Connecting plasma membrane function to lipid structure and organization with asym

描述（由申请人提供）：在过去的半个世纪里，随着分子生物学和生物化学的发展，我们对疾病的理解，以及这种理解推动了对生物技术和分子医学治疗方法的探索，解释分子行为的主要理论框架一直是结构-功能关系。然而，我们对分子生物学的结构-功能关系的理解在很大程度上仅限于中心法则的分子：蛋白质和寡核苷酸。在中心法则之外，特别是脂类和碳水化合物中，分子结构的巨大多样性表明，这些也可以从结构驱动功能的角度来理解。特别是，人们对脂质在质膜中可能发挥的功能作用非常感兴趣。该项目部署了一类新的合成脂质双分子层，开始绘制脂质分子结构之间的联系-无论是在单个分子结构还是超分子组织方面-以及质膜的功能。这里开发和部署的研究工具被称为不对称巨型单层囊泡（aguv），旨在独特地模拟细胞膜，比其他现有的合成脂质双分子层更好地捕获成分不对称和分子拥挤等特性。aguv可以帮助回答的许多问题之一涉及跨细胞膜的被动运输。被动转运是药物传递和环境毒素进入细胞的重要途径。最近的研究结果表明，这种转运的机制是复杂的，并且高度依赖于脂质行为。该项目部署了一种基于aguv的技术，用于系统地测量溶质分子与脂质双层相互作用和穿透的动力学，产生比其他方法能够提供的更丰富的机理数据。aguv还可用于研究细胞膜的力学特性。这些特性——尤其是抗弯曲性——控制着蛋白质的功能，在一系列生理过程中都很重要。虽然合成脂质双分子层已被用来探测这些性质，但人们对双分子层不对称对它们的影响知之甚少。本项目使用aguv来发现这些效果。脂质与整体膜蛋白的相互作用可能是脂质影响细胞行为的主要方式。然而，关于这些相互作用的起源或控制参数所知甚少。该项目通过使用荧光显微镜探测多肽如何模拟各种蛋白质的跨膜区域与分离的脂质结构域相关联，开始解决aguv中的这个问题。最后，aguv可以促进系统研究细胞内部大分子拥挤的影响。形成aguv的微流体技术允许在其中包含任意分子，从而导致独特的分子拥挤结构。

DESCRIPTION (provided by applicant): Over the past half century, as molecular biology and biochemistry have developed to inform our understanding of disease, and as this understanding has driven the search for treatments in biotechnology and molecular medicine, the dominant theoretical scaffold for interpreting molecular behavior has been the structure-function relationship. Our understanding of molecular biology's structure-function relationships is largely limited, however, to the molecules of the central dogma: proteins and oligonucleotides. The vast variety of molecular structure outside of the central dogma, particularly among lipids and carbohydrates, suggests that these, too, can be understood in terms of structure driving function. In particular, there has been intense interest in the functional role that lipids might play in the plasma membrane. This project deploys a new class of synthetic lipid bilayers to begin drawing connections between the structure of lipid molecules-both in terms of individual molecular structure and supermolecular organization-and the function of the plasma membrane. The research tools developed and deployed here, called asymmetric giant unilamellar vesicles (AGUVs), are designed to uniquely mimic the cell membrane, capturing properties such as compositional asymmetry and molecular crowding better than other existing synthetic lipid bilayers. One of the many questions that AGUVs can help answer involves passive transport across the cell membrane. Passive transport is an important route for drug delivery and passage of environmental toxins into cells. Recent results show that the mechanism of this transport is complex, and highly dependent on lipid behavior. This project deploys an AGUV-based technique for systematically measuring the dynamics of solute molecules interacting with and penetrating lipid bilayers, yielding richer mechanistic data than other approaches are capable of delivering. AGUVs can also be used to study the mechanical properties of the cell membrane. These properties- particularly resistance to bending-control protein function and are important in a range of physiological processes. While synthetic lipid bilayers have been used to probe these properties, little is known about the effects of bilayer asymmetry on them. This project uses AGUVs to discover these effects. Lipid interactions with integral membrane proteins are likely a major mode by which lipids influence cell behavior. Very little is known, however, about the origins or controlling parameters of these interactions. This project begins to untangle this problem in AGUVs by using fluorescence microscopy to probe how peptides modeling the transmembrane regions of various proteins associate with segregated lipid domains. Finally, AGUVs can facilitate the systematic study of the effects of macromolecular crowding in the cell interior. The microfluidic technique by which AGUVs are formed allows for the inclusion of arbitrary molecules within them, leading to unique molecularly crowded structures. PUBLIC HEALTH RELEVANCE: Cells are surrounded by membranes that consist of two layers of lipid molecules, and the chemical composition is different in these two layers. In this project, a new type of artificial cell membrane that is uniquely capable of mimicking this asymmetry is deployed to study a range of important biological processes, including drug transport into cells, mechanical deformation of the cell membrane, and interactions between lipid molecules and receptor proteins involved in cancer treatment and diabetes.