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

项目摘要 许多细胞过程，如扩散、运动、分裂和形态发生，都会产生膜张力。 渐变。这样的梯度驱动膜流动，从而松弛初始梯度。此外，静止的细胞 保持恒定的表面积和相对稳定的膜张力𝜎，通过平衡 膜被添加(通过胞吐作用)和移除(通过内吞作用)到细胞表面和从细胞表面移除。𝜎的变化 已经被提出提供快速、远距离的细胞信号。然而，质膜如何流动和 𝜎的梯度如何松弛还知之甚少，膜张力平衡时间的估计在 细胞数量从毫秒到几十分钟不等。造成这种缺乏的主要原因之一是 知识是缺乏合适的方法来测量活细胞的膜张力变化。在过去, 已经开发了两种膜张力测量方法，但都有严重的局限性。这个 第一类是基于小分子光学性质的变化。这些传感器探测本地属性 细胞膜。由于生物膜中的大的异质性，以及探针的潜在相互作用 不同的膜成分，将𝜎与这些小分子探测的局部性质相关联 传感器不是直截了当的。第二种方法依赖于从细胞中拉出一条薄膜系绳。 并使用光学捕捉器或原子力显微镜测量系缆力。系缆力是 与在线膜张力、膜弯曲模数和膜与膜之间的粘着能有关 质膜和细胞骨架。这种方法允许测量“真正的”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 最有可能出现的是张力梯度，并且仅提供局部测量。因此，尽管存在紧急情况 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 流程。我们建议通过开发一种全新的膜张力来弥合这一差距 基于DNA的传感器基于细胞表面弹性网络的自组装，称为 柠檬水，用于基于自组装DNA弹性网络的乐高类膜张力分析仪。 我们的目标是1)开发一个在细胞上自组装的DNA瓷砖和连接器弹簧的文库 表面形成具有可调特性的网络。各种DNA瓷砖和连接器弹簧设计 将产生并优化膜上的自组装。网络的连接性和弹性 网络将可以通过替换具有不同性质的组件来调节。扩张或收缩 将使用位于连接器上的FRET染料对来检测由于膜面积变化引起的网络- 弹簧模块。2)表征基于DNA的膜张力传感器对 控制各种细胞中的膜张力扰动。我们将使用巨型脂质体，红血 细胞，以及贴壁细胞，以校准柠檬水对膜张力受控变化的反应。

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.