Biomimetic Systems for Studying Nanoscale Structure Formation in Cell Membranes

研究细胞膜纳米级结构形成的仿生系统

基本信息

批准号：
7821480
负责人：
NOAH MALMSTADT
金额：
$ 15.77万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7821480
关键词：
Address Beds Behavior Biological Models Biomimetics Cell membrane Cells Cellular Structures Chemicals Coupling Cytoplasm Cytoskeletal Proteins Cytoskeleton Data Detection Development Diffusion Disease Elements Energy Transfer Engineering Environment Evaluation Event Fluorescence Fluorescence Microscopy Gel Health Human Hydrogels Image Imaging technology Indium Investigation Laboratories Lipid Bilayers Lipids Liquid substance Mechanics Mediating Membrane Membrane Microdomains Membrane Proteins Methods Microfluidics Modeling Molecular Monitor Nanostructures Nature Non-Insulin-Dependent Diabetes Mellitus Optics Pathway interactions Peptides Phase Physical condensation Physiological Physiological Processes Play Polymers Process Property Research Research Personnel Resolution Role Signal Transduction Structure System Techniques Technology Testing Transmembrane Domain Vesicle Viral Virus Diseases base density design fluorescence imaging in vitro Model lipid structure membrane model molecular scale nanoscale novel novel strategies novel therapeutic intervention novel therapeutics physical state public health relevance receptor research study single molecule tool

项目摘要

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.

描述（由申请人提供）：在过去的十年中，细胞膜中的脂质分子分离成多个液相、形成所谓的脂质筏的能力已被确定为重要的生理过程。脂筏是细胞膜中的控制元件，它们参与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。由合成双层构建的细胞膜的体外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，这些特性对于再现脂质的生理行为可能很重要。这些特性包括双层与下面的聚合物细胞骨架的机械附着以及细胞膜的组成不对称性。该提案呼吁制造新型人工双层结构，以更好地模拟细胞质膜的结构，并作为研究脂筏行为的研究平台。这将通过构建充满聚合物水凝胶的囊泡双层结构（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，膜将通过化学结合物理固定在其上。现有细胞膜人工双层模型的一个主要缺点是它们无法正确重现实际细胞中发现的纳米级脂筏，从而产生微米级的脂质域。有重要证据表明，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尺寸之间的关系。它还将提供一个多功能的研究平台，可用于研究各种与脂质结构相关的问题。纳米级脂质结构的研究需要开发能够解决这些微小结构的分析技术。该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，该技术将允许检测和评估具有空间和时间分辨率的纳米级筏。还提出了一种微流体技术，用于以逐层方式在 GUV 上组装双层，从而可以控制每层的成分，并促进非对称双层的制造，例如构成质膜的双层。与水凝胶细胞骨架一起，该技术将允许形成一种新型人造细胞，可以准确地模拟真核细胞质膜的最重要特性。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其环境的相互作用，因此是许多疾病状态的核心参与者，包括 2 型糖尿病和病毒感染。虽然模拟细胞膜的合成脂质双层一直是阐明脂质结构形成分子机制的重要工具，但这些系统无法重现真实细胞膜的重要特性。这里提出的新的人造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性，使它们能够作为研究平台来了解脂质纳米结构的行为以及新的治疗方法如何改变脂质介导的过程。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Excitation of Cy5 in self-assembled lipid bilayers using optical microresonators.

使用光学微谐振器激发自组装脂质双层中的 Cy5。

DOI：
10.1063/1.3576908
发表时间：
2011
期刊：
Applied physics letters
影响因子：
4
作者：
Freeman,LindsayM;Li,Su;Dayani,Yasaman;Choi,Hong-Seok;Malmstadt,Noah;Armani,AndreaM
通讯作者：
Armani,AndreaM

Microfluidic fabrication of asymmetric giant lipid vesicles.