光相关光谱（FCS）研究平面模型膜中的募集过程。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，而无需在细胞中发现的巨大复杂性。成像实验将对膜蛋白进行，这些膜蛋白被认为是永久筏外（转铁蛋白受体），永久筏相关[尿激酶纤溶酶原激活物受体（uPAR）]和暂时筏相关（avb3和a5b1整合素）。互补成像方法将允许PI研究蛋白质-筏共定位（epifluorescence microscopy），膜成分依赖的蛋白质横向迁移（宽视场单分子荧光显微镜），以及小蛋白质-脂质和蛋白质-蛋白质聚集体（FCS）的形成。该项目将成为PI目标的重要组成部分，该目标是在经典科学和工程学科之间开发创新的跨学科研究和教学计划。为了实现这一目标，研究成果将被落实到PI教授的课程中。此外，本项目将为1名本科生和2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德国优秀研究小组的学生一起参与合作研究。最后，作为新成立的IUPUI纳米成像中心的联合主任，PI还寻求通过提供生物分子成像的尖端设施，扩展到当地社区，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.