Self-assembled DNA elastic networks for measuring membrane tension in live cells

用于测量活细胞膜张力的自组装 DNA 弹性网络

• 批准号: 10196486
• 负责人: ERDEM KARATEKIN
• 金额: $ 25.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196486
• 关键词: Actins; Adhesions; Area; Atomic Force Microscopy; Biological; Cell Line; Cell membrane; Cell physiology; Cell surface; Cells; Cholesterol; Confocal Microscopy; Consensus; Cytoskeleton; DNA; DNA Library; Dyes; Elasticity; Endocytosis; Engineering; Entropy; Equilibrium; Equipment; Erythrocytes; Exocytosis; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Geometry; Heterogeneity; Knowledge; Lead; Libraries; Lipids; Liposomes; Liquid substance; Measurement; Measures; Membrane; Membrane Potentials; Methods; Modeling; Modulus; Molecular Probes; Monitor; Morphogenesis; Nanostructures; Oligonucleotides; Optics; Pharmaceutical Preparations; Physiological; Play; Property; Role; Secretory Cell; Signal Transduction; Single-Stranded DNA; Slide; Spectrin; Stretching; Surface; Tertiary Protein Structure; Testing; Thick; Thinness; Time; Transmembrane Domain; base; cell motility; design; fluorescence imaging; imaging modality; laser tweezer; mechanical properties; millisecond; novel strategies; optical traps; response; self assembly; sensor; small molecule; success; tool

Project Summary Many cellular processes, such as spreading, motility, division, and morphogenesis generate membrane tension gradients. Such gradients drive membrane flows, which relax the initial gradients. In addition, quiescent cells maintain a constant surface area and a relatively stable membrane tension, 𝜎, by balancing the rates at which membrane is added (via exocytosis) and removed (via endocytosis) to and from the cell surface. Changes in 𝜎 have been proposed to provide rapid, long-range cellular signaling. Yet, how the plasma membrane flows and how gradients of 𝜎 relax are very poorly understood, with estimates of membrane tension equilibration times in cells ranging from milliseconds to tens of minutes. One of the major reasons underlying this dearth of knowledge is the lack of suitable methods for measuring membrane tension changes in live cells. In the past, two classes of membrane tension measurements have been developed, but both have severe limitations. The first class is based on changes in optical properties of small molecules. These sensors probe local properties of cell membranes. Due to large heterogeneities in biological membranes, and potential interactions of the probes with various membrane components, correlating 𝜎 with the local properties probed by these small molecule sensors is not straightforward. The second approach relies on pulling a thin membrane tether from the cell surface and measuring the tether force using optical trapping or atomic force microscopy. The tether force is related to the in-line membrane tension, membrane bending modulus, and the adhesion energy between the plasma membrane and the cytoskeleton. This approach allows a "true" membrane tension to be measured, but requires specialized equipment, is very difficult to implement when cells undergo physiological changes when tension gradients are most likely to arise, and only provides a local measurement. Thus, despite the urgent need, there are no direct and convenient probes to quantify membrane tension gradients during cellular processes. We propose to close this gap by developing a radically new class of membrane tension sensors based on DNA-based self-assembly of an elastic network over cell surfaces, called LEMONADE, for Lego-like membrane tension analyzer based on self-assembled DNA elastic networks. We aim to 1) develop a library of DNA tiles and connector-springs that self-assemble on cell surfaces into a network with tunable properties. A variety of DNA tile and connector-spring designs will be generated and optimized for self-assembly on membranes. The connectivity and elasticity of the network will be tunable by substitution of components with different properties. Expansion or contraction of the network due to changes in membrane area will be detected using FRET dye pairs located on the connector- spring modules. 2) Characterize the response of the DNA-based membrane tension sensor to controlled membrane tension perturbations in various cells. We will use giant liposomes, red blood cells, and adhering cells to calibrate the response of LEMONADE to controlled changes in membrane tension.

项目概要 许多细胞过程，例如扩散、运动、分裂和形态发生都会产生膜张力 这种梯度驱动膜流，从而放松初始梯度。 通过平衡速率来保持恒定的表面积和相对稳定的膜张力𝜎 膜在细胞表面添加（通过胞吐作用）和去除（通过内吞作用）。 已被提议提供快速、远距离的细胞信号传导。然而，质膜如何流动和如何流动。 人们对 𝜎 松弛的梯度如何了解知之甚少，膜张力平衡时间的估计为 细胞周期从几毫秒到几十分钟不等，这是造成这种深度的主要原因之一。 过去，缺乏测量活细胞膜张力变化的合适方法。 已经开发出两类膜张力测量方法，但两者都有严重的局限性。 第一类是基于小分子光学特性的变化，这些传感器探测小分子的局部特性。 由于生物膜的巨大异质性以及探针之间潜在的相互作用。 与各种膜成分，将 𝜎 与这些小分子探测到的局部特性相关联 传感器并不简单。第二种方法依赖于从细胞中拉出薄膜系绳。 表面并使用光学捕获或原子力显微镜测量系链力。 与在线膜张力、膜弯曲模量以及膜之间的粘附能有关 这种方法可以测量“真实”的膜张力，但是 需要专门的设备，当细胞发生生理变化时很难实现 张力梯度最有可能出现，并且仅提供局部测量，尽管紧急。 需要，没有直接和方便的探针来量化细胞过程中的膜张力梯度 我们建议通过开发一种全新的膜张力来缩小这一差距。 基于 DNA 的细胞表面弹性网络自组装传感器，称为 LEMONADE，基于自组装 DNA 弹性网络的乐高式膜张力分析仪。 我们的目标是 1) 开发一个在细胞上自组装的 DNA 瓦片和连接器弹簧库 表面形成具有可调特性的网络。 将生成并优化膜上的自组装的连接性和弹性。 网络可以通过替换具有不同属性的组件来进行调整。 由于膜面积变化而产生的网络将使用位于连接器上的 FRET 染料对进行检测 - 2) 表征基于 DNA 的膜张力传感器的响应。 我们将使用巨型脂质体、红血来控制各种细胞的膜张力扰动。 细胞和粘附细胞来校准 LEMONADE 对膜张力受控变化的反应。