Biomimetic Systems for Studying Nanoscale Structure Formation in Cell Membranes

描述（由申请人提供）：细胞膜中的脂质分子分离成多个液相，形成所谓的脂筏的能力，在过去的十年中被认为是一个重要的生理过程。脂筏是细胞膜中的一个控制元件，它们参与了许多对人类健康有影响的分子途径，包括信号转导和病毒进入。合成双分子层构建的体外细胞膜模型在阐明筏形成的基本机制方面发挥了重要作用。然而，现有的人工双层模型缺乏细胞质膜固有的一些特性，而这些特性可能对再现脂质的生理行为很重要。这些特性包括双分子层与底层聚合细胞骨架的机械附着以及细胞膜组成的不对称性。这一建议要求制造新的人工双层结构，以更好地模拟细胞膜的结构，并作为研究脂质筏行为的研究平台。这将通过构建充满聚合物水凝胶的囊泡双层结构（即所谓的巨型单层囊泡，或guv）来实现。这种水凝胶将作为仿生细胞骨架，膜将通过化学偶联在物理上固定在其上。现有的人工双层细胞膜模型的一个主要缺点是它们不能正确地再现在实际细胞中发现的纳米尺度的脂筏，而是产生微米大小的脂结构域。有重要的证据表明，细胞中筏的大小受到膜与细胞骨架的机械附着的限制。构建仿生细胞骨架将允许精确控制膜-细胞骨架附着物的性质和密度，因此可以详细研究细胞骨架附着物和筏子大小之间的关系。它还将提供一个多功能的研究平台，可用于研究各种各样的脂质结构相关问题。脂质结构在纳米尺度上的研究需要能够解决这些微小结构的分析技术的发展。本提案概述了一套基于全内反射荧光显微镜和Fvrster共振能量转移的技术，该技术将允许以空间和时间分辨率检测和评估纳米级筏。还提出了一种微流控技术，用于在guv上逐层组装双层膜，允许控制每层的组成，并促进不对称双层膜的制造，如组成质膜的双层膜。与水凝胶细胞骨架一起，这项技术将允许一种新型的人造细胞，准确地模仿真核生物质膜的最重要特性。公共卫生相关性：细胞膜中的脂质纳米结构有助于控制细胞与环境的相互作用，因此是许多疾病状态（包括2型糖尿病和病毒感染）的核心行为者。虽然模拟细胞膜的合成脂质双层是阐明脂质结构形成的分子机制的重要工具，但这些系统无法再现真实细胞膜的重要特性。本文提出的新的人工细胞结构模拟了细胞膜中发现的细胞骨架附着和成分不对称，使它们成为了解脂质纳米结构如何表现以及新治疗方法如何改变脂质介导过程的研究平台。

DESCRIPTION (provided by applicant): The capacity of lipid molecules in cell membranes to separate into multiple liquid phases, forming so- called lipid rafts, has in the past decade been identified as an important physiological process. Lipid rafts are a control element in the cell membrane, and they take part in numerous molecular pathways with implications for human health, including signal transduction and viral entry. In vitro models of the cell membrane built from synthetic bilayers have played an important role in elucidating the fundamental mechanisms underlying raft formation. Existing artificial bilayer models, however, lack some properties that are inherent to the cell plasma membrane and that are likely important in reproducing the physiological behavior of lipids. These properties include the mechanical attachment of the bilayer to an underlying polymeric cytoskeleton and the compositional asymmetry of the cell membrane. This proposal calls for the fabrication of novel artificial bilayer constructs that will better mimic the structure of the cell plasma membrane and serve as research platforms for investigating lipid raft behavior. This will be accomplished by building vesicular bilayer structures (so called giant unilamellar vesicles, or GUVs) that are filled with a polymer hydrogel. This hydrogel will serve as a biomimetic cytoskeleton, and the membrane will be physically anchored to it via chemical conjugation. One major shortcoming of existing artificial bilayer models of the cell membrane is their inability to properly recapitulate the nanometer scale of lipid rafts found in actual cells, producing instead micrometer-sized lipid domains. There is significant evidence that the size of rafts in cells is limited by the mechanical attachment of the membrane to the cytoskeleton. Building a biomimetic cytoskeleton will allow for precise control over the nature and density of membrane-cytoskeleton attachments and therefore a detailed investigation of the relationship between cytoskeletal attachment and raft size. It will also provide a versatile research platform that can be used to investigate a wide variety of lipid structure-related questions. Investigation of lipid structures at the nanoscale requires the development of analytical techniques that can address these tiny structures. This proposal outlines a set of techniques based on total internal reflection fluorescence microscopy and Fvrster resonance energy transfer that will allow for the detection and evaluation of nanoscale rafts with spatial and temporal resolution. Also proposed is a microfluidic technology for assembling bilayers on GUVs in a layer-by-layer fashion, allowing for the composition of each layer to be controlled and facilitating the fabrication of asymmetric bilayers like those that compose the plasma membrane. Together with the hydrogel cytoskeleton, this technology will allow for a new type of artificial cell that mimics accurately most important properties of the eukaryotic plasma membrane. PUBLIC HEALTH RELEVANCE: Lipid nanostructures in cell membranes help control how cells interact with their environments and are therefore central actors in many disease states, including type-2 diabetes and viral infection. While synthetic lipid bilayers modeling the cell membrane have been important tools for elucidating the molecular mechanisms that underlie lipid structure formation, these systems fail to reproduce important properties of real cell membranes. The new artificial cell constructs proposed here mimic both the cytoskeletal attachment and compositional asymmetry found in cell membranes, allowing them to serve as research platforms for understanding how lipid nanostructures behave and how novel therapeutic approaches can alter lipid- mediated processes.